Phosphorus Assimilation and Control of the Phosphate Regulon
Chapter
87
BARRY L. WANNER
This chapter concerns the genes and gene products belonging to the phosphate (Pho) regulon of Escherichia coli and the regulation of these genes. By definition, all genes of a regulon are subject to a common molecular control. Genes of the Pho regulon are regulated by the two-component regulatory system composed of the response regulator PhoB (a transcriptional activator) and its partner, the sensor kinase PhoR. Altogether, 38 different Pho regulon genes have now been characterized in E. coli or closely related bacteria. Most of the corresponding gene products are known to have roles in the use of various phosphorus (P) compounds as sole P sources for growth. The roles of some gene products are unknown. The expression of the Pho regulon is inhibited when the preferred P source (inorganic orthophosphate [Pi]) is in excess; the expression of most Pho regulon genes is activated 100-fold or more during growth after exhaustion of environmental Pi. Also, no Pho regulon gene appears to be subject to an individual control, as no gene required for utilization of a specific P compound is regulated in response to the presence of that compound (134, 244, 246, 251).
Information in this chapter deals primarily with the Pho regulon of E. coli. Selected data on other bacterial systems are included only when they seem especially pertinent to the discussion. Chapter 82 in the first edition of this book was entitled "Phosphate Regulation of Gene Expression in Escherichia coli" and was written in December 1984 (242). This chapter was completed in December 1994. It was written with two goals in mind: (i) to provide the casual reader with a concise overview of P assimilation and Pho regulon control and (ii) to provide the more interested reader with a resource of additional information as well as selected citations for further reading on related topics.
Studies on the Pho regulon emanated from early observations that alkaline phosphatase synthesis increased upon Pi starvation (90, 223). Subsequently, numerous individuals have made important contributions to our understanding of the Pho regulon. Also, two books on phosphate metabolism in microorganisms have been published within the past 10 years. One (225) contains seminars of the Pho I Meeting held in Concarneau, France, in June 1986. The other (226) contains seminars of the Pho II Meeting held in Woods Hole, Mass., in September 1993. Although an attempt was made to present an as thorough and up-to-date chapter as possible, there are, undoubtedly, some omissions. I apologize for these beforehand.
In terms of cellular content, P is the third most abundant element (behind carbon and nitrogen). Glucose- or acetate-grown E. coli cells contain about 15 mg of P per g (dry weight) (46). P compounds serve as major building blocks of innumerable biomolecules: P is an essential component of membrane lipids, complex carbohydrates such as lipopolysaccharides, and nucleic acids; and P is incorporated into many proteins posttranslationally. P compounds are also especially important in energy metabolism because of the biological role of high-energy phosphoanhydride bonds. Therefore, P metabolism involves numerous metabolic pathways, in addition to those that may have a specific role in the process of assimilating P from the environment. Some pathways of P metabolism may be of primary importance for P assimilation during exponential growth, while others may be important for scavenging P compounds from the environment, for survival under conditions of P limitation, or for storage of high-energy P compounds under conditions of excess carbon and energy or limitation of biosynthetic capacity. Still others may be indispensable for energy production. This chapter deals primarily with those P metabolic pathways that have, or are likely to have, a role in the process of P assimilation per se.
E. coli uses three kinds of compounds as primary P sources: (i) inorganic phosphates, (ii) organophosphates (phosphate esters), and (iii) phosphonates (Table 1). Regardless of the P source, the overall process of P assimilation has at least two common steps (Fig. 1). The first step involves uptake of the P compound. Pi, a few organophosphates, and phosphonates are taken up intact directly; most organophosphates are hydrolyzed in the periplasm, and the Pi released is then taken up. A later step involves entry of Pi or a phosphoryl group of an alternative P compound into ATP (or a metabolizable organophosphate) via one of several steps in central metabolism (Fig. 2A).
Table 1Systems for uptake and degradation of environmental P sources in E. coli or S. typhimuriuma |
Inorganic phosphates exist in three chemical forms that are of biological importance: Pi, pyrophosphate (PPi), and metaphosphate (polyphosphate [poly(Pi)], also called volutin). Of these, only Pi appears to be taken up from the environment. Systems for transport of Pi are discussed in the section "Pi Uptake." Even though PPi and poly(Pi) are not taken up, both may serve as a sole P source for growth. PPi and poly(Pi) are apparently broken down in the periplasm by bacterial alkaline phosphatase (Bap) at a rate sufficient to serve as a sole P source. This is true because only cells capable of Bap synthesis use these compounds as a P source (186, 276; W. W. Metcalf and B. L. Wanner, unpublished data), even though neither is considered a good Bap substrate in vitro (73, 276).
Organophosphates may serve as a P source in one of two ways, depending on whether they are transportable (Fig. 1B). Most organophosphates are not taken up. Nontransportable ones may enter the periplasm (with or without the aid of a particular porin), where they may be hydrolyzed by a variety of periplasmic enzymes. Pi released in the periplasm is then taken up by a Pi transport system. Bap is a nonspecific phosphomonoesterase and is made at high levels when Pi is limited. Therefore, under these conditions, organophosphates entering the periplasm are likely to be hydrolyzed by Bap. Other periplasmic phosphatases are present under different conditions, and their synthesis is not under Pho regulon control. Particular esters may be substrates of these phosphatases. Individual periplasmic phosphatases are discussed in the sections "Bap" and "Other Periplasmic Phosphatases." A few organophosphates are transportable. In this regard, sn-glycerol-3-phosphate (G3P) is exceptional; a transporter specific for uptake of G3P (the Ugp system) is synthesized under Pho regulon control (12, 29). Accordingly, the expression of genes for the Ugp system is activated, and G3P is taken up intact by this system for use as a P source under conditions of Pi limitation. Other organophosphates may be taken up by transporters whose synthesis is not under Pho regulon control. Systems for transport of organophosphates are described in the section "Organophosphate Uptake."
Phosphonates are a large class of organophosphorus compounds that have a direct carbon-phosphorus (C-P) bond in place of the more familiar carbon-oxygen-phosphorus ester bond of organophosphates. Utilization of phosphonates as a P source requires cleavage of the C-P bond. There are two pathways for C-P bond cleavage for use of phosphonates as a P source (Fig. 1C). They differ in regard to their substrate specificities and mechanism of C-P bond fission. Genes for both of these pathways are members of the Pho regulon, and the expression of these genes is activated under conditions of Pi limitation in E. coli or closely related bacteria (96a, 249, 257). Genes and proteins for use of phosphonates as a P source are described in the section "Uptake and Breakdown of Phosphonates."
E. coli contains three kinds of inorganic phosphates: Pi, PPi, and poly(Pi). During logarithmic growth, the intracellular concentrations of these phosphates are about 10 mM for Pi (202), 0.5 mM for PPi (117), and 0.2 mM (on the basis of P residues) for poly(Pi) [2 μg of poly(Pi) per 1011 cells (45) is equivalent to 0.2 mM P residues if one assumes a cell volume of about 10–15 liter]. Whether there exists a "phosphate balance" for Pi and these high-energy inorganic phosphate compounds, as previously suggested (241), which may be analogous to the balance of AMP, ADP, and ATP in the energy charge (14, 74) has not been investigated. The constancy of intracellular Pi and PPi levels under many (but not all conditions) has been well established. It is unclear whether concomitant changes occur in the Pi, PPi, and poly(Pi) levels. Poly(Pi) levels have only recently been examined in strains in which poly(Pi) metabolism has been altered by mutation or by overexpression of genes for poly(Pi) synthesis or breakdown. Nevertheless, the available data are suggestive of an important (though still mysterious) role for poly(Pi) in physiology (45, 115). It may be for maintenance of a phosphate balance among Pi, poly(Pi), and related metabolites.
Numerous pathways involve Pi, PPi, or poly(Pi) metabolism and hence contribute to the levels of intracellular inorganic phosphates (Fig. 2). These include many well-known catabolic and anabolic pathways, of which only a few crucial ones are mentioned here. Pi is a substrate of enzymes in glycolysis, the tricarboxylic acid cycle, fermentation, and other processes (Table 2). Also, Pi is a coproduct of many biosynthetic reactions. Yet, intracellular Pi levels are maintained at a fairly constant level (about 10 mM) under conditions of aerobic or anaerobic growth on glucose with excess or limiting extracellular Pi (185, 202, 268). Under different growth conditions (especially in the presence of G3P), the intracellular Pi level may vary as much as fourfold (from 5 to 18 mM) (275). The intracellular PPi level is also generally invariant; it decreases from about 0.5 mM to an undetectable level (less than 0.1 mM) only under stressful situations (imposed by the presence of inhibitors or glucose limitation) (47, 117, 118, 119). Much less is known about the effects of growth conditions on poly(Pi) levels. Also, the abundance of poly(Pi) is lower in E. coli than in other cells, making it difficult to quantitate the amounts of poly(Pi) because of technical problems of measuring low levels accurately. The closely related bacterium Aerobacter aerogenes contains much larger amounts of poly(Pi). In A. aerogenes, it has been known for a long time that nutrient limitation (especially of sulfate) leads to increased poly(Pi) accumulation (80, 204). Therefore, it is thought that poly(Pi) may act as an energy storage polymer (reference 120 and references therein). Whether increased poly(Pi) accumulation is due to increased synthesis or decreased breakdown has not been determined.
Table 2Key enzymes involved in intracellular Pi metabolism |
Several enzymes are important in maintaining intracellular levels of PPi and poly(Pi) (Fig. 2). PPi is formed as a coproduct of nucleic acid biosynthesis and other biosynthetic pathways (Fig. 2B). Also, the hydrolysis of intracellular PPi to Pi is an essential driving force of many reactions that release PPi as a coproduct. PPi is broken down by a cytoplasmic pyrophosphatase (the ppa gene product), an essential enzyme in E. coli (38). Poly(Pi) is a linear polymer of tens or hundreds of metaphosphate residues. Poly(Pi) is synthesized from ATP by the enzyme polyphosphate kinase [the ppk gene product, a poly(Pi):ADP phosphotransferase; Fig. 2C], a peripheral membrane protein in E. coli (3). Ppk catalyzes poly(Pi) synthesis by transfer of the γ phosphoryl group of ATP to a poly(Pi) polymer in a freely reversible reaction. Ppk may not be the only enzyme for poly(Pi) synthesis. Mutants with a gene disruption of ppk show a greater than 10-fold reduction in poly(Pi) levels [from 2 μg to 0.16 μg of poly(Pi) per 1011 cells], yet there remains detectable poly(Pi) that appears to be predominantly short-chain polymers (with an average chain length of 60). It may be synthesized by an independent pathway (45).
Under certain conditions the breakdown of PPi and poly(Pi) may contribute to the intracellular Pi pool. Ppa is the only enzyme for PPi breakdown in E. coli. Although PPi may act as an energy source in other bacteria (274), no pathway(s) for conservation of the phosphoanhydride bond energy of PPi exists in E. coli. In contrast, intracellular poly(Pi) may serve as an energy (as well as a phosphate) storage molecule (115). By reversal of the biosynthetic reaction, Ppk catalyzes ATP synthesis by transfer of the terminal phosphoryl group of poly(Pi) to ADP (Fig. 2C). Therefore, poly(Pi) may be an important energy storage molecule and Ppk may be important for energy conservation. In other bacteria, poly(Pi) may act as a substrate of other energy conserving enzymes. Both poly(Pi) and ATP are efficient cosubstrates of glucokinase in Propionibacterium species (175). No similar energy-conserving reaction using poly(Pi) as a cosubstrate has been uncovered in E. coli. Poly(Pi) may also be broken down by two other cytoplasmic enzymes: a polyphosphatase (encoded by ppx of the ppk-ppx operon [4]) and guanosine pentaphosphate phosphohydrolase (encoded by gppA [106]). Poly(Pi) breakdown by Ppx or GppA leads to Pi release without energy conservation (Fig. 2C). Ppx acts as an exopolyphosphatase catalyzing release of terminal phosphate residues. GppA acts as an endopolyphosphatase producing metaphosphates about 40 residues in length and as an exopolyphosphatase releasing terminal phosphates until poly(Pi) is converted to tetraphosphate (106).
While Ppa, Ppk, Ppx, and GppA have no role in the process of environmental P assimilation per se, one or more may be connected to Pho regulon control involving energy metabolism. Also, it has been reported that the ppk-ppx operon is under Pho regulon control (105). The expression of multicopy lacZ fusions to the E. coli or Klebsiella aerogenes ppk promoter has been shown to be decreased in a Pho regulon mutant when starved of Pi (105). Unfortunately, it has been difficult to judge the significance of these observations because of the way in which Pi limitation was imposed. Expression was examined in cultures shifted from a complex medium containing yeast extract to a Pi-free minimal medium. Also, the putative Pho box sequences of the E. coli and K. aerogenes ppk promoters are poorly conserved. There is no other evidence that either ppa, ppk, ppx, or gppA is under Pho regulon control.
Bap is a nonspecific phosphomonoesterase with a pH optimum between 8 and 10. The induced synthesis of Bap (the phoA gene product) in response to Pi limitation is a classic example of enzyme induction in bacteria. Its differential rate of synthesis increases more than 1,000-fold when exponential growth ceases as a result of Pi limitation. Under these conditions, Bap may account for as much as 6% of the total protein synthesized. Because the enzyme is made in large amounts, is relatively stable, and is easy to assay and purify, a substantial amount of its biochemistry and structure is now understood (40, 111). Because of product inhibition by Pi, Bap activity is usually determined by measuring the transphosphorylation reaction in which Tris (generally 1 M) substitutes for H2O as a phosphoryl acceptor (272).
Bap is a periplasmic protein that is synthesized as a precursor protein of 471 residues with a 21-residue N-terminal signal peptide (125). It is secreted into the periplasm both co- and posttranslationally (98). Following removal of its signal peptide by leader peptidase I (the lepB gene product), which is located in the cytoplasmic membrane and cleaves proximal to Arg-22, the mature enzyme may be further processed by removal of Arg-22 by an aminopeptidase (the iap gene product) (94). Multiple isozymes are formed; these are (largely) attributable to the incomplete removal of Arg-22 of one or both subunits of the active enzyme, a 94.4-kDa dimer. Bap becomes active when the monomers are transported into the periplasmic space (157). Activation requires the formation of two intrachain disulfide bonds within each monomer (57). These features of the enzyme have made Bap an attractive model system for studying the mechanism of protein localization as well.
In particular, a truncated form of phoA (designated ‘phoA) lacking the region encoding its signal sequence has been especially useful for studying the localization of other proteins (86). A fusion joining ‘phoA sequences to the coding region of another gene may lead to formation of a hybrid protein in which N-terminal residues are contributed by the other gene and the C-terminal residues are contributed by the ‘phoA sequence. Such gene fusions synthesize a Bap fusion protein that is active only if the Bap moiety of the hybrid protein is localized to the cell surface. Genetic fusions of this sort have become a widely used tool for studying protein localization largely as a result of the development of the transposon TnphoA (143, 144, 145). This is because TnphoA allows for the simple in vivo construction of phoA gene fusions by transposition. Numerous improvements of this TnphoA methodology have led to the construction of alternative delivery vectors (55, 217), as well as modified TnphoA elements (called TnlacZ [142] or TnphoA ' [271] elements), for recombinational switching of TnphoA fusions (249, 271). Studies on protein localization using phoA have been recently reviewed (97, 227).
E. coli has at least six additional periplasmic enzymes with phosphatase activity (Table 1): a pH 2.5 acid phosphatase (the appA gene product [50]); a glucose-1-phosphatase (the agp gene product [180]); a uridine 5'-diphosphoglucose hydrolase (UDP-sugar hydrolase, the ushA gene product), which acts as a nonspecific 5' nucleotidase (32, 63); a 2',3' cyclic phosphodiesterase (the cpdB gene product, a 3' nucleotidase [126]); a hexose-6-phosphatase (62, 109); and an additional nonspecific acid phosphatase (which may be more than one species [177]). All of these except AppA and CpdB have a pH optimum for phosphatase activity of between 4 and 6. AppA and CpdB have pH optima of 2.5 and 7.5, respectively. The verification of multiple phosphatases with similar activities has required the purification and biochemical characterization of individual enzymes, the isolation of mutants lacking a particular enzyme, or the molecular cloning of the corresponding structural genes. Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) has two nonspecific acid phosphatases (the phoN gene product and a second one named AphA, a 3' nucleotidase), which are absent in E. coli (102, 228, 229). No pH 2.5 acid phosphatase or glucose-1-phosphatase has been characterized in S. typhimurium. Like E. coli, most species of Salmonella (S. typhimurium is an exception) contain UDP-sugar hydrolase. UshA catalyzes the degradation of extracellular UDP-glucose to uridine, glucose 1-phosphate, and Pi. Also, both E. coli and S. typhimurium contain Agp, CpdB, and a periplasmic acid hexose-6-phosphatase. Genes corresponding to AphA, the periplasmic hexose-6-phosphatases, or other acid phosphatases have not been characterized. All of these enzymes likely contribute to extracellular (periplasmic) Pi levels under certain conditions, even though none of them appears to have a primary role in P assimilation because none of the respective genes is under Pho regulon control.
AppA has an extremely low pH optimum (ca. 2.5) for activity, suggesting that its physiological role may involve breakdown of strongly acidic substrates. Interestingly, AppA acts on poly(Pi) as a substrate. Further, its catalytic rate constant with poly(Pi) substrates increases with increasing chain length up to five phosphoryl residues (48). Whether AppA has greater activity toward other strongly acidic substrates (such as inositol polyphosphates) has not been determined. AppA is thought to belong to a family of histidine phosphatases with phosphohistidine as an enzyme intermediate (168, 169, 170).
AppA synthesis is induced under conditions of oxygen deprivation, upon entering the stationary phase, and Pi limitation (reference 49 and references therein). Induction is due to increased transcription of an upstream promoter, the appC promoter of the appCBA operon; it is dependent on the RNA polymerase sigma factor RpoS (also known as AppR or KatF and by other acronyms). A minor promoter immediately preceding appA appears to be insignificant in regard to the regulation of AppA synthesis. On the basis of the regulatory characteristics of appA and the location of appA in an operon encoding cytochrome d oxidase-like subunits, it would appear that AppA has a primary role in fermentation or respiration. AppC and AppB have 75 and 70% sequence similarities with the CydA and CydB subunits of E. coli cytochrome d oxidase, respectively.
PhoN is a nonspecific acid phosphatase with a pH optimum near pH 5.5. Its synthesis is induced about two- to fivefold under conditions of nutrient limitations (for carbon, nitrogen, phosphate, or sulfur), mildly acidic pH, and slow or stationary-phase growth (5, 108). phoN is positively regulated by the two-component regulatory system composed of the response regulator PhoP and its partner sensor kinase PhoQ (75, 158). It is uncertain what environmental stimulus leads to activation of the PhoP-PhoQ regulatory system. Interestingly, gene activation by PhoP is much greater (50-fold or more versus 2- to 5-fold) within macrophages than in laboratory media (5). It should be pointed out that (despite the similar designations) phoN, phoP, and phoQ are unrelated to the Pho regulon or its control in E. coli or S. typhimurium. Also, even though phoN is absent in E. coli, phoP and phoQ are present in both species (76, 103). This finding is consistent with results showing that the PhoP-PhoQ system acts as a global regulatory system in the control of many genes other than phoN. Some of these are likely to be present in E. coli. Whether PhoP or phospho-PhoP acts directly at the promoter of phoN, or of other PhoP-regulated genes, has not been established. The PhoP-PhoQ two-component regulatory system is discussed in chapter 154.
Much less is understood concerning the synthesis of Agp, UshA, CpdB, and other acid phosphatases. Although small effects have been noted in regard to the amounts of these enzymes made under various growth conditions, they appear to be largely synthesized constitutively. Agp synthesis (as judged by using an agp-phoA gene fusion) is unaffected by limitations of Pi, nitrogen or sulfur or by an rpoS (appR) mutation (178). Its synthesis is positively regulated by cyclic 3',5'-adenosine monophosphate (cAMP) and the catabolite activator protein (CAP, encoded by crp), since a cya mutation reduces agp expression six- to sevenfold. UshA activity increases about twofold upon entry of cells into stationary phase (165). Whether this increase is due to transcriptional control of ushA has not been determined (32). Interestingly, UshA activity is inhibited by an intracellular protein inhibitor (165), whose synthesis closely parallels synthesis of UshA. The role of the inhibitor protein is unknown. It may prevent UshA from degrading intracellular UDP-glucose or UDP-galactose prior to export; or it may play a role (perhaps as a chaperonin) in the export process. Transcription of cpdB is modulated (up to 3.5-fold) by the carbon source in a manner that is partially dependent upon cAMP and CAP (126). Early studies showed that the amounts of the acid hexose phosphatase and 2',3' cyclic phosphodiesterase are similarly regulated by cAMP and CAP (108). These results are consistent with roles of these periplasmic enzymes capable of degrading sugar phosphates and nucleotides for degradation of such compounds for use as carbon and energy sources. Accordingly, the release of Pi by these enzymes may be of secondary importance.
E. coli has two major Pi transporters: the Pst and Pit systems (Table 1). Pst is a high-affinity, low-velocity system: Pst has a Km of about 0.4 μM and a V max of 15.9 nmol of Pi per min per mg of protein. In contrast, Pit is a low-affinity, high-velocity system: Pit has a Km of 38.2 μM and a V max of 55 nmol of Pi per min per mg of protein (268, 269). When Pi is in excess, Pi is (apparently) taken up by the Pit transporter, which appears to be made constitutively and whose synthesis is not under Pho regulon control. Under conditions of Pi limitation, synthesis of the Pst system is activated more than 100-fold and Pi is (primarily) taken up by the Pst transporter. Whether the amount of the Pst system present as a basal level contributes to Pi uptake when Pi is in excess has not been established. Nevertheless, it is reasonable to suppose that a functional Pst system is present in normal cells under Pi-excess conditions. This is because the Pst system has a negative regulatory role in Pho regulon control; an intact Pst system is required for Pi inhibition of Pho regulon gene expression. The role of the Pst system in this inhibition is described in the section "Pi Control by Transmembrane Signal Transduction." In addition, two organophosphate transport systems (GlpT and UhpT) are capable of Pi uptake as a secondary substrate (81, 176, 267, 273). These are described in the section "Organophosphate Uptake." Also, the PhnCDE phosphonate transporter appears to be capable of Pi uptake (153). The PhnCDE transporter is described in the section "Uptake and Breakdown of Phosphonates."
The Pst system is a periplasmic protein-dependent transporter similar to those for histidine, maltose, and ribose (59). These transporters belong to the superfamily of ABC (ATP-binding cassette) transporters, which are present in a wide variety of cells, including those of mammals (84). An ABC family transporter (also called a traffic ATPase) is composed of a membrane-associated complex containing four proteins (or protein domains). Two of these are transmembrane (integral membrane) proteins that probably form a transport channel. Each of the other two proteins contains an ATP-binding motif; these probably provide the energy for transport via ATP hydrolysis. Furthermore, bacterial ABC transporters having a role in solute uptake usually contain an additional component, a periplasmic binding protein. Accordingly, PstA and PstC are the integral membrane channel proteins, PstB is the ABC family traffic ATPase (sometimes called the permease), and PstS (formerly called PhoS) is the periplasmic binding protein (Table 3). Since PstB has a single ATP-binding motif, PstB probably exists as a dimer in the Pst transporter complex.
Table 3Sequenced psi genes of the E. coli or S. typhimurium Pho regulona |
Genes for each of the Pst components are arranged in an operon (the pstSCAB-phoU operon) together with a gene for a protein called PhoU. It has recently been shown that, as expected, a deletion of phoU has no effect on Pi uptake by the Pst system (211). However, a Δ phoU mutation has an extremely deleterious effect on growth (Table 4). The severe growth defect of a Δ phoU mutation is largely alleviated by a Δ pstB or Δ pstSCAB mutation, as well as by other compensatory mutations (including ones that reduce expression of the pst genes). That is, the growth defect is due to synthesis of a functional Pst system in the absence of PhoU. This finding implies that PhoU may have an auxiliary role in the overall process of Pi assimilation via the Pst system. Once Pi is taken up, Pi is incorporated into ATP for use as a P source. PhoU may be an enzyme for ATP synthesis (Fig. 1A). Accordingly, Pi taken up by the Pst transporter in the absence of PhoU may accumulate, and a high intracellular Pi concentration may lead to growth inhibition. This would explain why Pst+ Δ phoU mutants are Pi sensitive whereas Pst– Δ phoU mutants are not (211). Growth inhibition by Pi has also been observed among Pst mutants in which the Pi transport channel is thought to be permanently switched on (open) by mutation, although the growth defect is much less severe in this case (264). Another laboratory reported contradictory results on the effect of a Δ phoU mutation (161); these workers, unexpectedly, found that a Δ phoU mutation abolished Pi uptake. Because they had not observed a growth defect, they probably studied Pi uptake in a Δ phoU mutant with a secondary mutation in the Pst system. This inference has been substantiated by showing that their mutant is not complemented by a phoU + gene alone (P. M. Steed, W. Jiang, and B. L. Wanner, unpublished data).
Table 4Effects of mutations on Pi control of the Pho regulon |
The Pst system is highly specific for Pi; it recognizes the inorganic oxyanions arsenate and sulfate poorly or not at all (19, 151). This high specificity resides in PstS, a 34.4-kDa protein with a single Pi-binding site (Kd ca. 1 μM [116]). PstS binds directly to both the monovalent (H2PO4 –) and divalent (HPO4 2–) anion, with a slight preference for the divalent anion. The structure of PstS at 1.7 Å (1 Å = 0.1 nm) shows 12 strong hydrogen bonds between the four oxygens of Pi and polar side chains within a binding pocket (129, 236). PstS probably contacts the channel proteins (PstA and PstC) on the periplasmic face of the inner membrane. Residues that may comprise a Pi binding site on the channel proteins or that may be important for translocation of Pi have been identified in PstA and PstC (44, 264). Generally, mutations that abolish Pi transport also abolish inhibition by Pi of Pho regulon gene expression. An exceptional mutation (an R220E change in PstA) abolishes only transport (43). This residue may lie on the periplasmic side of the membrane, where it might interact with PstS (263).
Pit is a single-component transporter analogous to LacY. Also, the substrates of the Pst and Pit systems differ. Whereas Pst transports free Pi, as cited above, Pit transports metal phosphates with similar kinetics for MgHPO4 and CaHPO4 (231). These results raise a question about the physiological role of the Pit system. Pit may actually be a divalent metal transporter, for which Pi is an effective anion. Also, the absence of Pit has no effect on growth with Pi or on Pho regulon control. Whether other metal transporters for Mg2+, Mn2+, or Ca2+ transport Pi as a counterion may have been overlooked because of the frequent use of Pi as a buffer in transport assays. In a study on Ca2+ transport in E. coli, an effect of Pi may simply have not been tested (164). Pit is also capable of arsenate transport; and Pit mutants have been isolated as arsenate-resistant mutants (270). Unfortunately, only a few pit mutants have been characterized. Also, one widely used pit mutation was discovered to exist "naturally" in a derivative of E. coli K-12 called K10 (15). It should be mentioned that many mutants used in early studies of the Pho regulon and the Pst system are descendents of this Pit– strain. Consequently, many of these strains contain compensatory mutations, allowing them to grow on Pi as a P source (211). New studies on the Pit system should be aided by the recent availability of the pit gene sequence (206).
E. coli has three inducible organophosphate uptake systems: Ugp and GlpT for G3P (13, 81) and UhpT for hexose 6-phosphates (176, 273). S. typhimurium also has these systems (64, 83, 95) and, in addition, an uptake system (PgtP) for phosphoenolpyruvate and 2- or 3-phosphoglycerate (191), which is absent in E. coli (Table 1). Synthesis of the Ugp system is inducible by Pi limitation, for uptake of G3P as an alternative P source. Transcription of the ugpBAECQ operon is also activated by carbon starvation (256) via a mechanism involving cAMP and its receptor protein (101, 213). Synthesis of GlpT, UhpT, and PgtP is inducible by a substrate(s) specific for that transporter: GlpT synthesis is inducible by exogenous G3P, UhpT synthesis is inducible by exogenous hexose 6-phosphates, and PgtP synthesis is inducible by exogenous phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate. Also, synthesis of GlpT, UhpT, and PgtP is subject to catabolite repression, in agreement with a primary role of these transporters in the uptake of the respective substrates for use as carbon and energy sources. In addition, the PhnCDE transporter is apparently capable of organophosphate uptake (153). The PhnCDE system is described in the section "Uptake and Breakdown of Phosphonates."
Two systems transport G3P. GlpT transports G3P for use as a carbon source, and as mentioned above, its synthesis is inducible by G3P. The Ugp system transports G3P for use as a P source; its synthesis is activated by Pi limitation and is under Pho regulon control. When present, GlpT is, as expected, capable of G3P uptake for use as a P source as well. Paradoxically, the Ugp system cannot transport G3P for use as a carbon source, even in a mutant synthesizing the Ugp system constitutively (195); the probable explanation is discussed below. The existence of a second uptake system for use of G3P as a P source had been noted earlier (268). The Ugp system itself was discovered by the isolation of second-site revertants of a GlpT– mutant capable of G3P uptake (13). Those mutants turned out to be ones that synthesized the Ugp system constitutively because they were constitutive for expression of the Pho regulon (12).
The Ugp system is a periplasmic binding protein-dependent transport system. The Ugp transporter has a Km of ca. 1.5 μM and a V max of ca. 3 nmol of G3P per min per mg of protein (195). UgpB is the periplasmic binding protein; UgpA and UgpC are the integral membrane channel proteins, and UgpC is the ATPase (Table 3). Genes for each of these components are arranged in an operon (the ugpBAECQ operon) together with the gene for UgpQ, a glycerophosphoryl phosphodiesterase (Table 3). Curiously, this enzyme hydrolyzes only diesters that are in the process of being transported by the Ugp system (28). The inability of G3P to serve as a carbon source when transported exclusively by the Ugp system has been a paradox (195), as the Km and V max values of the Ugp and GlpT systems are similar. Apparently, inhibition of the Ugp system by intracellular Pi interferes with uptake by this system during growth on G3P as a carbon source as a result of accumulation of excess intracellular Pi under these conditions (29).
GlpT, UhpT, and PgtP belong to a family of anion exchangers (141). Each of these is a single-component system that transports its respective organophosphate substrate(s) in an unaltered form. Each is capable of heterologous (organophosphate:Pi) exchanges with its organophosphate substrate or of homologous (Pi:Pi) exchanges (6, 207, 233). An anion exchange mechanism for uptake of organophosphates is an economical one in terms of energy costs. Because the P requirement for growth is only a fraction (about 1/20) of the carbon requirement, a mechanism for Pi export is expected to be required during growth on a phosphorylated compound as a carbon and energy source. This requirement may be met by exchanging Pi. As expected, organophosphate uptake by these transporters is competitively inhibited by Pi. Arsenate is also an exchangeable substrate of these transporters. The existence of a common transport mechanism for GlpT, UhpT, and PgtP is in agreement with the high degree of sequence similarity at the protein level (about 30% amino acid identity) over their entire lengths (65, 71, 279). As pointed out elsewhere (99), they are even more similar if one assumes a frameshift (resulting from a DNA sequencing error or translational frame shifting) within the published pgtP coding region (279).
In any case, none of these anion exchangers (GlpT, UhpT, and PgtP) is thought to play a role in Pi uptake in the absence of a specific inducer for that transporter. Mutants that synthesize either of these systems constitutively have been isolated; in a few cases, such constitutive mutations have apparently allowed for Pi uptake by one of these transporters in the absence of the Pst and Pit systems (66). Further, one laboratory (179) showed that UhpT activity or synthesis may be enhanced during Pi limitation. Under conditions of Pi limitation (but not under conditions of Pi excess), glucose 1-phosphate (a secondary substrate of the UhpT system) may be taken up by the UhpT transporter. Whether the UhpT, GlpT, or PgtP system contributes to Pi uptake under conditions of Pi limitation has not been examined. Net Pi uptake by these anion exchangers may result from nonstoichiometric exchange, as observed for glucose 6-phosphate uptake in an analogous exchanger of Streptococcus lactis (7), or from heterologous exchange of Pi with another anion.
Phosphonates are commonly found in organisms as diverse as Bdellovibrio, Bacteroides, Streptomyces, Tetrahymena, and Trypanosoma species, mollusks, insects, and others. Phosphonates occur principally in the form of phosphonolipids, in which 2-aminoethylphosphonate is found in place of its phosphate ester analog ethanolamine phosphate. Phosphonates exist as constituents of glycolipids, glycoproteins, and polysaccharides as well. Two treatises have focused on phosphonates in biology (85, 89). Also, several newer studies concern the existence of these unusual compounds in nature (18, 54, 79, 110). In certain instances, phosphonates are quite abundant. Phosphonates may account for as much as 30% of the P content of phospholipids in Tetrahymena species (107) and 50% of the total P content in sea anemones (183) and may be the main P compounds in the hemolymph of locusts (110). Despite their widespread occurrence, the biological role of phosphonates in nature is poorly understood.
No doubt because of the abundance of phosphonates in nature, bacteria have evolved pathways for phosphonate breakdown. Two of these pathways are of particular concern in this chapter because they are designed for use of phosphonates as sole P sources. Genes for both of these pathways are under Pho regulon control in E. coli, Enterobacter aerogenes, and S. typhimurium. These pathways are commonly referred to as the phosphonatase and the C-P lyase pathways (Fig. 3). They differ in the catalytic mechanism whereby they break the C-P bond. Genes for both pathways include those for an uptake system that may be specific for a phosphonate degraded by that pathway; also, genes for both pathways include those for degradative enzymes or an enzyme complex necessary for intracellular phosphonate breakdown by that pathway. The phosphonatase pathway acts only on phosphonates with a two-carbon substitution (such as the natural compound 2-aminoethylphosphonate). In a two-step process, it leads to hydrolytic cleavage of the C-P bond by a reaction requiring an adjacent carbonyl group. In contrast, the C-P lyase pathway has a broad substrate specificity; it leads to cleavage of both substituted phosphonates (such as 2-aminoethylphosphonate) and unsubstituted phosphonates (including alkyl- and arylphosphonates) by a reaction mechanism involving redox or radical chemistry. Whereas the P product of the phosphonatase pathway is Pi, the P product of the C-P lyase pathway is unknown. As pointed out elsewhere (122), the P product need not be Pi to allow a phosphonate to serve as a P source; it may be one of many phosphorylated compounds capable of entering cellular metabolism and eventually donating a phosphoryl group to ATP (Fig. 1C). Recent studies have provided a wealth of new information on the molecular biology and molecular genetics of genes for both of these pathways. Our current understanding of the phosphonatase and C-P lyase pathways is summarized below. The more interested reader should consult one of several recent reviews for more detailed information and additional primary literature citations (247, 250, 257).
It has been known for more than 30 years (34, 281) that E. coli is capable of growth on unsubstituted phosphonates such as orthophosphite (phosphonic acid), methylphosphonate, and ethylphosphonate. Compounds with a single H-P bond as in orthophosphite are chemically similar to ones with a single C-P bond. Use of these substrates is characteristic of C-P lyase activity. The carbon product of C-P bond cleavage by a lyase is the corresponding hydrocarbon (Fig. 3B) (51). Numerous other bacteria also break phosphonates down by a C-P lyase pathway. There has been considerable interest in C-P lyases because of their broad substrate specificities. This is largely attributable to the potential use of a C-P lyase in biodegradation processes. Synthetic phosphonates include herbicides, insecticides, and nerve agents as well as antibiotic and antiviral agents (250). Yet, despite intensive efforts, nobody has been able to detect a C-P lyase activity in a cell extract. An in vitro activity reported by one laboratory (162) was subsequently shown to be artifactual by several others (150), because the activity detected (release of Pi) was proven to be independent of substrate addition. As a result, the mechanism of C-P bond cleavage by a lyase is poorly understood.
The finding that E. coli K-12 is capable of using methylphosphonate as a P source after a prolonged lag (235) led to detailed studies of the genes and gene products involved in the C-P lyase pathway. Further, this study showed that the in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the Pho regulon and is abolished in phn (psiD) mutants. The prolonged lag (ca. 36 h) observed with E. coli K-12 is a characteristic of the cryptic nature of the phn(EcoK) locus of E. coli K-12 (253). Earlier studies had used different E. coli strains (34, 281). Yet, most natural isolates of E. coli (including most members of the ECOR collection [198] as well as E. coli B) contain a functional phn locus (253). The functional phn(EcoB) locus of E. coli B was therefore cloned. This was done by complementation of an E. coli K-12 phn (psiD) mutant (253). Its DNA sequence has been determined, and its genes and gene products have been analyzed (37). The phn(EcoB) locus has also been subjected to detailed mutational studies (152, 153, 154, 155; W. W. Metcalf, W. Jiang, and B. L. Wanner, unpublished data).
The phn (psiD) locus comprises 14 genes (named alphabetically phnC to phnP) in an operon with a single promoter upstream of phnC. PhnC, PhnD, and PhnE probably comprise a periplasmic binding protein-dependent phosphonate uptake system (Table 3). Accordingly, PhnC is the ABC family traffic ATPase, PhnD is the periplasmic binding protein, and PhnE is the integral membrane channel protein. This determination is based on sequence similarities (37), periplasmic localization of PhnD determined by TnphoA analysis (Metcalf et al., unpublished data), and requirements of phnC, phnD, and phnE (but none of the other phn genes) in uptake of Pi or an organophosphate in appropriate mutants (153). Presumably, Pi and phosphoserine, as well as 5-bromo-4-chloro-3-indolylphosphate-p-toluidine (the blue dye commonly used to detect phosphatase activities), are nonspecific substrates of the PhnCDE transporter. Direct evidence of a transport function has been hampered by the commercial unavailability of suitable radiolabeled phosphonate substrates. In addition, the cryptic phn(EcoK) locus has been cloned and sequenced (135). Its cryptic nature is due to a frameshift mutation resulting from a short repeated sequence near the 3' end of phnE. The functional phnE(EcoB) gene has a tandem 8-bp duplication within its coding region; the cryptic phnE(EcoK) gene has three tandem copies of these 8 bp, resulting in a frameshift mutation. Further, activation of the cryptic phn(EcoK) locus occurs by loss of one copy of this repeated sequence.
Seven gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, and PhnM) are essential for catalysis, and these proteins are therefore likely to be components of a C-P lyase pathway or C-P lyase enzyme complex (37, 154). Mutations of these genes abolish phosphonate utilization without affecting uptake. In agreement with this finding, it has been shown that these seven gene products are sufficient for methane production with methylphosphonate as a substrate (unpublished data cited in reference 37). PhnN and PhnP also appear to be important, although they may be dispensable. Mutations of these dramatically reduce (but do not abolish) growth on phosphonates (154). PhnN and PhnP may be important for derivatization of the P product of the C-P lyase. PhnM is extremely hydrophobic and so is a strong candidate for an integral membrane protein. Accordingly, a C-P lyase (composed of PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, and PhnM) may exist in the form of an enzyme complex in the membrane, and PhnN and PhnP may be accessory proteins of the complex. The inability to detect C-P lyase activity in vitro may be due to the fragile nature of the complex.
Two additional proteins (PhnF and PhnO) appear to be regulatory proteins. Neither PhnF nor PhnO is required for transport or catalysis. Also, both proteins show sequence similarities with other regulatory proteins. PhnF, like PhnR (encoded by the phosphonatase gene cluster), appears to be a member of the GntR family of transcriptional regulators (82), which includes GntR (280), FadR (58), KorA (96), FarR (182), and others. No information is available on how PhnF (or PhnR) acts, however. PhnO has a helix-turn-helix motif that is commonly found in regulatory proteins. There is also no information on the mode of action of PhnO.
In similar ways, genes for phosphonate degradation were cloned from S. typhimurium (96a). This was done because S. typhimurium appeared to contain genes only for the phosphonatase pathway. An earlier study revealed the presence of genes for both a phosphonatase and C-P lyase pathway in Enterobacter aerogenes (122); the former but not the latter was shown to be homologous to genes for phosphonate degradation of S. typhimurium (96a). Therefore, the S. typhimurium genes were mutated and sequenced (Metcalf et al., unpublished data). The S. typhimurium phn locus is composed of seven genes, named phnR to phnX (Table 3). The expression of these genes is under Pho regulon control, as determined by examination of various phn-lacZ transcriptional fusions. Translation of its DNA sequence indicates that PhnR may be a transcriptional regulator; it has sequence similarities at the protein level with members of the GntR family of regulatory proteins. PhnS, PhnT, PhnU, and PhnV probably comprise a periplasmic binding protein-dependent phosphonate uptake system. Accordingly, PhnS is the periplasmic binding protein, PhnT is the ABC family traffic ATPase, and PhnU and PhnV are the integral membrane channel proteins. This conclusion is based on sequence similarities, periplasmic localization of PhnS determined by TnphoA analysis, and complementation studies among transport and catalysis genes of the E. coli and S. typhimurium phosphonate degradation pathways. Also, the S. typhimurium PhnSTUV transporter appears to be specific for 2-aminoethylphosphonate, in contrast to the E. coli PhnCDE transporter. Further, the sequence similarity between these two transporters is no greater than it is between periplasmic binding protein-dependent transporters having different substrates. PhnW and PhnX are phosphonate-degrading enzymes (Fig. 3A); they correspond to a 2-aminoethylphosphonate:pyruvate aminotransaminase and a phosphonoacetaldehyde hydrolase (trivial name, phosphonatase), respectively.
PhoE was discovered as a new outer membrane protein (originally called protein e, Ic, or E) in compensatory mutants of strains lacking the porins OmpF and OmpC. Because of their slow growth and sensitivity to hydrophobic compounds, ompF and ompC mutants give rise to compensatory mutants synthesizing new membrane proteins (69, 123). PhoE synthesis was shown to be activated under conditions of Pi limitation (172) and subject to Pho regulon control (221). The compensatory mutants were shown to synthesize PhoE because they are constitutive for expression of the Pho regulon. The appearance of an additional outer membrane protein in a Pho regulon constitutive mutant had also been noted earlier (258). The expression of homologous phoE genes is activated by Pi limitation in E. coli (171, 222), S. typhimurium (16, 208), and many closely related bacteria (210, 212, 230).
E. coli PhoE is composed of 330 amino acid residues and is synthesized in a precursor form with a 21-residue amino-terminal signal peptide (171). It is exported into the periplasm, where PhoE forms its tertiary structure, the monomers are assembled into trimers, and the trimers eventually enter the outer membrane (53). Porin proteins are atypical integral membrane proteins since they are not hydrophobic, unlike integral membrane proteins of the cytoplasmic (inner) membrane. Also, they do not contain long α helices of hydrophobic amino acid residues found in typical membrane proteins. High-resolution crystal structures of PhoE and OmpF are quite similar (41). This was expected, as PhoE and OmpF (as well as OmpC) are about 60% identical at the protein level (159, 171). Both PhoE and OmpF form trimers of identical subunits. The polypeptide chain of each monomer forms a 16-stranded antiparallel β-barrel structure surrounding a large channel (41). Since each subunit forms a channel, the trimer contains three channels. The outer surface of the trimeric structure reveals a hydrophobic boundary as well as other features that are likely important for tight association of these porin trimers with membrane lipid head groups and lipopolysaccharides of the outer membrane.
A central channel in each monomer has pore dimensions between 11 and 19 Å at the mouth and 11 by 7 Å in a constricted zone called the eyelet (41). OmpF and OmpC show a slight cation selectivity, presumably due to solute interactions with amino acid side chains near the eyelet. In contrast, PhoE has a preference for anions such as organophosphates and Pi. This selectivity is due to the presence of several lysine residues in its primary sequence. In particular, Lys-125 is crucial for the anion selectivity of PhoE (212). Changing this residue from lysine to glutamate changes its selectivity from anionic to cationic (17). This residue lies within a loop extending into the eyelet region of the constriction zone (41) and so is likely to interact with solutes. Despite this selectivity, PhoE (like OmpF and OmpC) is considered a nonspecific porin. It has only a slight preference for anions and is therefore not truly a "phosphoporin." In particular, PhoE shows no preference for phosphates over other anions (20). Nevertheless, its synthesis may be especially important for diffusion of anions across the outer membrane, including Pi, organophosphates, polyphosphates, teichoic acids, nucleic acids, and others. In contrast, Pseudomonas aeruginosa contains a phosphoporin (OprP) synthesized under conditions of Pi limitation (21) which, like the E. coli maltodextran porin LamB (22) or the nucleoside porin Tsx (the phage T6 receptor [23, 131]), has high specificity toward its respective substrate, Pi.
Pi control of the Pho regulon involves two processes: inhibition when Pi is in excess and activation under conditions of Pi limitation. It is a paradigm of a signal transduction pathway in which occupancy of a cell surface receptor regulates gene expression in the cytoplasm (Fig. 4). PhoR acts as the Pi sensor in this process. Whether PhoR detects Pi directly via a Pi regulatory site (as shown) or indirectly solely via an interaction with the Pst transporter or PhoU is unknown. PhoR is thought to exist in two forms, which were named PhoRA and PhoRR long before it was understood how they may act (255). Expression of the Pho regulon is activated several hundred-fold under conditions of extracellular Pi limitation. This activation requires PhoRA and PhoB, the response regulator. The expression of Pho regulon genes is inhibited when extracellular Pi is in excess (ca. 4 μM). This inhibition requires an intact Pst transporter, a protein called PhoU, and PhoRR; Pi inhibition of Pho regulon gene expression is independent of Pi transport per se (43). Since all of these components are membrane associated, inhibition probably involves formation of a "repression complex" in the membrane (244). As depicted in Fig. 4, interconversion of PhoRA and PhoRR is thought to involve protein-protein interactions between PhoR, PhoU, and the Pst transporter in response to the extracellular Pi level. In any case, PhoRA may predominate under conditions of Pi limitation. Accordingly, PhoRA may correspond to autophosphorylated PhoR, a phosphoryltransferase, and PhoB kinase. A conformational change leading to formation of PhoRA may occur within the repression complex as a result of low Pi occupancy of the Pst system, low Pi occupancy of a PhoR regulatory site, or release of PhoR from the repression complex. A conformational change leading to formation of PhoRR may be brought about by full Pi occupancy of the Pst system, Pi binding to a PhoR regulatory site, or association of PhoR with the repression complex. Further, Pi saturation of the Pst system may facilitate Pi binding to the regulatory site. Either way, PhoRR may predominate when Pi is in excess and may act by facilitating the dephosphorylation of phospho-PhoB. PhoB and PhoR are depicted in contact in Fig. 4, even though such interactions are likely to be transient. They are shown to interact solely in order to indicate that PhoRA and PhoRR may have a direct role in the phosphorylation of PhoB and dephosphorylation of phospho-PhoB, respectively. More detailed mechanisms for how PhoR, PhoU, and the Pst transporter may act in Pi control of the Pho regulon are described elsewhere (251).
PhoB is 229 amino acids in length (138) and is composed of two domains, a highly conserved N-terminal receiver (phosphorylation) domain of ca. 100 amino acids and a C-terminal regulatory domain with a helix-turn-helix motif beginning near residue 195 (data not shown). Its N-terminal domain has sequence similarities with the superfamily of response regulators (174, 234), containing over 170 members (E. C. Kofoid, personal communication). PhoB is a member of the OmpR family of response regulators (234). Its phosphorylation site is Asp-53; also, Thr-83 is important in phosphotransfer (133). A C-terminal subdomain including Glu-177 may be important for interaction with σ 70 (RpoD) of RNA polymerase holoenzyme (132). Both PhoB and phospho-PhoB bind DNA in a site-specific manner (136, 137); however, DNA binding is enhanced by phosphorylation. All phoB alleles behave as null mutations because they result in a negative phenotype (Table 4); a few are leaky in regard to activation of certain promoters such as phoB or pstS. Commonly used alleles include phoB19 (a T158K R201C double mutation), phoB23 (E9K), phoB62 (R201H), and phoB63 (G185R) (277). Among these, the best null allele appears to be phoB23, which is a charge change at a residue highly conserved in other family members.
PhoR is a cytoplasmic membrane sensor 431 amino acids in length (139). It is probably composed of three domains. It has an N-terminal domain of about 50 amino acids with two α-helical transmembrane segments capable of forming a helical hairpin and a 5- to 7-amino-acid region that may be exposed to the periplasm. This conclusion is inferred on the basis of its sequence and is consistent with topology studies (193). The bulk of PhoR is cytoplasmic and consists of two additional domains. It has an unusually large linker domain of about 150 amino acids immediately following its membrane domain and a highly conserved C-terminal histidine kinase domain beginning near residue 159 (166) in common with transmitter domains of the large family of sensor kinases (174). Other membrane sensor kinases typically have a linker domain of about 50 amino acids within the beginning of the C-terminal conserved domain. A truncated form of PhoR (lacking its N-terminal 83 amino acids) is autophosphorylated by the γ-phosphoryl group of ATP in vitro (136) on His-213 (201), and phospho-’phoR rapidly transfers the phosphoryl group to PhoB. Although it is reasonable to suppose that PhoR may facilitate the dephosphorylation of phospho-PhoB, no such activity has been demonstrated.
Most commonly used phoR mutants contain null mutations, including the phoR17 (Y155 opal), phoR19 (E141 amber), phoR20 (W135 amber), phoR68 (Y225 ochre), and phoR78 (K98 ochre) alleles (277). Two phoR null alleles were also sequenced in this laboratory: phoR68 and phoR70 (a T183N Q186 amber double mutation [B.-D. Chang and B. L. Wanner, unpublished data]). All of these mutations abolish inhibition due to excess Pi and activation due to Pi limitation. Nevertheless, all Pho regulon genes are expressed even in the absence of PhoR. Typically, a phoR null mutation results in low-level expression of the Pho regulon during growth on a glucose minimal medium (Table 4). The actual level is highly regulated by carbon and energy sources. Under these conditions, expression of the Pho regulon is activated by two other signaling pathways; one involves CreC (formerly called PhoM), and the other involves acetyl phosphate synthesis (245, 262). Activation by these pathways is described in the section "Pi-Independent Controls of the Pho Regulon."
Several other phoR alleles are more informative. A classic one, phoR69 (T220N [277; Chang and Wanner, unpublished data]), leads to constitutive signaling. This mutation results in high-level expression of the Pho regulon in the absence of CreC (255) or acetyl phosphate synthesis (M. R. Wilmes-Riesenberg and B. L. Wanner, unpublished data). Indeed, activation due to phoR69 (mutant C3) provided the first evidence of positive control in gene regulation (72, 199). Additional mutations that may lead to constitutive signaling include R204W, T217M, P218S, and E371K (201). Also, truncated forms of PhoR lacking 83 or 158 N-terminal amino acids behave as weak constitutively active PhoR proteins (278). Additional altered-function phoR alleles (Chang and Wanner, unpublished data) include three partially dominant ones (L146P, L147P, and R148C) and six with primary defects in inhibition but not activation (T217A, P218L, P218S, Y225C, L263P, and R380C). The former lie within the PhoR linker region and may define residues important for protein-protein interactions. The latter appear to be specifically defective in the ability to facilitate the dephosphorylation of phospho-PhoB.
PhoU is peripherally associated with the cytoplasmic membrane (214), even though it lacks features of a typical membrane protein. Its membrane association may be due to interaction with the Pst system, PhoR, or both. A sole point mutation (the phoU35 allele) results in an A147E change (263) in a relatively long predicted α-helical region. Because this mutation abolishes Pi inhibition without adversely affecting growth (unlike a Δ phoU mutation; Table 4), PhoU may have distinct functions as a negative regulator and as an enzyme in Pi metabolism (Table 3) (211). PhoU may act together with PhoRR in the dephosphorylation of phospho-PhoB.
The Pst transporter is described in the section "Pi Uptake." That the Pi-binding protein (PstS) is involved in Pi control is supported by the finding that missense changes altering Pi binding (236) abolish Pi inhibition of Pho regulon gene expression (134). Numerous additional Pst mutations, including mutations in the ABC motif of PstB (44) and in the channel proteins PstA and PstC (43, 44, 264), simultaneously abolish Pi uptake and Pi inhibition of gene expression. Deletion of the pst genes leads to high-level expression of the Pho regulon in a manner dependent upon PhoRA and PhoB (Table 4) (211). Two Pst alleles are especially notable in regard to gene regulation. An R220E change in PstA abolishes Pi transport without affecting inhibition of gene expression (43), which shows that these processes may be uncoupled. Curiously, a nonsense mutation (W92 opal; the pstA2 allele [43]) had been previously shown to lead to only partial relief of Pi inhibition (42, 266); however, this effect may depend on the strain (43).
It is interesting that both the Pst system and PhoU are required to inhibit expression of the Pho regulon, yet their synthesis is turned on only under conditions of Pi limitation. In this regard, an unusual mutant (termed a phase mutant [240]) has been shown to be blocked in activation of the pstSCAB-phoU operon. The mutation itself (pstS463, formerly called pho-463) is due to an IS2 in the pstS promoter region (C. S. Schmellik-Sandage and B. L. Wanner, unpublished data). It results in low constitutive level expression of the pstSCAB-phoU operon under all conditions (as determined by examination of pstS-lacZ and pstS463-lacZ transcriptional fusions). Pi limitation of the PstS463 mutant leads to an "induced state" in which cells remain turned on even when subsequently grown under excess Pi conditions. It appears as though inhibition is slowed following a period of Pi limitation. Eventually, these cells are able to turn off expression of the Pho regulon, and hence they display a metastable phase variation-like phenotype. Apparently, increased amounts of the Pst system or PhoU are required in order to reestablish normal inhibition after Pi-limited growth.
An additional class of Pho regulon constitutive mutants have lesions in the phoF locus (240, 242). In some ways, phoF mutants behave like pst mutants, as they abolish both Pi uptake and Pi inhibition of gene expression. They are unlinked to the pst region. Also, phoF mutants have other phenotypes in common that pst mutants do not share. Further studies of phoF have been difficult in part because of an inability to transfer phoF lesions to new strains. Therefore, how "PhoF" may act is unknown. Also, the possibility of double mutations has not been ruled out.
As expected, PhoB and PhoR of other members of the family Enterobacteriaceae are highly conserved. PhoB proteins of Shigella dysenteriae, Shigella flexneri, and Klebsiella pneumoniae are 96% or greater identical (at the amino acid level) with E. coli PhoB (124; GenBank accession number X81001 for Shigella flexneri). PhoR proteins of Shigella dysenteriae and K. pneumoniae are, respectively, 98 and 87% identical (at the amino acid level) with E. coli PhoR (124). S. typhimurium phoB and phoR are also homologous to the respective E. coli genes (96a).
There are indications that similar controls by Pi probably operate in a number of other bacterial species as well. E. coli PhoB has 59 and 48% identity (at the amino acid level) with PhoB of P. aeruginosa (GenBank accession number A37775 [11] and Rhizobium meliloti (GenBank accession number M96261 [unpublished submission]), respectively. Also, P. aeruginosa phoB mutants abolish activation under conditions of Pi limitation. E. coli PhoR has 31% identity to a PhoR candidate of P. aeruginosa (11). Also, E. coli PhoU has 46% identity with P. aeruginosa PhoU (GenBank accession number D28587 [104]) and 42% identity with the partial sequence of an R. meliloti open reading frame (GenBank accession number M96261). Further, phoB and phoR homologs as well as pstA, pstB, and phoU homologs of P. aeruginosa are adjacent, while phoB and phoU homologs of R. meliloti homologs may be adjacent. Mycobacterium leprae also has an open reading frame highly similar to E. coli PhoR (GenBank accession number U00018). In addition, PhoB and PhoR homologs have been identified in Agrobacterium tumefaciens, Bacillus subtilis, and Synechococcus sp. strain PCC7942. ChvI of A. tumefaciens has 35% identity with E. coli PhoB and is required for gene activation during Pi limitation (36, 146). PhoP and PhoR of B. subtilis are 40 and 27% identical with E. coli PhoB and PhoR (121, 196, 197), respectively; these proteins are also involved in gene activation in response to Pi limitation (91). SphR and SphS of Synechococcus sp. strain PCC7942 are 38 and 26% identical to PhoB and PhoR (2), respectively. SphR is required for gene activation during Pi limitation (1, 163). Whether these two-component regulatory systems control gene expression in similar ways (with involvement of a Pi-specific transporter and PhoU homolog) has not been determined.
The E. coli Pho regulon has 31 or more genes arranged in eight transcriptional units (Table 3; Fig. 5). The phnR to phnX gene cluster of S. typhimurium includes at least two additional transcriptional units that are absent in E. coli. All of these genes have been sequenced and appear to be transcribed solely from a single promoter upstream of the respective gene or operon. S. typhimurium has 13 or more of these genes, including phoBR, phoE, pstSCAB-phoU, and ugpBAECQ. The phoA-psiF and phnC to phnP genes are known to be absent in S. typhimurium. Whether phoH and psiE are present is unknown. In addition, S. typhimurium contains the phnR-to-phnX gene cluster, which is absent in E. coli. This locus appears to contain three promoters, which precede phnR, phnS, and phnW; phnR and phnW are divergently transcribed from an intergenic region between them; one transcript may be for phnWX, a second may be for phnR, and a third may be for phnSTUV, which is immediately distal to phnR. Only phoE (208) and phnR to phnX (Metcalf et al., unpublished data) of S. typhimurium have been sequenced. S. typhimurium phoBR has also been cloned but not sequenced (96a).
The E. coli phoBR operon is subject to multiple controls. It is autogenously regulated by PhoB in a positive manner (77). It may also be subject to an antisense (negative) RNA control (254; Chang and Wanner, unpublished data). A promoter near the 5' end of the phoB coding region leads to lacZ transcription in the antisense orientation. In addition, an A-to-G change 22 bp upstream of its translation start reduces PhoB translation more than 100-fold without affecting transcription, possibly as a result of an effect on antisense RNA control (Chang and Wanner, unpublished data). Another laboratory showed that phoR is expressed in the absence of the phoB promoter on a multicopy plasmid and inferred the existence of a promoter preceding phoR (193, 218). Since chromosomal insertions in phoB abolish expression of phoR due to polarity (254), it is unlikely that the putative phoR promoter has a physiological role in single copy.
Many phosphate starvation-inducible (psi) genes correspond to various members of the Pho regulon (Table 3), yet others do not (152, 242). More than 20 psi promoters were identified in a set of 54 mutants carrying random psi-lacZ transcriptional fusions (259). Several of these promoters are turned on by other limitations (for carbon, nitrogen, or oxygen [anaerobiosis]), by growth on a complex medium, or by UV irradiation as well as by Pi limitation. Some are subject to additional genetic regulatory controls. The ugpB (psiB or psiC) promoter is activated by PhoB and by cAMP and CAP, acting at different sites (101, 213). Also, psiE and phoH (psiH) may be controlled by other factors in addition to PhoB (237, 240, 256). No new studies on Pi control of pepN, psiG, psiI, psiJ, psiL, psiN, psiO, and psiR exist beyond those summarized previously (242). Several psi promoters are clearly controlled by other regulatory systems and are not subject to Pho regulon control. These include promoters identified as psiP for the integration host factor (himA) gene, psiQ for the glutamate synthase (gltBDF) operon, psi-51 (an unassigned psi promoter) for the anaerobically regulated G3P dehydrogenase (glpABC) operon, and perhaps others (152; P. M. Steed and B. L. Wanner, unpublished data). Therefore, psi gene members of the Pho regulon correspond only to a subset of genes activated under conditions of Pi limitation. Other psi genes may correspond to those controlled by the PhoP-PhoQ two-component regulatory system (158), the stationary-phase sigma factor RpoS (127), or other factors. For example, phosphate starvation has also been shown to lead to activation of the LexA-controlled gene sfiA in a PhoB- and RpoS-independent manner (60).
In a similar way, four psi loci (named psiA, psiB, psiC, and psiD) were identified in S. typhimurium (68). Only two of these (psiB and psiC) appear to be members of the Pho regulon (96a). Also, the psiR locus (which was identified as a negative regulatory mutation of psiC [68]) corresponds to the E. coli pstSCAB-phoU operon (96a). One (psiD) has been shown to be activated by the response regulator PhoP, for the S. typhimurium acid phosphatase gene phoN (75); it is clearly not a member of the Pho regulon. Importantly, the psiC locus corresponds to the phnR-to-phnX gene cluster (96a). None of the other S. typhimurium psi loci has an assigned function.
Corroborative evidence for different kinds of psi genes has been obtained by examining E. coli proteins made during Pi limitation. On the basis of two-dimensional gel analysis, more than 80 Psi proteins are made in increased amounts in response to Pi limitation. These include Psi proteins that are members of the Pho regulon as well as proteins that are members of other regulons, including those that are members of the heat shock (HtpR, σ 32) regulon, leucine response (Lrp) regulon, LexA-controlled SOS regulon, and OxyR-controlled regulon. Many of the same proteins are synthesized under conditions of carbon, nitrogen, or amino acid (isoleucine) limitation, in response to a temperature shift from 37 to 50°C (heat shock) or from 37 to 10°C (cold shock), or upon treatment with nalidixic acid, cadmium chloride, or a quinone. These studies are summarized in chapter 115.
Global regulation in response to phosphate starvation (as well as various other stimuli or genetic defects) has also been examined by measuring mRNA levels of different chromosomal segments (39). Signals for psi mRNAs were detected for chromosomal regions corresponding to phoA, phoBR, phoE, phnC to phnP, and pstSCAB-phoU. In addition, strong signals were detected for six other regions that may encode new psi genes. Although further studies are necessary to determine which genes in these regions correspond to psi genes, one region near 81.2 min includes gpsA (for G3P dehydrogenase). It is interesting that this enzyme acts on the substrate of the Ugp transporter (Table 3). Thus, gpsA may be a new member of the Pho regulon. Another region near 27.8 min contains the oppABCDE operon (encoding the oligopeptide transport system), which may also be a new member. Despite this finding, synthesis of OppA has been shown to be more greatly inhibited during Pi limitation (205). Other RNA signals were detected for regions near 14.4, 23, 56.8, and 87.5 min (39). New studies are necessary in order to show which of these mRNAs are subject to genetic control by regulatory genes of the Pho regulon.
All Pho regulon genes or operons are preceded by a promoter containing an upstream activation site with a consensus Pho box sequence for transcriptional activation by phospho-PhoB (Fig. 6) (138). In vitro transcription of the phnC, phoA, phoB, phoH, pstS, and ugpB promoters requires phospho-PhoB. Regions of these promoters protected by phospho-PhoB have been determined on the basis of DNase I protection and methylation interference studies. In vitro studies of the phoE promoter have not been carried out. In vivo mRNA start sites have also been determined for all of these promoters. Further, it has been shown that the Pho box is required for transcriptional activation by examining the effects of 5' deletions on the expression of the phnC (D. K. Agrawal and B. L. Wanner, unpublished data), phoE (220), and pstS (113) promoters. A single point mutation [the pho-1003(Bin) allele] has been examined. A G-to-T change at –5 with respect to the mRNA start of phoA bypasses the PhoB requirement for transcriptional activation and leads to a low constitutive level of expression of phoA. Therefore, this mutation has created a PhoB-independent promoter. Whether transcription initiates at the same start site has not been determined (242). Unlike other Pho regulon promoters, the phoE promoter has an additional Pho box in the opposite orientation more than 100 bases upstream of its mRNA start site (not shown), which may act as a transcriptional enhancer (220). Optimization of the –10 region of the phoE promoter toward consensus has led to an elevated basal level in the presence of excess Pi and decreased dependency on its upstream activation site for expression under conditions of Pi limitation (194).
All Pho regulon promoters have a number of features in common. The Pho box is composed of two 7-bp direct repeats with the well-conserved consensus CTGTCAT separated by a 4-bp segment that is part of the –35 region (Fig. 6). The occurrence of a 7-bp unit every 11 bp may allow binding of multiple phospho-PhoB molecules to the same face of the helix. This pattern is extended further upstream for promoters such as pstS and ugpB that have additional copies of the 7-bp repeat. The –35 region itself is AT rich and is far from consensus –35 elements of σ 70 promoters. The role of phospho-PhoB may be to provide a substitute contact point for RNA polymerase (137). The first 7-bp repeat is positioned 10 bp upstream of the –10 promoter element, and the major transcript initiates with a G residue.
Computational analysis indicates that an additional Pho box sequence lies within the phnW-to-phnR intergenic region, before phnS, and that there may be a weak one before psiE (Metcalf et al., unpublished data); also, transcription of these promoters is dependent on PhoB, as determined by studies using lacZ transcriptional fusions. Whether a Pho box precedes both phnW and phnX is uncertain, as no phnR-lacZ fusion has been examined. Gel mobility shift and DNase I protection assays show that sequences in the phnW-to-phnR intergenic region and preceding phnS are bound by phospho-PhoB (S.-K. Kim and B. L. Wanner, unpublished data). Putative Pho box sequences preceding phnA (37) and ppk (105) have also been predicted. Subsequent studies of the phnA to phnP genes revealed that phnA and phnB are unnecessary for phosphonate utilization and that a PhoB-dependent psi promoter precedes phnC (155). Whether phnA and phnB are regulated by a Pho box preceding phnA has not been determined. It is also unclear, for reasons mentioned in the section "Intracellular Inorganic Phosphates," whether the ppk-ppx operon is a member of the Pho regulon.
The Pho regulon is highly regulated in phoR mutants even though its control by Pi is abolished as a result of the absence of PhoR. Instead, it is regulated by the carbon and energy source via controls that are not apparent under conditions of Pi limitation in the presence of PhoR (243, 260, 261, 262). Those controls that are observed (primarily) in the absence of PhoR are referred to as Pi-independent controls in order to distinguished them from Pi control. These Pi-independent controls involve two (or more) signaling pathways. One pathway requires the sensor kinase CreC (255) and results in activation by phosphorylation of PhoB by CreC (10); the other depends upon acetyl phosphate synthesis (245, 262). Activation by CreC is discussed in the next section, "Control by the Sensor Kinase CreC." Activation by acetyl phosphate is discussed in the section, "Control by Acetyl Phosphate as an Effector Molecule." Whether CreC or acetyl phosphate also has a role in the control of the Pho regulon in the presence of PhoR is considered in the section "Is There Cross-Regulation by CreC or Acetyl Phosphate?"
The discovery of multiple controls for activation of the Pho regulon resulted entirely from genetic studies (248, 255, 262; B. L. Wanner, J. Bernstein, and M. Lyster, unpublished data). Table 4 shows effects due to mutations that alter its control by Pi, PhoR, the Pst transporter, and PhoU; these are described in the section "Pi Control by Transmembrane Signal Transduction." Table 5 shows effects due to mutations that alter its Pi-independent controls by CreC and acetyl phosphate synthesis. In all cases, activation depends on PhoB and is believed to require phospho-PhoB as a transcriptional activator. phoA expression has been examined primarily as a means to assess Pho regulon gene expression. Similar qualitative results have also been obtained when other Pho regulon genes have been studied (which has usually involved examination of expression of the respective promoter-lacZ transcriptional fusions in single copy).
Table 5Effects of mutations on Pi-independent controls of the Pho regulon |
A null phoR mutation totally abolishes Pi control of the Pho regulon and leads to an intermediate level of expression that is between the inhibited level observed in the presence of PhoR and excess Pi and the high activated level observed in the presence of PhoR under conditions of Pi limitation (Table 4). The actual level observed in a phoR mutant varies with the carbon source. A twofold effect is seen for glucose- versus acetate-grown cells; this expression requires CreC (Table 5). Much greater differences are observed when other carbon sources are used (Wilmes-Riesenberg and Wanner, unpublished data). Activation due to CreC (in the absence of acetyl phosphate synthesis) may lead to ca. 30 to 150% as much phoA expression as activation by PhoR under conditions of Pi limitation (data not shown). Pyruvate leads to high-level activation of phoA expression; this activation is attributable in part to activation by CreC and in part to activation by acetyl phosphate (Table 5). Also, these signaling pathways are genetically separable (262).
CreC (originally named PhoM) is a cytoplasmic membrane sensor kinase 474 amino acids in length (9). Like many membrane sensors of two-component regulatory systems, CreC has an N-terminal domain with two α-helical transmembrane segments (residues 7 to 27 and 184 to 207) flanking a large periplasmic domain (as verified by TnphoA analysis [271]). It has a near-canonical transmitter domain beginning near residue 210, including a typical linker domain of about 50 amino acids followed by a highly conserved histidine kinase domain (174). CreC is encoded by the creABCD operon (Fig. 7A) together with the response regulator family member CreB (also called PhoM-Orf2). A LacZ-CreC fusion protein (in which LacZ is joined to residue 206 of CreC) is autophosphorylated by the γ-phosphoryl group of ATP in vitro (10) on His-265 (on the basis of a conserved sequence motif with this residue), and the phosphorylated fusion protein is capable of transferring its phosphoryl group to CreB or PhoB, although phosphotransfer to PhoB may be less efficient. The N-3 position of the imidazole ring in a histidinyl residue appears to be the site of autophosphorylation. Full-length membrane-bound CreC has also been shown to be autophosphorylated by ATP in vitro.
A role for CreC in activation of PhoB was revealed by finding null creC (phoM) mutations in regulatory mutants of the Pho regulon, which had been fortuitously isolated from a null phoR mutant (258). They were originally named PhoM because their mutational effects appeared to be masked in the presence of PhoR (255), under conditions of Pi limitation with glucose as a carbon source. At the time, it was mistakenly thought that a null phoR mutant resulted in low constitutive, i.e., unregulated, level of expression and that CreC was therefore responsible for constitutive synthesis. This turned out not to be entirely correct; it was later shown that (most) early studies of the Pho regulon had been carried out in strains carrying a mutation in creC (244). Activation resulting from CreC in a phoR mutant is highly regulated, especially by the carbon source (261; Wilmes-Riesenberg and Wanner, unpublished data). The "constitutivity" of null phoR mutants is actually due to the presence of a mutation (creC510; originally called pho-510) within the creABCD (phoM) operon (243, 260).
The creC510 mutation responsible for constitutive activation of PhoB results in an R77P change in CreC, as determined in the E. coli genome sequencing project (31). This residue is expected to lie within the periplasmic domain of CreC (Fig. 7B) and, on the basis of its phenotype, probably results in "constitutive signaling." Hence, CreC510 is constitutively active. In addition, (nonpolar) null creB mutations lead to constitutivity, but for a different reason. It was partly because of the similar behaviors of creC510 and null creB alleles that creC510 had been thought to lie in creB (244) and therefore on occasion referred to as creB510. The location of creC510 (R77P) is fully compatible with our earlier restriction mapping data (261) and DNA sequencing of creB on the appropriate plasmids (Kim and Wanner, unpublished data).
CreC interacts with both CreB and PhoB, and so competition between them for CreC may occur in vivo (244). Constitutivity may result from loss of this competition due to absence of CreB. An effect due to competition may be especially apparent when there is a low level of phospho-CreC due to absence of a signaling stimulus. Consequently, in the absence of CreB, a low level of phospho-CreC may lead to constitutive activation of PhoB. In the presence of CreB, a high level of phospho-CreC resulting from signal sensing leads to phosphorylation of PhoB in response to carbon sources that are thought to act as signals for CreC. Also, a high level of phospho-CreC resulting the creC510 mutation may lead to constitutive activation of PhoB. Therefore, activation of PhoB by CreC in phoR mutants with a creC510 or null creB allele occurs as a result of constitutive signaling or absence of competition by CreB, respectively.
Competition between CreB and PhoB for a low level of phospho-CreC may also account for the metastable "clonal variation" phenotype associated with null phoR mutants (239, 243, 260, 261). The autogenous activation of PhoB synthesis (77) brought about by phosphorylation of PhoB is probably partly responsible as well. The clonal variation phenotype is especially apparent when phoR mutants that are otherwise wild type [in particular, cre(BC)+] are grown on complex media lacking carbohydrates. Under these conditions, phoR mutants may form Bap+ or Bap– colonies, both of which may give rise to sectored colonies and cells of the opposite Bap phenotype (239). Three conditions overcome this metastable character. Signaling (e.g., due to the presence of glucose) leads to the formation entirely of cells in an on state that is subsequently maintained in the absence of an inducing signal. Both creC510 and (nonpolar) null creB alleles abolish clonal variation and result in constitutive activation of PhoB by CreC (S.-K. Kim, M. R. Wilmes-Riesenberg, and B. L. Wanner, unpublished data).
Accordingly, in the absence of a signal, a low level of phospho-CreC may on occasion phosphorylate PhoB. This in turn leads to increased PhoB synthesis, and under these conditions, PhoB may be better able to compete with CreB for a low level of phospho-CreC. Hence, PhoB continues to be phosphorylated, and expression of the Pho regulon remains turned on. Similarly, a high level of phospho-CreC brought about by signaling results in phosphorylation of PhoB, leading to increased PhoB synthesis (due to its autogenous control [77]). Again, PhoB is phosphorylated, and expression of the Pho regulon remains turned on in the absence of the signal. Eventually, phospho-PhoB or PhoB may decay, leading to decreased PhoB synthesis, and return to an off state. In this regard, stationary-phase growth invariably leads to formation of some cells in the off state, even though no condition leads to the formation entirely of Bap– cells (243).
The creC510 (R77P) mutation probably arose in the E. coli K-12 descendent 58F+ after X-ray mutagenesis about 50 years ago (243). Because 58F+ is a progenitor of many other strains, including some classic Hfr strains (see chapter 133), creC510 is now widespread among common laboratory strains of E. coli K-12 (243, 260). Strains carrying creC510 probably also carry a mutant form of the nearby gene robA (for right oriC binding protein), the robA1 (E15K) allele (203). robA lies immediately upstream of the creABCD operon in the opposite orientation (Fig. 7A). The robA1 (E15K) mutation is associated with strains carrying the creC510 mutation (260).
CreB is a response regulator 229 amino acids in length (9) that is likely phosphorylated on Asp-54 (as judged from its sequence). It has an N-terminal conserved receiver domain (174) of ca. 100 and (presumably) a C-terminal regulatory domain with a weak helix-turn-helix motif near its C terminus (data not shown). CreB or phospho-CreB may act as a transcriptional regulator of an unknown set of target genes, though no direct evidence for this exists. The CreC-CreB two-component regulatory system may be involved in the control of genes encoding enzymes for carbon catabolism. This is because phosphorylation of PhoB by CreC is regulated in an unknown way by the presence of different carbon sources (261; Wilmes-Riesenberg and Wanner, unpublished data). Also, expression of a creA-lacZ transcriptional fusion is subject to carbon source regulation in a manner that appears to be negatively controlled by cAMP and CAP (J. Cui, D. C. Young, and B. L. Wanner, unpublished data).
CreA is a 157-amino-acid periplasmic protein of unknown function, as judged from its sequence (9) and TnphoA analysis (271). CreD is a 450-amino-acid inner membrane protein with multiple predicted α-helical transmembrane segments, mostly in its C-terminal region (9; data not shown); it has an unorthodox transmitter domain about 200 residues in length immediately following a predicted N-terminal transmembrane segment (114). CreD corresponds to cet, which had been so named because creD (cet) mutations lead to colicin E2 tolerance (61).
Expression of the creABCD operon has been examined by using lacZ fusions and by Northern (RNA) hybridization. Two early reports of studies using cre-lacZ fusions are probably incorrect. One used a cre DNA fragment that was later shown to lack the creA promoter (9, 140). Another showed a threefold effect due to the phoU35 allele on β-galactosidase activity encoded by a creC-lacZ gene fusion (128). This study may be incorrect because a lacZ translational fusion to a membrane protein (CreC) was used to study gene regulation. The same phoU allele (as well as a phoB, phoR, or pst allele) has no effect on expression of a creA-lacZ transcriptional fusion, yet expression of a creA-lacZ fusion is highly regulated by carbon sources (Cui et al., unpublished data), as mentioned above. Three mRNAs of the creABCD operon were detected in studies on creD (cet) mutants. Two probably correspond to a transcript of the entire operon and its breakdown product; the third is only 1.5 kb and may correspond to creD alone, indicating the presence of an internal promoter. No mRNA start site(s) has been determined. Nevertheless, except for its role in catabolite regulation of Pho regulon genes in phoR mutants, the biological function of the creABCD operon and its gene products remains a mystery.
Acetyl phosphate is an intermediate of the phosphotransacetylase-acetate kinase (Pta-AckA) pathway (Fig. 8). The first evidence that acetyl phosphate may act as an effector of gene regulation resulted from the discovery of ackA mutants as Bap+ revertants of a null phoR creC (phoM) mutant (262). An ackA mutant is expected to result in acetyl phosphate accumulation during growth on glucose or pyruvate and decreased synthesis during growth on acetate. In a phoR creC mutant, an ackA mutation leads to high-level expression of phoA on glucose, an elevated level on pyruvate, and no effect on acetate (Table 5). Conversely, a pta mutant is expected to result in decreased synthesis of acetyl phosphate during growth on glucose or pyruvate and in accumulation of acetyl phosphate on acetate. In a phoR creC mutant, a pta mutation has no effect on glucose, abolishes phoA expression on pyruvate, and leads to low-level expression on acetate (Table 5). Growth of a wild-type (ackA + pta +) strain is expected to result in higher amounts of acetyl phosphate synthesis on pyruvate than on glucose or acetate as a result of elevated acetyl coenzyme A (acetyl-CoA) levels on pyruvate. A phoR creC mutant displays low-level expression of phoA on pyruvate and no expression on glucose or acetate (Table 5). A Δ(ackA pta) mutation is expected to block acetyl phosphate synthesis under all conditions. A phoR creC ackA pta mutant shows no expression of phoA on these carbon sources (Table 5).
Earlier studies indicated that an additional control of the Pho regulon existed, beyond its controls by PhoR and by CreC (252; Wanner et al., unpublished data). A historical account of these studies has been published (248). In brief, two lines of evidence existed: (i) second-site Bap+ revertants of phoR creC mutants were readily selectable, and (ii) growth of phoR creC mutants on complex media containing high concentrations of fermentable carbon sources such as glucose led to activation of phoA expression. To study this additional control, transposon-induced Bap+ pseudorevertants of a phoR creC mutant were isolated (262). By sequencing DNA adjacent to the transposon insertion sites and by searching GenBank for these sequences, we discovered in 1989, when the ackA sequence was deposited (147), that four of our Bap+ mutants had an insertion in ackA (262). Subsequent experiments carried out with these and other ackA and pta mutations resulted in showing that activation of the Pho regulon in the absence of both PhoR and CreC resulted from acetyl phosphate synthesis (262). Also, this activation requires PhoB and is inhibited by Pi in the presence of PhoR. Hence, activation of PhoB by acetyl phosphate, PhoR, and CreC appeared to be due to a common biochemical mechanism, activation of PhoB by phosphorylation.
Acetyl phosphate may lead to activation of PhoB in one of two ways (245, 262). Acetyl phosphate may act directly as a chemical phosphorylating (or acetylating) agent by virtue of its role as a high-energy compound, or it may act indirectly via an unknown sensor kinase. In accordance with the latter mechanism, elevated levels of acetyl phosphate (or a closely related molecule) may lead to phosphorylation of PhoB by a sensor (previously designated X [245, 262]). Acetyl phosphate may lead to activation of other systems in a similar way(s). Subsequent studies showed that activation of the nitrogen response regulator NtrC (NRI) in the absence of its partner sensor kinase NtrB (NRII) also occurs in vivo under conditions expected to result in increased acetyl phosphate synthesis (67), essentially in accordance with results originally obtained in studies of the Pho regulon (262). Further, as in the case of the Pho regulon (238, 261), earlier reports indicated a mechanism for activation of NtrC in the absence of NtrB existed in vivo (30, 188).
The finding that acetyl phosphate acts as a chemical phosphorylating agent in vitro in the autophosphorylation of the response regulators CheY (24), AlgR (56), NtrC (NRI; [67, 148]), ComA (190), VanR (87), PhoB (85a, 148), and OmpR (148) provides evidence in favor of a direct effect. However, it should be noted that the concentrations of acetyl phosphate required for efficient autophosphorylation in vitro (24, 56, 67, 85a, 87, 148) are much greater (10 mM or more) than those found in vivo (26, 92, 93, 148, 181), which have been shown to range from ca. 20 μM to 1 mM, depending on the strain and growth conditions. Acetyl phosphate also acts as chemical acetylating agent for CheY, but not VanR, at the same concentration that is capable of phosphorylating these response regulators (24, 87), although a role for acetylation by acetyl phosphate has been ruled out for CheY (264a). Further, we have recently obtained evidence for the involvement of a sensor kinase for in vivo activation of PhoB due to acetyl phosphate, at least under certain conditions (S.-K. Kim, M. Wilmes-Riesenberg, and B. L. Wanner, unpublished data). Our inability to find mutations of a sensor kinase that block activation of PhoB by acetyl phosphate in an earlier study (262) may have resulted from the presence of multiple signaling kinases capable of activating PhoB under those conditions. Therefore, whether acetyl phosphate acts as a direct phosphorylating agent of PhoB in vivo remains an open question. Importantly, acetyl phosphate levels are subject to considerable variation in vivo, making acetyl phosphate an attractive candidate as an effector molecule (149, 245, 246, 262).
Pta catalyzes the formation of acetyl phosphate from acetyl-CoA and Pi with release of free CoA, and AckA catalyzes the formation of ATP and acetate from acetyl phosphate and ADP, in freely reversible reactions (70, 78, 190, 200). Consequently, the Pta-AckA pathway (Fig. 8) operates in the direction of ATP and acetate formation during growth on glucose or pyruvate and in the opposite direction during growth on acetate. Pta and AckA appear to be largely synthesized constitutively (as judged from activity measurements), although about twofold-higher levels have been observed under conditions of anaerobic growth (78). Larger effects on Pta and AckA synthesis are seen under conditions of glucose starvation, however (167). Acetate is also metabolized by acetyl-CoA synthetase (27), the acs gene product (25), allowing for growth of pta and ackA mutants on acetate, albeit at a slower rate (78).
Acetyl phosphate has also been implicated in other processes. The ability to synthesize acetyl phosphate appears to be important for survival under conditions of glucose starvation (167). Also, earlier studies indicated that acetyl phosphate may act as an energy source for binding protein-dependent (ABC family) transporters (88). Further studies are necessary to assess the role of acetyl phosphate as an effector of gene regulation (262), its mechanism(s) of activation of PhoB in vivo, and its role in other processes as well.
All effects due to CreC or acetyl phosphate on the expression of Pho regulon genes are inhibited by PhoR when Pi is in excess. There also appears to be no requirement for these alternative signaling pathways in the presence of PhoR, because under conditions of Pi limitation, PhoR leads to full activation of PhoB. However, there may be a role for CreC or acetyl phosphate in coordinating Pho regulon control with the overall process of cell growth. I previously suggested that CreC and acetyl phosphate may be involved in an overall global control that I referred to as cross-regulation. I will briefly describe a few reasons for cross-regulation. Since proof of this concept is lacking, my discussion will by its nature be a bit speculative.
To maximize growth, it is fundamental that cells be able to coordinate all the diverse aspects of metabolism. This coordination likely involves many transcriptional controls as well as other controls. It also likely involves innumerable input signals. Different genes, operons, and regulons may have a large degree of independent control(s). They may also share controls to create an overlapping network for specific metabolic purposes and eventually for global regulation. It may be especially important to have regulatory coupling(s) between genes for enzymes in central pathways of carbon, energy, and Pi metabolism. A regulatory coupling between these and the Pho regulon (Fig. 9) may be a form of cross-regulation (245) among related metabolic pathways.
Cross-regulation was defined as the control of a response regulator of one two-component regulatory system by a different regulatory system (245). I adopted this term in order to distinguish interactions that may be especially significant as biological regulatory processes from those interactions (commonly called cross talk) that may be insignificant in vivo. By definition, cross-regulation controls the activity of the response regulator. Also, it probably always involves phosphorylation (or dephosphorylation) of the response regulator, because these are the only mechanisms known to activate or inactivate a response regulator. However, cross-regulation may involve different mechanisms. Besides phosphorylation by a nonpartner sensor, it may involve a different covalent modification, the binding of an effector molecule, or a different mechanism altogether.
The most compelling evidence in favor of a role for cross-regulation is seen in the Pho regulon (262). The activation of PhoB by acetyl phosphate provides a teleological basis for cross-regulation, because the Pta-AckA pathway for acetyl phosphate synthesis leads (during growth on glucose or pyruvate) to the incorporation of Pi into ATP, the primary phosphoryl donor in metabolism (245). Therefore, the Pta-AckA pathway is formally a pathway both in carbon and energy metabolism and in Pi metabolism (Fig. 2). Also, activation of PhoB by CreC may connect the control of the Pho regulon to the control of other central pathways of carbon and energy metabolism. Further, the activation of PhoB by these Pi-independent controls may allow for coordinating the overall process of P assimilation under certain conditions in the presence of PhoR (244, 248).
Additional evidence indicates that Pho regulon control by CreC or acetyl phosphate connects activation of PhoB to central pathways of carbon and energy metabolism. More than 90 independent mutants that affect Pi-independent control of the Pho regulon have been isolated (references 238, 248, 261, and 262 and unpublished data cited therein; S. Yamagata, L. L. Daniels, and B. L. Wanner, unpublished data). Some result in activation due to CreC, while others result in activation due to acetyl phosphate synthesis. These mutants have lesions in 1 of more than 15 loci, including the creABCD and ackA-pta operons as well as genes for aerobic respiratory control (arcA), adenylate cyclase (cya), CAP (crp), guanine biosynthesis (guaAB), isocitrate dehydrogenase (icd), malate dehydrogenase (mdh), osmoregulation (ompR), exopolysaccharide production (ops), a ribulose 5'-phosphate epimerase homolog (orf221), phosphotransferase system (ptsHI), poly(Pi) kinase (ppk), purine biosynthesis (pur), ribose phosphate isomerase (rpiA), and others (unidentified). These loci include those that had been tentatively named phoG or phoH (242) prior to their identification. Mutations of many of these genes likely affect a central pathway directly, while others do so indirectly.
Two additional conditions for control of the Pho regulon are notable because they act in the presence of Pi and in the presence of PhoR. It has been known for a long time that pyrimidine and purine limitations cause activation of phoA expression in the presence of PhoR and excess Pi (265). Indeed, these results were the primary basis for the hypothesis that an unknown nucleotide may act as an effector of the Pho regulon (160, 242, 255). No convincing evidence for the existence of such an effector exists, however. Also, none has been identified despite deliberate searches (184, 187, 224). Further, as I discussed elsewhere, activation by these limitations may be entirely due to indirect effects involving acetyl phosphate synthesis (248). Hence, activation of PhoB by acetyl phosphate under these conditions may occur in the presence of PhoR and excess Pi.
In addition, increased osmolarity or procaine causes inhibition of Pho regulon gene expression, in the presence or absence of PhoR (33, 156, 233). Curiously, this inhibition affects some promoters (phoA and phoE) more than others (phoB and pstS). These inhibitions may decrease the level of phospho-PhoB, and different amounts of phospho-PhoB may be necessary for activation of these promoters in vivo. Activation by CreC or acetyl phosphate may also result in different effects on individual Pho regulon promoters in the presence of PhoR and excess Pi (245). Such effects may be brought about by partial phosphorylation of PhoB under conditions of nutrient shifts when Pi is in excess.
Much has been learned about Pi regulation of gene expression since I wrote about it for the first edition of this book. Yet, many new questions now await future investigations. Only a few of these are mentioned here. There is now a much better understanding of how cells cope under conditions of Pi limitation. Clearly, not one but several regulatory systems respond to Pi limitation and lead to activation of different psi genes. Only a subset of these psi genes are members of the Pho regulon and are controlled by the two-component regulatory system composed of PhoR and PhoB. Others may be regulated by the PhoP-PhoQ regulatory system, the sigma factor RpoS, or other systems. Do these systems interact? If so, how?
Activation and inhibition of Pho regulon gene expression may be entirely regulated by phosphorylation and dephosphorylation reactions. Activation is brought about by phosphorylation of PhoB by PhoR; inhibition is (presumably) brought about by PhoR facilitating the dephosphorylation of phospho-PhoB. The control of these processes is a form of transmembrane signal transduction. What is the mechanism of transmembrane signaling? Inhibition involves detection of environmental Pi by a mechanism requiring an intact Pst system, a protein called PhoU, and PhoR. Is the Pst system solely responsible for detecting extracellular Pi? If so, how is this signal communicated? What is the role of PhoU in this process? Of PhoR? Does PhoR detect environmental Pi directly? If not, why is PhoR an integral membrane protein? Is there an additional control of PhoB that does not involve its phosphorylation? If so, what is the nature of that control(s)? Is there an additional control of PhoR? What is its nature?
Of the 38 genes of the Pho regulon that have been characterized, 35 are known to have a role in P assimilation or Pho regulon control. Twenty-one of these genes are involved in the utilization of phosphonates. All studies of these genes were carried out subsequent to publication of the first edition of this book. Consequently, many new questions concern phosphonate utilization. In regard to the C-P lyase pathway, new studies await development of a cell-free system for determination of the proteins involved in degradation, the degradative pathway, and the catalytic mechanism of C-P bond cleavage. Genes of both the C-P lyase and phosphonatase pathways encode potential regulatory proteins. What is the role of these proteins? How do they interact in the overall control of these and other Pho regulon genes by PhoR and PhoB?
Intracellular Pi metabolism involves many well-known central pathways. Is a specific pathway involved in Pi assimilation under particular growth conditions? PhoU may act as a Pi enzyme. Does PhoU have an activity for incorporation of Pi into an organophosphate? Do other Pho regulon gene products have a similar activity?
Two additional controls of the Pho regulon lead to its activation in the absence of PhoR. Does activation by CreC or acetyl phosphate have a role in Pho regulon control in the presence of PhoR? If it does, then a mechanism may be necessary for inactivation of PhoR (in order to avoid inhibition by PhoR). How might PhoR be inactivated? CreC leads to activation in response to some unknown catabolite. What does CreC sense? CreC and its partner CreB likely control the expression of other (unknown) genes. What are the target genes of the CreC-CreB two-component regulatory system? Increased acetyl phosphate synthesis leads to activation of PhoB. What is the mechanism of activation by acetyl phosphate? Under certain conditions, a sensor appears to be involved in the activation of PhoB by acetyl phosphate. Is a different sensor involved under other conditions? Which one(s)?
Cross-regulation refers to a mechanism for the control of a response regulator by a means not involving its partner sensor kinase. How important is cross-regulation in the control of the Pho regulon? In the control of other response regulators? In the overall control of cell growth and central metabolism? Searching for answers to these and other questions is likely to continue to entertain many investigators for a long time in the future.
This laboratory is supported by NIH grant GM35392 and NSF grant MCB 9405929. L. L. Daniels, A. Haldimann, W. Jiang, S.-K. Kim, and S. Yamagata have contributed to recent unpublished results from this laboratory. I thank A. Haldimann and B. Magasanik for careful reading of the manuscript and helpful criticisms.
References
1. Aiba, H., and T. Mizuno. 1994. A novel gene whose expression is regulated by the response-regulator, SphR, in response to phosphate limitation in Synechococcus species PCC7942. Mol. Microbiol. 13:25–34.
2. Aiba, H., M. Nagaya, and T. Mizuno. 1993. Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal-transduction protein families: implication in the adaptive response to phosphate limitation. Mol. Microbiol. 8:81–91.
3. Akiyama, M., E. Crooke, and A. Kornberg. 1992. The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein. J. Biol. Chem. 267:22556–22561.
4. Akiyama, M., E. Crooke, and A. Kornberg. 1993. An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J. Biol. Chem. 268:633–639.
5. Alpuche-Aranda, C. M., J. A. Swanson, W. P. Loomis, and S. I. Miller. 1992. Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes. Proc. Natl. Acad. Sci. USA 89:10079–10083.
6. Ambudkar, S. V., T. J. Larson, and P. C. Maloney. 1986. Reconstitution of sugar phosphate transport systems of Escherichia coli. J. Biol. Chem. 261:9083–9086.
7. Ambudkar, S. V., L. A. Sonna, and P. C. Maloney. 1986. Variable stoichiometry of phosphate-linked anion exchange in Streptococcus lactis: implications for the mechanism of sugar phosphate transport in bacteria. Proc. Natl. Acad. Sci. USA 83:280–284.
8. Amemura, M., K. Makino, H. Shinagawa, A. Kobayashi, and A. Nakata. 1985. Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli. J. Mol. Biol. 184:241–250.
9. Amemura, M., K. Makino, H. Shinagawa, and A. Nakata. 1986. Nucleotide sequence of the phoM region of Escherichia coli: four open reading frames may constitute an operon. J. Bacteriol. 168:294–302.
10. Amemura, M., K. Makino, H. Shinagawa, and A. Nakata. 1990. Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2. J. Bacteriol. 172:6300–6307.
11. Anba, J., M. Bidaud, M. L. Vasil, and A. Lazdunski. 1990. Nucleotide sequence of the Pseudomonas aeruginosa phoB gene, the regulatory gene for the phosphate regulon. J. Bacteriol. 172:4685–4689.
12. Argast, M., and W. Boos. 1980. Coregulation in Escherichia coli of a novel transport system for sn-glycerol-3-phosphate and outer membrane protein Ic (e, E) with alkaline phosphatase and phosphate-binding protein. J. Bacteriol. 143:142–150.
13. Argast, M., D. Ludtke, T. J. Silhavy, and W. Boos. 1978. A second transport system for sn-glycerol-3-phosphate in Escherichia coli. J. Bacteriol. 136:1070–1083.
14. Atkinson, D. E. 1977. Cellular Energy Metabolism and Its Regulation. Academic Press, Inc., New York.
15. Bachmann, B. J. 1987. Derivations and genotypes of some mutant derivatives of Escherichia coli K-12, p. 1190–1219. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 2. American Society for Microbiology, Washington, D.C.
16. Bauer, K., R. Benz, J. Brass, and W. Boos. 1985. Salmonella typhimurium contains an anion-selective outer membrane porin induced by phosphate starvation. J. Bacteriol. 161:813–816.
17. Bauer, K., M. Struyve, D. Bosch, R. Benz, and J. Tommassen. 1989. One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli. J. Biol. Chem. 264:16393–16398.
18. Baumann, H., A. O. Tzianabos, J.-R. Brisson, D. L. Kasper, and H. J. Jennings. 1992. Structural eludidation of two capsular polysaccharides from one strain of Bacteriodes fragilis using high-resolution NMR spectroscopy. Biochemistry 31:4081–4089.
19. Bennett, R. L., and M. H. Malamy. 1970. Arsenate resistant mutants of Escherichia coli. Biochem. Biophys. Res. Commun. 40:496–503.
20. Benz, R., R. P. Darveau, and R. E. W. Hancock. 1984. Outer-membrane protein PhoE from Escherichia coli forms anion-selective pores in lipid-bilayer membranes. Eur. J. Biochem. 140:319–324.
21. Benz, R., C. Egli, and R. E. W. Hancock. 1993. Anion transport through the phosphate-specific OprP-channel of the Pseudomonas aeruginosa outer membrane: effects of phosphate, di- and tribasic anions and of negatively-charged lipids. Biochim. Biophys. Acta Bio-Membr. 1149:224–230.
22. Benz, R., G. Francis, T. Nakae, and T. Ferenci. 1992. Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site. Biochim. Biophys. Acta Bio-Membr. 1104:299–307.
23. Benz, R., A. Schmid, C. Maier, and E. Bremer. 1988. Characterization of the nucleoside binding site inside the Tsx channel of Escherichia coli outer membrane. Eur. J. Biochem. 176:699–705.
24. Blat, Y., and M. Eisenbach. 1994. Phosphorylation-dependent binding of the chemotaxis signal molecule CheY to its phosphatase, CheZ. Biochemistry 33:902–906.
25. Blattner, F. R., V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels. 1993. Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 minutes. Nucleic Acids Res. 21:5408–5417.
26. Bochner, B. R., and B. N. Ames. 1982. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J. Biol. Chem. 257:9759–9769.
27. Brown, T. D. K., M. C. Jones-Mortimer, and H. L. Kornberg. 1977. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J. Gen. Microbiol. 102:327–336.
28. Brzoska, P., and W. Boos. 1988. Characteristics of a ugp-encoded and phoB-dependent glycerophosphoryl diester phosphodiesterase which is physically dependent on the Ugp transport system of Escherichia coli. J. Bacteriol. 170:4125–4135.
29. Brzoska, P., M. Rimmele, K. Brzostek, and W. Boos. 1994. The pho regulon-dependent Ugp uptake system for glycerol-3-phosphate in Escherichia coli is trans inhibited by Pi. J. Bacteriol. 176:15–20.
30. Bueno, R., G. Pahel, and B. Magasanik. 1985. Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli. J. Bacteriol. 164:816–822.
31. Burland, V., G. Plunkett III, H. J. Sofia, D. L. Daniels, and F. R. Blattner. 1995. Analysis of the Escherichia coli genome VI: DNA sequence of the region from 92.8 through 100 minutes. Nucleic Acids Res. 23:2105–2119.
32. Burns, D. M., and I. R. Beacham. 1986. Nucleotide sequence and transcriptional analysis of the E. coli ushA gene, encoding periplasmic UDP-sugar hydrolase (5'-nucleotidase): regulation of the ushA gene, and the signal sequence of its protein product. Nucleic Acids Res. 14:4325–4342.
33. Case, C. C., B. Bukau, S. Granett, M. R. Villarejo, and W. Boos. 1986. Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli. J. Bacteriol. 166:706–712.
34. Casida, L. E. 1960. Microbial oxidation and utilization of orthophosphite during growth. J. Bacteriol. 80:237–241.
35. Chang, C. N., W.-J. Kuang, and E. Y. Chen. 1986. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. Gene 44:121–125.
36. Charles, T. C., and E. W. Nester. 1993. A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens. J. Bacteriol. 175:6614–6625.
37. Chen, C.-M., Q. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh. 1990. Molecular biology of carbon-phosphorus bond cleavage: cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B. J. Biol. Chem. 265:4461–4471.
38. Chen, J., A. Brevet, M. Fromant, F. Lévêque, J.-M. Schmitter, S. Blanquet, and P. Plateau. 1990. Pyrophosphatase is essential for growth of Escherichia coli. J. Bacteriol. 172:5686–5689.
39. Chuang, S.-E., D. L. Daniels, and F. R. Blattner. 1993. Global regulation of gene expression in Escherichia coli. J. Bacteriol. 175:2026–2036.
40. Coleman, J. E. 1992. Structure and mechanism of alkaline phosphatase. Annu. Rev. Biophys. Biophys. Chem. 21:441–483.
41. Cowan, S. W., T. Schirmer, G. Rummel, M. Steiert, R. Ghosh, R. A. Pauptit, J. N. Jansonius, and J. P. Rosenbusch. 1992. Crystal structures explain functional properties of two E. coli porins. Nature (London) 358:727–733.
42. Cox, G. B., H. Rosenberg, J. A. Downie, and S. Silver. 1981. Genetic analysis of mutants affected in the Pst inorganic phosphate transport system. J. Bacteriol. 148:1–9.
43. Cox, G. B., D. Webb, J. Godovac-Zimmermann, and H. Rosenberg. 1988. Arg-220 of the PstA protein is required for phosphate transport through the phosphate-specific transport system in Escherichia coli but not for alkaline phosphatase repression. J. Bacteriol. 170:2283–2286.
44. Cox, G. B., D. Webb, and H. Rosenberg. 1989. Specific amino acid residues in both the PstB and PstC proteins are required for phosphate transport by the Escherichia coli Pst system. J. Bacteriol. 171:1531–1534.
45. Crooke, E., M. Akiyama, N. N. Rao, and A. Kornberg. 1994. Genetically altered levels of inorganic polyphosphate in Escherichia coli. J. Biol. Chem. 269:6290–6295.
46. Damoglou, A. P., and E. A. Dawes. 1968. Studies on the lipid content and phosphate requirement for glucose- and acetate-grown Escherichia coli. Biochem. J. 110:775–781.
47. Danchin, A., L. Dondon, and J. Daniel. 1984. Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12. Mol. Gen. Genet. 193:473–478.
48. Dassa, E., and P. L. Boquet. 1981. Is the acid phosphatase of Escherichia coli with pH optimum of 2.5 a polyphosphate depolymerase? FEBS Lett. 135:148–150.
49. Dassa, J., H. Fsihi, C. Marck, M. Dion, M. Kieffer-Bontemps, and P. L. Boquet. 1991. A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA). Mol. Gen. Genet. 229:341–352.
50. Dassa, J., C. Marck, and P. L. Boquet. 1990. The complete nucleotide sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase. J. Bacteriol. 172:5497–5500.
51. Daughton, C. G., A. M. Cook, and M. Alexander. 1979. Biodegradation of phosphonate toxicants yields methane or ethane on cleavage of the C-P bond. FEMS Microbiol. Lett. 5:91–93.
52. Davis, E. O., and P. J. F. Henderson. 1987. The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12. J. Biol. Chem. 262:13928–13932.
53. De Cock, H., W. Overeem, and J. Tommassen. 1992. Biogenesis of outer membrane protein PhoE of Escherichia coli. Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein. J. Mol. Biol. 224:369–379.
54. de Lederkremer, R. M., C. Lima, M. I. Ramirez, M. A. J. Ferguson, S. W. Homans, and J. Thomas-Oates. 1991. Complete structure of the glycan of lipopeptidophosphoglycan from Trypanosoma cruzi epimastigotes. J. Biol. Chem. 266:23670–23675.
55. De Lorenzo, V., M. Herrero, U. Jakubzik, and K. N. Timmis. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 172:6568–6572.
56. Deretic, V., J. H. J. Leveau, C. D. Mohr, and N. S. Hibler. 1992. In vitro phosphorylation of AlgR, a regulator of mucoidy in Pseudomonas aeruginosa, by a histidine protein kinase and effects of small phospho-donor molecules. Mol. Microbiol. 6:2761–2767.
57. Derman, A. I., and J. Beckwith. 1991. Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. J. Bacteriol. 173:7719–7722.
58. DiRusso, C. C., A. K. Metzger, and T. L. Heimert. 1993. Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichia coli by FadR. Mol. Microbiol. 7:311–322.
59. Doige, C. A., and G. F.-L. Ames. 1993. ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance. Annu. Rev. Microbiol. 47:291–319.
60. Dri, A. M., and P. L. Moreau. 1993. Phosphate starvation and low temperature as well as ultraviolet irradiation transcriptionally induce the Escherichia coli LexA-controlled gene sfiA. Mol. Microbiol. 8:697–706.
61. Drury, L. S., and R. S. Buxton. 1988. Identification and sequencing of the Escherichia coli cet gene which codes for an inner membrane protein, mutation of which causes tolerance to colicin E2. Mol. Microbiol. 2:109–119.
62. Dvorak, H. F., R. W. Brockman, and L. A. Heppel. 1967. Purification and properties of two acid phosphatase fractions isolated from osmotic shock fluid of Escherichia coli. Biochemistry 6:1743–1751.
63. Edwards, C. J., D. J. Innes, D. M. Burns, and I. R. Beacham. 1993. UDP-sugar hydrolase isozymes in Salmonella enterica and Escherichia coli: silent alleles of ushA in related strains of group I Salmonella isolates, and of ushB in wild-type and K12 strains of E. coli, indicate recent and early silencing events, respectively. FEMS Microbiol. Lett. 114:293–298.
64. Eidels, L., P. D. Rick, N. P. Stimler, and M. J. Osborn. 1974. Transport of d-arabinose-5-phosphate and d-sedoheptulose-7-phosphate by the hexose phosphate transport system of Salmonella typhimurium. J. Bacteriol. 119:138–143.
65. Eiglmeier, K., W. Boos, and S. T. Cole. 1987. Nucleotide sequence and transcriptional startpoint of the glpT gene of Escherichia coli: extensive sequence homology of the glycerol-3-phosphate transport protein with components of the hexose 6-phosphate transport system. Mol. Microbiol. 1:251–258.
66. Elvin, C. M., C. M. Hardy, and H. Rosenberg. 1985. Pi exchange mediated by the GlpT-dependent sn-glycerol-3-phosphate transport system in Escherichia coli. J. Bacteriol. 161:1054–1058.
67. Feng, J., M. R. Atkinson, W. McCleary, J. B. Stock, B. L. Wanner, and A. J. Ninfa. 1992. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J. Bacteriol. 174:6061–6070.
68. Foster, J. W., and M. P. Spector. 1986. Phosphate starvation regulon of Salmonella typhimurium. J. Bacteriol. 166:666–669.
69. Foulds, J., and T. Chai. 1978. Chromosomal location of a gene (nmpA) involved in the expression of a major outer membrane protein in Escherichia coli. J. Bacteriol. 136:501–506.
70. Fox, D. K., and S. Roseman. 1986. Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 261:13487–13497.
71. Friedrich, M. J., and R. J. Kadner. 1987. Nucleotide sequence of the uhp region of Escherichia coli. J. Bacteriol. 169:3556–3563.
72. Garen, A., and H. Echols. 1962. Properties of two regulating genes for alkaline phosphatase. J. Bacteriol. 83:297–300.
73. Garen, A., and C. Levinthal. 1960. A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim. Biophys. Acta 38:470–483.
74. Glembotski, C. C., A. G. Chapman, and D. E. Atkinson. 1981. Adenylate energy charge in Escherichia coli CR341T28 and properties of heat-sensitive adenylate kinase. J. Bacteriol. 145:1374–1385.
75. Groisman, E. A., E. Chiao, C. J. Lipps, and F. Heffron. 1989. Salmonella typhimurium phoP virulence gene is a transcriptional regulator. Proc. Natl. Acad. Sci. USA 86:7077–7081.
76. Groisman, E. A., F. Heffron, and F. Solomon. 1992. Molecular genetic analysis of the Escherichia coli phoP locus. J. Bacteriol. 174:486–491.
77. Guan, C.-D., B. L. Wanner, and H. Inouye. 1983. Analysis of regulation of phoB expression using a phoB-cat fusion. J. Bacteriol. 156:710–717.
78. Guest, J. R. 1979. Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants. J. Gen. Microbiol. 115:259–271.
79. Hård, K., J. M. Van Doorn, J. E. Thomas-Oates, J. P. Kamerling, and D. J. Van der Horst. 1993. Structure of the Asn-linked oligosaccharides of apolipophorin III from the insect Locusta migratoria. Carbohydrate- linked 2-aminoethylphosphonate as a constituent of a glycoprotein. Biochemistry 32:766–775.
80. Harold, F. M., and S. Sylvan. 1963. Accumulation of inorganic polyphosphate in Aerobacter aerogenes. II. Environmental control and the role of sulfur compounds. J. Bacteriol. 86:222–231.
81. Hayashi, S.-I., J. P. Koch, and E. C. C. Lin. 1964. Active transport of l-α-glycerophosphate in Escherichia coli. J. Biol. Chem. 239:3098–3105.
82. Haydon, D. J., and J. R. Guest. 1991. A new family of bacterial regulatory proteins. FEMS Microbiol. Lett. 79:291–296.
83. Hengge, R., T. J. Larson, and W. Boos. 1983. sn-Glycerol-3-phosphate transport in Salmonella typhimurium. J. Bacteriol. 155:186–195.
84. Higgins, C. F. 1992. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8:67–113.
85. Hilderbrand, R. L. 1983. The Role of Phosphonates in Living Systems. CRC Press, Inc., Boca Raton, Fla.
85a. Hiratsu, K., K. Makino, A. Nakata, and H. Shinagawa. 1995. Autophosphorylation and activation of transcriptional activator PhoB of Escherichia coli by acetyl phosphate in vitro. Gene 161:7–10.
86. Hoffman, C. S., and A. Wright. 1985. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc. Natl. Acad. Sci. USA 82:5107–5111.
87. Holman, T. R., Z. Wu, B. L. Wanner, and C. T. Walsh. 1994. Identification of the DNA-binding site for the phosphorylated VanR protein required for vancomycin resistance. Biochemistry 33:4625–4631.
88. Hong, J.-S., A. G. Hunt, P. S. Masters, and M. A. Lieberman. 1979. Requirement of acetyl phosphate for the binding protein-dependent transport system in Escherichia coli. Proc. Natl. Acad. Sci. USA 76:1213–1217.
89. Hori, T., M. Horiguchi, and A. Hayashi. 1984. Biochemistry of Natural C-P Compounds. Maruzen, Ltd., Kyoto, Japan.
90. Horiuchi, T., S. Horiuchi, and D. Mizuno. 1959. A possible negative feedback phenomenon controlling formation of alkaline phosphomonoesterase in Escherichia coli. Nature (London) 183:1529–1530.
91. Hulett, F. M., J. Lee, L. Shi, G. Sun, R. Chesnut, E. Sharkova, M. F. Duggan, and N. Kapp. 1994. Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis. J. Bacteriol. 176:1348–1358.
92. Hunt, A. G. 1980. Micromethod for the measurement of acetyl phosphate and acetyl coenzyme A. Methods Enzymol. 122:43–50.
93. Hunt, A. G., and J.-S. Hong. 1980. A micromethod for the measurement of acetyl phosphate and acetyl coenzyme A. Anal. Biochem. 108:290–294.
94. Ishino, Y., H. Shinagawa, K. Makino, M. Amemura, and A. Nakata. 1987. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169:5429–5433.
95. Island, M. D., B.-Y. Wei, and R. J. Kadner. 1992. Structure and function of the uhp genes for the sugar phosphate transport system in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 174:2754–2762.
96. Jagura-Burdzy, G., and C. M. Thomas. 1994. KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer. Proc. Natl. Acad. Sci. USA 91:10571–10575.
96a. Jiang, W., W. W. Metcalf, K.-S. Lee, and B. L. Wanner. 1995. Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2. J. Bacteriol. 177:6411–6421.
97. Johnson, K., C. K. Murphy, and J. Beckwith. 1992. Protein export in Escherichia coli. Curr. Opin. Biotechnol. 3:481–485.
98. Josefsson, L. G., and L. L. Randall. 1981. Different exported proteins in E. coli show differences in the temporal mode of processing in vivo. Cell 25:151–157.
99. Kadner, R. J., C. A. Webber, and M. D. Island. 1993. The family of organo-phosphate transport proteins includes a transmembrane regulatory protein. J. Bioenerg. Biomembr. 25:637–645.
100. Kasahara, M., K. Makino, M. Amemura, and A. Nakata. 1989. Nucleotide sequence of the ugpQ gene encoding glycerophosphoryl diester phosphodiesterase of Escherichia coli K12. Nucleic Acids Res. 17:2854.
101. Kasahara, M., K. Makino, M. Amemura, A. Nakata, and H. Shinagawa. 1991. Dual regulation of the ugp operon by phosphate and carbon starvation at two interspaced promoters. J. Bacteriol. 173:549–558.
102. Kasahara, M., A. Nakata, and H. Shinagawa. 1991. Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase. J. Bacteriol. 173:6760–6765.
103. Kasahara, M., A. Nakata, and H. Shinagawa. 1992. Molecular analysis of the Escherichia coli phoP-phoQ operon. J. Bacteriol. 174:492–498.
104. Kato, J., Y. Sakai, T. Nikata, and H. Ohtake. 1994. Cloning and characterization of a Pseudomonas aeruginosa gene involved in the negative regulation of phosphate taxis. J. Bacteriol. 176:5874–5877.
105. Kato, J., T. Yamamoto, K. Yamada, and H. Ohtake. 1993. Cloning, sequence and characterization of the polyphosphate kinase-encoding gene (ppk) of Klebsiella aerogenes. Gene 137:237–242.
106. Keasling, J. D., L. Bertsch, and A. Kornberg. 1993. Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase. Proc. Natl. Acad. Sci. USA 90:7029–7033.
107. Kennedy, K. E., and G. A. Thompson. 1970. Phosphonolipids: localization in surface membranes of Tetrahymena. Science 168:989–991.
108. Kier, L. D., R. Weppelman, and B. N. Ames. 1977. Regulation of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium. J. Bacteriol. 130:420–428.
109. Kier, L. D., R. Weppelman, and B. N. Ames. 1977. Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium. J. Bacteriol. 130:399–410.
110. Kilby, P. M., and G. K. Radda. 1991. 2-Aminoethylphosphonic acid is the main phosphorus compound in locust hemolymph. Naturwissenschaften 78:514–517.
111. Kim, E. E., and H. W. Wyckoff. 1991. Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis. J. Mol. Biol. 218:449–464.
112. Kim, S.-K., K. Makino, M. Amemura, H. Shinagawa, and A. Nakata. 1993. Molecular analysis of the phoH gene, belonging to the phosphate regulon in Escherichia coli. J. Bacteriol. 175:1316–1324.
113. Kimura, S., K. Makino, H. Shinagawa, M. Amemura, and A. Nakata. 1989. Regulation of the phosphate regulon of Escherichia coli: characterization of the promoter of the pstS gene. Mol. Gen. Genet. 215:374–380.
114. Kofoid, E. C., and J. S. Parkinson. 1988. Transmitter and receiver modules in bacterial signaling proteins. Proc. Natl. Acad. Sci. USA 85:4981–4985.
115. Kornberg, A. 1995. Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J. Bacteriol. 177:491–496.
116. Kubena, B. D., H. Luecke, H. Rosenberg, and F. A. Quiocho. 1986. Crystallization and X-ray diffraction studies of a phosphate-binding protein involved in active transport in Escherichia coli. J. Biol. Chem. 261:7995–7996.
117. Kukko, E., and J. Heinonen. 1982. The intracellular concentration of pyrophosphate in the batch culture of Escherichia coli. Eur. J. Biochem. 127:347–349.
118. Kukko, E., and H. Saarento. 1984. Diphosphate concentration does not correlate with the level of inorganic diphosphatase in Escherichia coli. Folia Microbiol. 29:282–287.
119. Kukko-Kalske, E., M. Lintunen, M. K. Inen, R. Lahti, and J. Heinonen. 1989. Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells. J. Bacteriol. 171:4498–4500.
120. Kulaev, I. S., and V. M. Vagabov. 1983. Polyphosphate metabolism in micro-organisms. Adv. Microb. Physiol. 24:84–171.
121. Lee, J. W., and F. M. Hulett. 1992. Nucleotide sequence of the phoP gene encoding PhoP, the response regulator of the phosphate regulon of Bacillus subtilis. Nucleic Acids Res. 20:5848.
122. Lee, K.-S., W. W. Metcalf, and B. L. Wanner. 1992. Evidence for two phosphonate degradative pathways in Enterobacter aerogenes. J. Bacteriol. 174:2501–2510.
123. Lee, R. L., C. A. Schnaitman, and A. P. Pugsley. 1979. Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli. J. Bacteriol. 138:861–870.
124. Lee, T.-Y., K. Makino, H. Shinagawa, M. Amemura, and A. Nakata. 1989. Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae. J. Bacteriol. 171:6593–6599.
125. Li, P., J. Beckwith, and H. Inouye. 1988. Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli. Proc. Natl. Acad. Sci. USA 85:7685–7689.
126. Liu, J., and I. R. Beacham. 1990. Transcription and regulation of the cpdB gene in Escherichia coli K12 and Salmonella typhimurium LT2: evidence for modulation of constitutive promoters by cyclic AMP-CRP complex. Mol. Gen. Genet. 222:161–165.
127. Loewen, P. C., and R. Hengge-Aronis. 1994. The role of the sigma factor σS (KatF) in bacterial global regulation. Annu. Rev. Microbiol. 48:53–80.
128. Ludtke, D., J. Bernstein, C. Hamilton, and A. Torriani. 1984. Identification of the phoM gene product and its regulation in Escherichia coli K-12. J. Bacteriol. 159:19–25.
129. Luecke, H., and F. A. Quiocho. 1990. High specificity of a phosphate transport protein determined by hydrogen bonds. Nature (London) 347:402–406.
130. Magota, K., N. Otsuji, T. Miki, T. Horiuchi, S. Tsunasawa, J. Kondo, F. Sakiyama, M. Amemura, T. Morita, H. Shinagawa, and A. Nakata. 1984. Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli. J. Bacteriol. 157:909–917.
131. Maier, C., E. Bremer, A. Schmid, and R. Benz. 1988. Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli. Demonstration of a nucleoside-specific binding site. J. Biol. Chem. 263:2493–2499.
132. Makino, K., M. Amemura, S.-K. Kim, A. Nakata, and H. Shinagawa. 1993. Role of the σ70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli. Genes Dev. 7:149–160.
133. Makino, K., M. Amemura, S.-K. Kim, A. Nakata, and H. Shinagawa. 1994. Mechanism of transcriptional activation of the phosphate regulon in Escherichia coli, p. 5–12. In A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in Microorganisms: Cellular and Molecular Biology. ASM Press, Washington, D.C.
134. Makino, K., M. Amemura, S.-K. Kim, H. Shinagawa, and A. Nakata. 1992. Signal transduction of the phosphate regulon in Escherichia coli mediated by phosphorylation, p. 191–200. In S. Papa, A. Azzi, and J. M. Tager (ed.), Adenine Nucleotides in Cellular Energy Transfer and Signal Transduction. Birkhaeuser Verlag, Basel.
135. Makino, K., S.-K. Kim, H. Shinagawa, M. Amemura, and A. Nakata. 1991. Molecular analysis of the cryptic and functional phn operons for phosphonate use in Escherichia coli K-12. J. Bacteriol. 173:2665–2672.
136. Makino, K., H. Shinagawa, M. Amemura, T. Kawamoto, M. Yamada, and A. Nakata. 1989. Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins. J. Mol. Biol. 210:551–559.
137. Makino, K., H. Shinagawa, M. Amemura, S. Kimura, A. Nakata, and A. Ishihama. 1988. Regulation of the phosphate regulon of Escherichia coli: activation of pstS transcription by PhoB protein in vitro. J. Mol. Biol. 203:85–95.
138. Makino, K., H. Shinagawa, M. Amemura, and A. Nakata. 1986. Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12. J. Mol. Biol. 190:37–44.
139. Makino, K., H. Shinagawa, M. Amemura, and A. Nakata. 1986. Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli. J. Mol. Biol. 192:549–556.
140. Makino, K., H. Shinagawa, and A. Nakata. 1984. Cloning and characterization of the alkaline phosphatase positive regulatory gene (phoM) of Escherichia coli. Mol. Gen. Genet. 195:381–390.
141. Maloney, P. C., S. V. Ambudkar, V. Anantharam, L. A. Sonna, and A. Varadhachary. 1990. Anion-exchange mechanisms in bacteria. Microbiol. Rev. 54:1–17.
142. Manoil, C. 1990. Analysis of protein localization by use of gene fusions with complementary properties. J. Bacteriol. 172:1035–1042.
143. Manoil, C., and J. Beckwith. 1985. TnphoA: a transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82:8129–8133.
144. Manoil, C., and J. Beckwith. 1986. A genetic approach to analyzing membrane protein topology. Science 233:1403–1408.
145. Manoil, C., J. J. Mekalanos, and J. Beckwith. 1990. Alkaline phosphatase fusions: sensors of subcellular location. J. Bacteriol. 172:515–518.
146. Mantis, N. J., and S. C. Winans. 1993. The chromosomal response regulatory gene chvI of Agrobacterium tumefaciens complements an Escherichia coli phoB mutation and is required for virulence. J. Bacteriol. 175:6626–6636.
147. Matsuyama, A., H. Yamamoto, and E. Nakano. 1989. Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene. J. Bacteriol. 171:577–580.
148. McCleary, W. R., and J. B. Stock. 1994. Acetyl phosphate and the activation of two-component response regulators. J. Biol. Chem. 269:31567–31572.
149. McCleary, W. R., J. B. Stock, and A. J. Ninfa. 1993. Is acetyl phosphate a global signal in Escherichia coli. J. Bacteriol. 175:2793–2798.
150. McMullan, G., R. Watkins, D. B. Harper, and J. P. Quinn. 1991. Carbon-phosphorus bond cleavage activity in cell-free extracts of Enterobacter aerogenes ATCC 15038 and Pseudomonas sp. 4ASW. Biochem. Int. 25:271–279.
151. Medveczky, N., and H. Rosenberg. 1971. Phosphate transport in Escherichia coli. Biochim. Biophys. Acta 241:494–506.
152. Metcalf, W. W., P. M. Steed, and B. L. Wanner. 1990. Identification of phosphate-starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions. J. Bacteriol. 172:3191–3200.
153. Metcalf, W. W., and B. L. Wanner. 1991. Involvement of the Escherichia coli phn (psiD) gene cluster in assimilation of phosphorus in the form of phosphonates, phosphite, Pi esters, and Pi. J. Bacteriol. 173:587–600.
154. Metcalf, W. W., and B. L. Wanner. 1993. Mutational analysis of an Escherichia coli fourteen-gene operon for phosphonate degradation using TnphoA ' elements. J. Bacteriol. 175:3430–3442.
155. Metcalf, W. W., and B. L. Wanner. 1993. Evidence for a fourteen-gene, phnC to phnP, locus for phosphonate metabolism in Escherichia coli. Gene 129:27–32.
156. Meyer, S. E., S. Granett, J. U. Jung, and M. R. Villarejo. 1990. Osmotic regulation of PhoE porin synthesis in Escherichia coli. J. Bacteriol. 172:5501–5502.
157. Michaelis, S., H. Inouye, D. Oliver, and J. Beckwith. 1983. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J. Bacteriol. 154:366–374.
158. Miller, S. I., A. M. Kukral, and J. J. Mekalanos. 1989. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc. Natl. Acad. Sci. USA 86:5054–5058.
159. Mizuno, T., W.-Y. Chou, and M. Inouye. 1983. A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene. J. Biol. Chem. 258:6932–6940.
160. Morris, H., M. J. Schlesinger, M. Bracha, and E. Yagil. 1974. Pleiotropic effects of mutations involved in the regulation of Escherichia coli K-12 alkaline phosphatase. J. Bacteriol. 119:583–592.
161. Muda, M., N. N. Rao, and A. Torriani. 1992. Role of PhoU in phosphate transport and alkaline phosphatase regulation. J. Bacteriol. 174:8057–8064.
162. Murata, K., N. Higaki, and A. Kimura. 1989. A microbial carbon-phosphorus bond cleavage enzyme requires two protein components for activity. J. Bacteriol. 171:4504–4506.
163. Nagaya, M., H. Aiba, and T. Mizuno. 1994. The sphR product, a two-component system response regulator protein, regulates phosphate assimilation in Synechococcus sp. strain PCC 7942 by binding to two sites upstream from the phoA promoter. J. Bacteriol. 176:2210–2215.
164. Nakamura, T., C. Hsu, and B. P. Rosen. 1986. Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters. J. Biol. Chem. 261:678–683.
165. Neu, H. C. 1967. The 5'-nucleotidase of Escherichia coli. II. Surface localization and purification of the Escherichia coli 5'-nucleotidase inhibitor. J. Biol. Chem. 242:3905–3911.
166. Nixon, B. T., C. W. Ronson, and F. M. Ausubel. 1986. Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc. Natl. Acad. Sci. USA 83:7850–7854.
167. Nyström, T. 1994. The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival. Mol. Microbiol. 12:833–843.
168. Ostanin, K., E. H. Harms, P. E. Stevis, R. Kuciel, M.-M. Zhou, and R. L. Van Etten. 1992. Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. J. Biol. Chem. 267:22830–22836.
169. Ostanin, K., A. Saeed, and R. L. Van Etten. 1994. Heterologous expression of human prostatic acid phosphatase and site-directed mutagenesis of the enzyme active site. J. Biol. Chem. 269:8971–8978.
170. Ostanin, K., and R. L. Van Etten. 1993. Asp304 of Escherichia coli acid phosphatase is involved in leaving group protonation. J. Biol. Chem. 268:20778–20784.
171. Overbeeke, N., H. Bergmans, F. V. Mansfeld, and B. Lugtenberg. 1983. Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12. J. Mol. Biol. 163:513–532.
172. Overbeeke, N., and B. Lugtenberg. 1980. Expression of outer membrane protein e of Escherichia coli K12 by phosphate limitation. FEBS Lett. 112:229–232.
173. Overduin, P., W. Boos, and J. Tommassen. 1988. Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system. Mol. Microbiol. 2:6:767–775.
174. Parkinson, J. S., and E. C. Kofoid. 1992. Communication modules in bacterial signaling proteins. Annu. Rev. Genet. 26:71–112.
175. Phillips, N. F. B., P. J. Horn, and H. G. Wood. 1993. The polyphosphate- and ATP-dependent glucokinase from Propionibacterium shermanii: both activities are catalyzed by the same protein. Arch. Biochem. Biophys. 300:309–319.
176. Pogell, B. M., B. R. Maity, S. Frumkin, and S. Shapiro. 1966. Induction of an active transport system for glucose 6-phosphate in Escherichia coli. Arch. Biochem. Biophys. 116:406–415.
177. Pradel, E., and P. L. Boquet. 1988. Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase. J. Bacteriol. 170:4916–4923.
178. Pradel, E., and P. L. Boquet. 1989. Mapping of the Escherichia coli acid glucose-1-phosphatase gene agp and analysis of its expression in vivo by use of an agp-phoA protein fusion. J. Bacteriol. 171:3511–3517.
179. Pradel, E., and P. L. Boquet. 1991. Utilization of exogenous glucose-1-phosphate as a source of carbon or phosphate by Escherichia coli K12: respective roles of acid glucose-1-phosphatase, hexose-phosphate permease, phosphoglucomutase and alkaline phosphatase. Res. Microbiol. 142:37–45.
180. Pradel, E., C. Marck, and P. L. Boquet. 1990. Nucleotide sequence and transcriptional analysis of the Escherichia coli agp gene encoding periplasmic acid glucose-1-phosphatase. J. Bacteriol. 172:802–807.
181. Prüss, B. M., and A. J. Wolfe. 1994. Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli. Mol. Microbiol. 12:973–984.
182. Quail, M. A., C. E. Dempsey, and J. R. Guest. 1994. Identification of a fatty acyl responsive regulator (FarR) in Escherichia coli. FEBS Lett. 356:183–187.
183. Quin, L. D. 1965. The presence of compounds with a carbon-phosphorus bond in some marine invertebrates. Biochemistry 4:324–330.
184. Rao, N. N., A. Kar, M. F. Roberts, J. Yashphe, and A. Torriani-Gorini. 1994. Phosphate, phosphorylated metabolites, and the Pho regulon of Escherichia coli, p. 22–29. In A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in Microorganisms: Cellular and Molecular Biology. ASM Press, Washington, D.C.
185. Rao, N. N., M. F. Roberts, A. Torriani, and J. Yashphe. 1993. Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli. J. Bacteriol. 175:74–79.
186. Rao, N. N., and A. Torriani. 1988. Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins. J. Bacteriol. 170:5216–5223.
187. Rao, N. N., E. Wang, J. Yashphe, and A. Torriani. 1986. Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli. J. Bacteriol. 166:205–211.
188. Reitzer, L. J., and B. Magasanik. 1985. Expression of glnA in Escherichia coli is regulated at tandem promoters. Proc. Natl. Acad. Sci. USA 82:1979–1983.
189. Roggiani, M., and D. Dubnau. 1993. ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. J. Bacteriol. 175:3182–3187.
190. Rose, I. A. 1962. Acetate kinase, p. 115–118. In P. D. Boyer, H. Lardy, and K. Myrbäck (ed.), The Enzymes. Academic Press, Inc., New York.
191. Saier, M. H., Jr., D. L. Wentzel, B. U. Feucht, and J. J. Judice. 1975. A transport system for phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in Salmonella typhimurium. J. Biol. Chem. 250:5089–5096.
192. Schneider, T. D., G. D. Stormo, and L. Gold. 1986. Information content of binding sites on nucleotide sequences. J. Mol. Biol. 188:415–431.
193. Scholten, M., and J. Tommassen. 1993. Topology of the PhoR protein of Escherichia coli and functional analysis of internal deletion mutants. Mol. Microbiol. 8:269–275.
194. Scholten, M., and J. Tommassen. 1994. Effect of mutations in the –10 region of the phoE promoter in Escherichia coli on regulation of gene expression. Mol. Gen. Genet. 245:218–223.
195. Schweizer, H., M. Argast, and W. Boos. 1982. Characteristics of a binding protein-dependent transport system for sn-glycerol-3-phosphate in Escherichia coli that is part of the pho regulon. J. Bacteriol. 150:1154–1163.
196. Seki, T., H. Yoshikawa, H. Takahashi, and H. Saito. 1987. Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis. J. Bacteriol. 169:2913–2916.
197. Seki, T., H. Yoshikawa, H. Takahashi, and H. Saito. 1988. Nucleotide sequence of the Bacillus subtilis phoR gene. J. Bacteriol. 170:5935–5938.
198. Selander, R. K., D. A. Caugant, and T. S. Whittam. 1987. Genetic structure and variation in natural populations of Escherichia coli, p. 1625–1648. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 2. American Society for Microbiology, Washington, D.C.
199. Sheppard, D., and E. Englesberg. 1966. Positive control in the l-arabinose gene-enzyme complex of Escherichia coli B/r as exhibited with stable merodiploids. Cold Spring Harbor Symp. Quant. Biol. 31:345–347.
200. Shimizu, M., T. Suzuki, K.-Y. Kameda, and Y. Abiko. 1991. Phosphotransacetylase of Escherichia coli B, purification and properties. Biochim. Biophys. Acta 191:550–558.
201. Shinagawa, H., K. Makino, M. Yamada, M. Amemura, T. Sato, and A. Nakata. 1994. Signal transduction in the phosphate regulon of Escherichia coli: dual functions of PhoR as a protein kinase and a protein phosphatase, p. 285–289. In A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in Microorganisms: Cellular and Molecular Biology. ASM Press, Washington, D.C.
202. Shulman, R. G., T. R. Brown, K. Ugurbil, S. Ogawa, S. M. Cohen, and J. A. den Hollander. 1979. Cellular applications of 31P and 13C nuclear magnetic resonance. Science 205:160–166.
203. Skarstad, K., B. Thöny, D. S. Hwang, and A. Kornberg. 1993. A novel binding protein of the origin of the Escherichia coli chromosome. J. Biol. Chem. 268:5365–5370.
204. Smith, I. W., J. F. Wilkinson, and J. P. Duguid. 1954. Volutin production in Aerobacter aerogenes due to nutrient imbalance. J. Bacteriol. 68:450–463.
205. Smith, M. W., and J. W. Payne. 1992. Expression of periplasmic binding proteins for peptide transport is subject to negative regulation by phosphate limitation in Escherichia coli. FEMS Microbiol. Lett. 100:183–190.
206. Sofia, H. J., V. Burland, D. L. Daniels, G. Plunkett III, and F. R. Blattner. 1994. Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes. Nucleic Acids Res. 22:2576–2586.
207. Sonna, L. A., S. V. Ambudkar, and P. C. Maloney. 1988. The mechanism of glucose 6-phosphate transport by Escherichia coli. J. Biol. Chem. 263:6625–6630.
208. Spierings, G., R. Elders, B. Van Lith, H. Hofstra, and J. Tommassen. 1992. Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes. Gene 122:45–52.
209. Spierings, G., C. Ockhuijsen, H. Hofstra, and J. Tommassen. 1992. Characterization of the Citrobacter freundii phoE gene and development of C. freundii-specific oligonucleotides. FEMS Microbiol. Lett. 99:199–204.
210. Spierings, G., C. Ockhuijsen, H. Hofstra, and J. Tommassen. 1993. Polymerase chain reaction for the specific detection of Escherichia coli/Shigella. Res. Microbiol. 144:557–564.
211. Steed, P. M., and B. L. Wanner. 1993. Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon. J. Bacteriol. 175:6797–6809.
212. Struyvé, M., J. Visser, H. Adriaanse, R. Benz, and J. Tommassen. 1993. Topology of PhoE porin: the ‘eyelet’ region. Mol. Microbiol. 7:131–140.
213. Su, T.-Z., H. P. Schweizer, and D. L. Oxender. 1991. Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli. Mol. Gen. Genet. 230:28–32.
214. Surin, B. P., N. E. Dixon, and H. Rosenberg. 1986. Purification of the PhoU protein, a negative regulator of the pho regulon of Escherichia coli K-12. J. Bacteriol. 168:631–635.
215. Surin, B. P., D. A. Jans, A. L. Fimmel, D. C. Shaw, G. B. Cox, and H. Rosenberg. 1984. Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene. J. Bacteriol. 157:772–778.
216. Surin, B. P., H. Rosenberg, and G. B. Cox. 1985. Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships. J. Bacteriol. 161:189–198.
217. Taylor, R. K., C. Manoil, and J. J. Mekalanos. 1989. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J. Bacteriol. 171:1870–1878.
218. Tommassen, J., P. De Geus, and B. Lugtenberg. 1982. Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products. J. Mol. Biol. 157:265–274.
219. Tommassen, J., K. Eiglmeier, S. T. Cole, P. Overduin, T. J. Larson, and W. Boos. 1991. Characterization of two genes, glpQ and ugpQ, encoding glycerophosphoryl diester phosphodiesterases of Escherichia coli. Mol. Gen. Genet. 226:321–327.
220. Tommassen, J., M. Koster, and P. Overduin. 1987. Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene: identification of an element, upstream from the promoter, required for efficient expression of PhoE protein. J. Mol. Biol. 198:633–641.
221. Tommassen, J., and B. Lugtenberg. 1980. Outer membrane protein e of Escherichia coli K-12 is coregulated with alkaline phosphatase. J. Bacteriol. 143:151–157.
222. Tommassen, J., and B. Lugtenberg. 1981. Localization of phoE, the structural gene for outer membrane protein e in Escherichia coli K-12. J. Bacteriol. 147:118–123.
223. Torriani, A. 1960. Influence of inorganic phosphate in the formation of phosphatases by Escherichia coli. Biochim. Biophys. Acta 38:460–469.
224. Torriani, A. 1990. From cell membrane to nucleotides: the phosphate regulon in Escherichia coli. Bioessays 12:371–376.
225. Torriani-Gorini, A., F. G. Rothman, S. Silver, A. Wright, and E. Yagil (ed.). 1987. Phosphate Metabolism and Cellular Regulation in Microorganisms. American Society for Microbiology, Washington, D.C.
226. Torriani-Gorini, A., E. Yagil, and S. Silver (ed.). 1994. Phosphate in Microorganisms: Cellular and Molecular Biology. ASM Press, Washington, D.C.
227. Traxler, B., D. Boyd, and J. Beckwith. 1993. The topological analysis of integral cytoplasmic membrane proteins. J. Membr. Biol. 132:1–11.
228. Uerkvitz, W. 1988. Periplasmic nonspecific acid phosphatase II from Salmonella typhimurium LT2. Crystallization, detergent reactivation, and phosphotransferase activity. J. Biol. Chem. 263:15823–15830.
229. Uerkvitz, W., and C. F. Beck. 1981. Periplasmic phosphatases in Salmonella typhimurium LT2. A biochemical, physiological, and partial genetic analysis of three nucleoside monophosphate dephosphorylating enzymes. J. Biol. Chem. 256:382–389.
230. Van der Ley, P., A. Bekkers, J. van Meersbergen, and J. Tommassen. 1987. A comparative study on the phoE genes of three enterobacterial species: implications for structure-function relationships in a pore-forming protein of the outer membrane. Eur. J. Biochem. 164:469–475.
231. Van Veen, H. W., T. Abee, G. J. J. Kortstee, W. N. Konings, and A. J. B. Zehnder. 1994. Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli. Biochemistry 33:1766–1770.
232. Varadhachary, A., and P. C. Maloney. 1991. Reconstitution of the phosphoglycerate transport protein of Salmonella typhimurium. J. Biol. Chem. 266:130–135.
233. Villarejo, M., J. L. Davis, and S. Granett. 1983. Osmoregulation of alkaline phosphatase synthesis in Escherichia coli K-12. J. Bacteriol. 156:975–978.
234. Volz, K. 1993. Structural conservation in the CheY superfamily. Biochemistry 32:11741–11753.
235. Wackett, L. P., B. L. Wanner, C. P. Venditti, and C. T. Walsh. 1987. Involvement of the phosphate regulon and the psiD locus in the carbon-phosphorus lyase activity of Escherichia coli K-12. J. Bacteriol. 169:1753–1756.
236. Wang, Z., A. Choudhary, P. S. Ledvina, and F. A. Quiocho. 1994. Fine tuning the specificity of the periplasmic phosphate transport receptor. Site-directed mutagenesis, ligand binding, and crystallographic studies. J. Biol. Chem. 269:25091–25094.
237. Wanner, B. L. 1983. Overlapping and separate controls on the phosphate regulon in Escherichia coli K-12. J. Mol. Biol. 166:283–308.
238. Wanner, B. L. 1985. Phase mutants: evidence of a physiologically regulated "change-in-state" gene system in Escherichia coli. UCLA Symp. Mol. Cell. Biol. New Ser. 20:103–122.
239. Wanner, B. L. 1986. Bacterial alkaline phosphatase clonal variation in some Escherichia coli K-12 phoR mutant strains. J. Bacteriol. 168:1366–1371.
240. Wanner, B. L. 1986. Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12. J. Mol. Biol. 191:39–58.
241. Wanner, B. L. 1987. Bacterial alkaline phosphatase gene regulation and the phosphate response in Escherichia coli, p. 12–19. In A. Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright, and E. Yagil (ed.), Phosphate Metabolism and Cellular Regulations in Microorganisms. American Society for Microbiology, Washington, D.C.
242. Wanner, B. L. 1987. Phosphate regulation of gene expression in Escherichia coli, p. 1326–1333. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 2. American Society for Microbiology, Washington, D.C.
243. Wanner, B. L. 1987. Control of phoR-dependent bacterial alkaline phosphatase clonal variation by the phoM region. J. Bacteriol. 169:900–903.
244. Wanner, B. L. 1990. Phosphorus assimilation and its control of gene expression in Escherichia coli, p. 152–163. In G. Hauska and R. Thauer (ed.), The Molecular Basis of Bacterial Metabolism. Springer-Verlag, Heidelberg.
245. Wanner, B. L. 1992. Minireview. Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? J. Bacteriol. 174:2053–2058.
246. Wanner, B. L. 1993. Gene regulation by phosphate in enteric bacteria. J. Cell. Biochem. 51:47–54.
247. Wanner, B. L. 1994. Phosphate-regulated genes for the utilization of phosphonates in members of the family Enterobacteriaceae, p. 215–221. In A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in Microorganisms: Cellular and Molecular Biology. ASM Press, Washington, D.C.
248. Wanner, B. L. 1994. Multiple controls of the Escherichia coli Pho regulon by the Pi sensor PhoR, the catabolite regulatory sensor CreC, and acetyl phosphate, p. 13–21. In A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in Microorganisms: Cellular and Molecular Biology. ASM Press, Washington, D.C.
249. Wanner, B. L. 1994. Gene expression in bacteria using TnphoA and TnphoA ' elements to make and switch phoA gene, lacZ (op), and lacZ (pr) fusions, p. 291–310. In K. W. Adolph (ed.), Methods in Molecular Genetics, vol. 3. Academic Press, Orlando, Fla..
250. Wanner, B. L. 1994. Molecular genetics of carbon-phosphorus bond cleavage in bacteria. Biodegradation 5:175–184.
251. Wanner, B. L. 1995. Signal transduction and cross regulation in the Escherichia coli phosphate regulon by PhoR, CreC, and acetyl phosphate, p. 203–221. In J. A. Hoch and T. J. Silhavy (ed.), Two-Component Signal Transduction. American Society for Microbiology, Washington, D.C.
252. Wanner, B. L., and J. Bernstein. 1982. Determining the phoM map location in Escherichia coli K-12 by using a nearby transposon Tn10 insertion. J. Bacteriol. 150:429–432.
253. Wanner, B. L., and J. A. Boline. 1990. Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli. J. Bacteriol. 172:1186–1196.
254. Wanner, B. L., and B.-D. Chang. 1987. The phoBR operon in Escherichia coli K-12. J. Bacteriol. 169:5569–5574.
255. Wanner, B. L., and P. Latterell. 1980. Mutants affected in alkaline phosphatase expression: evidence for multiple positive regulators of the phosphate regulon in Escherichia coli. Genetics 96:242–266.
256. Wanner, B. L., and R. McSharry. 1982. Phosphate-controlled gene expression in Escherichia coli using Mud1-directed lacZ Fusions. J. Mol. Biol. 158:347–363.
257. Wanner, B. L., and W. W. Metcalf. 1992. Molecular genetic studies of a 10.9-kbp operon in Escherichia coli for phosphonate uptake and biodegradation. FEMS Microbiol. Lett. 100:133–140.
258. Wanner, B. L., A. Sarthy, and J. R. Beckwith. 1979. Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins. J. Bacteriol. 140:229–239.
259. Wanner, B. L., S. Wieder, and R. McSharry. 1981. Use of bacteriophage transposon Mu d1 to determine the orientation for three proC-linked phosphate-starvation-inducible (psi) genes in Escherichia coli K-12. J. Bacteriol. 146:93–101.
260. Wanner, B. L., M. R. Wilmes, and E. Hunter. 1988. Molecular cloning of the wild-type phoM operon in Escherichia coli K-12. J. Bacteriol. 170:279–288.
261. Wanner, B. L., M. R. Wilmes, and D. C. Young. 1988. Control of bacterial alkaline phosphatase synthesis and variation in an Escherichia coli K-12 phoR mutant by adenyl cyclase, the cyclic AMP receptor protein, and the phoM operon. J. Bacteriol. 170:1092–1102.
262. Wanner, B. L., and M. R. Wilmes-Riesenberg. 1992. Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in the control of the phosphate regulon in Escherichia coli. J. Bacteriol. 174:2124–2130.
263. Webb, D. C., and G. B. Cox. 1994. Proposed mechanism for phosphate translocation by the phosphate-specific transport (Pst) system and role of the Pst system in phosphate regulation, p. 37–42. In A. Torriani-Gorini, E. Yagil, and S. Silver (ed.), Phosphate in Microorganisms: Cellular and Molecular Biology. ASM Press, Washington, D.C.
264. Webb, D. C., H. Rosenberg, and G. B. Cox. 1992. Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices. J. Biol. Chem. 267:24661–24668.
264a. Welch, M., K. Oosawa, S. -I. Aizawa, and M. Eisenbach. 1994. Effects of phosphorylation, Mg2+, and conformation of the chemotaxis protein CheY on its binding to the flagellar switch protein FliM. Biochemistry 33:10470–10476.
265. Wilkins, A. S. 1972. Physiological factors in the regulation of alkaline phosphatase synthesis in Escherichia coli. J. Bacteriol. 110:616–623.
266. Willsky, G. R., R. L. Bennett, and M. H. Malamy. 1973. Inorganic phosphate transport in Escherichia coli: involvement of two genes which play a role in alkaline phosphatase regulation. J. Bacteriol. 113:529–539.
267. Willsky, G. R., and M. H. Malamy. 1974. The loss of the phoS periplasmic protein leads to a change in the specificity of a constitutive inorganic phosphate transport system in Escherichia coli. Biochem. Biophys. Res. Commun. 60:226–233.
268. Willsky, G. R., and M. H. Malamy. 1976. Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli. J. Bacteriol. 127:595–609.
269. Willsky, G. R., and M. H. Malamy. 1980. Characterization of two genetically separable inorganic phosphate transport system in Escherichia coli. J. Bacteriol. 144:356–365.
270. Willsky, G. R., and M. H. Malamy. 1980. Effect of arsenate on inorganic phosphate transport in Escherichia coli. J. Bacteriol. 144:366–374.
271. Wilmes-Riesenberg, M. R., and B. L. Wanner. 1992. TnphoA and TnphoA ' elements for making and switching fusions for study of transcription, translation, and cell surface localization. J. Bacteriol. 174:4558–4575.
272. Wilson, I. B., J. Dayan, and K. Cyr. 1964. Some properties of alkaline phosphatase from Escherichia coli. Transphosphorylation. J. Biol. Chem. 239:4182–4185.
273. Winkler, H. H. 1966. A hexose-phosphate transport system in Escherichia coli. Biochim. Biophys. Acta 117:231–240.
274. Wood, H. G. 1977. Some reactions in which inorganic pyrophosphate replaces ATP and serves as a source of energy. Fed. Proc. 36:2197–2205.
275. Xavier, K. B., M. Kossmann, H. Santos, and W. Boos. 1995. Kinetic analysis by in vivo 31P nuclear magnetic resonance of internal Pi during uptake of sn-glycerol-3-phosphate by the pho regulon-dependent Ugp system and the glp regulon-dependent GlpT uptake GlpT system. J. Bacteriol. 177:699–704.
276. Yagil, E. 1975. Derepression of polyphosphatase in Escherichia coli by starvation for inorganic phosphate. FEBS Lett. 55:124–127.
277. Yamada, M., K. Makino, M. Amemura, H. Shinagawa, and A. Nakata. 1989. Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes. J. Bacteriol. 171:5601–5606.
278. Yamada, M., K. Makino, H. Shinagawa, and A. Nakata. 1990. Regulation of the phosphate regulon of Escherichia coli: properties of phoR deletion mutants and subcellular localization of PhoR protein. Mol. Gen. Genet. 220:366–372.
279. Yang, Y., D. Goldrick, and J.-S. Hong. 1988. Nucleotide sequence and transcription start point of the phosphoglycerate transporter gene of Salmonella typhimurium. J. Bacteriol. 170:3421–3426.
280. Yoshida, K.-I., Y. Fujita, and A. Sarai. 1993. Missense mutations in the Bacillus subtilis gnt repressor that diminish operator binding ability. J. Mol. Biol. 231:167–174.
281. Zeleznick, L. D., T. C. Myers, and E. B. Titchener. 1963. Growth of Escherichia coli on methyl- and ethylphosphonic acids. Biochim. Biophys. Acta 78:546–547.
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