4. The response to high levels of extracellular Pi

Clues to understanding how E. coli and other bacteria cope with high levels of extracellular Pi came from studies on Pi remediation [12, 74, 75]. Excess Pi in natural water sources is a major cause of eutrophication [76]. Toxic cyanobacterial blooms are frequently attributed to Pi accumulation in water sources resulting from agricultural runoff [77]. Normally, Pi is removed from wastewaters by chemical precipitation, which is an expensive process [78]. Biological Pi removal is an alternative to chemical treatments in which bacteria accumulate excess Pi as polyphosphate (polyP) [79, 80]. The bacteria can then be retained as sludge, which can be separated from the wastewater, which now has a much reduced phosphorous concentration.

4.1. The Tn-seq experiment—identifying the players of the high-Pi response

insertion frequency under one particular condition) (see Figure 4).

essential and those with reduced frequency were classified as important for fitness.

In order to further investigate cellular processes involved in Pi homeostasis when cells are grown in conditions of high environmental Pi, we performed a Tn-seq experiment. Tn-seq relies on the ability to saturate a bacterial genome by transposon mutagenesis. Cells are grown in a selective environment and individual transposon insertions are mapped using next-generation-sequencing protocols. The frequency of insertions in each gene is used to analyze the importance of each gene under those growth conditions. Those genes that receive few or no insertions are identified as essential (no insertions under any conditions), conditionally essential (no or few insertions under one condition), or conditionally important for fitness (reduced

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Wild-type E. coli strain MG1655 that harbors a rpsL mutation conferring streptomycin resistance was mutagenized with a mini-Tn5 transposon delivered from a conjugative plasmid that required the lambda Π protein for replication. The donor strain could be counter-selected because it contained a mutation in the dapA gene and required supplementation with diaminopimelic acid. By selecting transconjugants that were kanamycin-resistant and that did not require diaminopimelic acid we were able to obtain a library of about 200,000 independent mutants. Such a library would be predicted to give about 30–50 random insertions per gene. This mutant library was then grown in duplicate in one of three different defined media containing variable Pi concentrations. We used media containing 0.1, 2.0, and 400 mM Pi. Preliminary experiments showed that growth of the wild-type strain in 400 mM Pi was significantly slower than in the other media. This high-Pi medium was also of a significantly

Figure 4. The design of a Tn-seq experiment. A library of transposon insertion mutants was grown in duplicate in liquid cultures containing either 0.1 mM Pi, 2.0 mM Pi, or 400 mM Pi. Chromosomal DNA was prepared from each sample for deep sequencing to identify the sites of insertion and their frequencies. If an insertion does not affect the growth of a strain, then it is assumed that that gene is not required for growth. Genes with no insertions under any conditions were classified as essential genes. Genes with no or very few insertions under one condition were classified as conditionally

PolyP is found in all kingdoms of life [81, 82]. It is a linear chain of variable length of Pi residues that are linked by phosphoanhydride bonds. The cellular amounts of polyP are controlled through its polymerization and depolymerization, presumably to meet cellular needs for free Pi. PolyP can be synthesized from ATP by polyP kinase, encoded by the ppk gene [83]. It is degraded by exopolyphosphatase, encoded by the ppx gene. The Ppk reaction is fully reversible and cells can also use polyP to synthesize ATP.

To enhance biological removal of Pi from wastewater, Kato et al. cloned the pstSCAB and ppk genes on plasmids [74]. They found that an E. coli strain harboring these plasmids could accumulate up to 16% of their dry weight as phosphorus with over 60% of the cellular phosphorous stored as polyP. They also noted that these strains grew very poorly. Subsequent work from this group showed that phoU mutants also accumulated high levels of polyP [75]. It was known that phoU mutants expressed the transporter at high levels, even when environmental Pi levels were high. As an additional contribution to understanding the phenotype of a phoU mutant, our group showed that PhoU also negatively regulates the activity of the Pst transporter [23]. Those experiments were performed by uncoupling expression of the Pst transporter from its normal PhoB-dependent mechanism through a technique called promoter swapping [84]. We felt that it was important to keep the pstSCAB-phoU operon at its normal location in the E. coli chromosome, so we developed a technique using Lambda-Red recombineering methodology to swap the Ptac promoter for the wild-type PpstS promoter [85]. As we held expression levels constant with an exogenous promoter, we demonstrated that a phoU deletion mutant accumulated Pi at a higher rate than cells expressing the pstSCAB genes and phoU. Other ABC transporters, such as the methionine transporter, have regulatory domains that respond to the cytoplasmic concentrations of transported substrates and function as sites of allosteric inhibition of transport [86–88]. We proposed that PhoU plays a similar role for Pi transport in E. coli. We learned from these observations that E. coli cells tightly control the amounts of the Pst transporter as well as its activity. When intracellular amounts of Pi become too high,E. coli cells store excess Pi as polyP.

In addition to its role as a Pi and energy store, PolyP has many other important functions in E. coli [89]. For example, it is involved in metal detoxification and can function as a primitive chaperone to protect against oxidative damage [90–92]. Of importance to our discussion here, Keasling hypothesized that E. coli cells could detoxify metals by sequestering them with intracellular polyP. Following hydrolysis of polyP to Pi, the metal/Pi complexes would be exported through the Pit transporters. PolyP is also involved in cell signaling, respiratory chain gene expression, bacterial persistence, and in stress response networks [93–96]. It has recently been shown that when external Pi levels are very high, polyP can even activate PhoB during the stationary phase of growth through the small molecule acetyl phosphate [97]. It is then postulated that phospho-PhoB inhibits the synthesis of c-di-GMP, blocking the production of AI-2, leading to the inhibition of biofilm formation.

### 4.1. The Tn-seq experiment—identifying the players of the high-Pi response

excess Pi as polyphosphate (polyP) [79, 80]. The bacteria can then be retained as sludge, which can be separated from the wastewater, which now has a much reduced phosphorous

PolyP is found in all kingdoms of life [81, 82]. It is a linear chain of variable length of Pi residues that are linked by phosphoanhydride bonds. The cellular amounts of polyP are controlled through its polymerization and depolymerization, presumably to meet cellular needs for free Pi. PolyP can be synthesized from ATP by polyP kinase, encoded by the ppk gene [83]. It is degraded by exopolyphosphatase, encoded by the ppx gene. The Ppk reaction is

To enhance biological removal of Pi from wastewater, Kato et al. cloned the pstSCAB and ppk genes on plasmids [74]. They found that an E. coli strain harboring these plasmids could accumulate up to 16% of their dry weight as phosphorus with over 60% of the cellular phosphorous stored as polyP. They also noted that these strains grew very poorly. Subsequent work from this group showed that phoU mutants also accumulated high levels of polyP [75]. It was known that phoU mutants expressed the transporter at high levels, even when environmental Pi levels were high. As an additional contribution to understanding the phenotype of a phoU mutant, our group showed that PhoU also negatively regulates the activity of the Pst transporter [23]. Those experiments were performed by uncoupling expression of the Pst transporter from its normal PhoB-dependent mechanism through a technique called promoter swapping [84]. We felt that it was important to keep the pstSCAB-phoU operon at its normal location in the E. coli chromosome, so we developed a technique using Lambda-Red recombineering methodology to swap the Ptac promoter for the wild-type PpstS promoter [85]. As we held expression levels constant with an exogenous promoter, we demonstrated that a phoU deletion mutant accumulated Pi at a higher rate than cells expressing the pstSCAB genes and phoU. Other ABC transporters, such as the methionine transporter, have regulatory domains that respond to the cytoplasmic concentrations of transported substrates and function as sites of allosteric inhibition of transport [86–88]. We proposed that PhoU plays a similar role for Pi transport in E. coli. We learned from these observations that E. coli cells tightly control the amounts of the Pst transporter as well as its activity. When intracellular amounts of Pi become

In addition to its role as a Pi and energy store, PolyP has many other important functions in E. coli [89]. For example, it is involved in metal detoxification and can function as a primitive chaperone to protect against oxidative damage [90–92]. Of importance to our discussion here, Keasling hypothesized that E. coli cells could detoxify metals by sequestering them with intracellular polyP. Following hydrolysis of polyP to Pi, the metal/Pi complexes would be exported through the Pit transporters. PolyP is also involved in cell signaling, respiratory chain gene expression, bacterial persistence, and in stress response networks [93–96]. It has recently been shown that when external Pi levels are very high, polyP can even activate PhoB during the stationary phase of growth through the small molecule acetyl phosphate [97]. It is then postulated that phospho-PhoB inhibits the synthesis of c-di-GMP, blocking the production of AI-2, leading to the inhibition of biofilm

fully reversible and cells can also use polyP to synthesize ATP.

344 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

too high,E. coli cells store excess Pi as polyP.

formation.

concentration.

In order to further investigate cellular processes involved in Pi homeostasis when cells are grown in conditions of high environmental Pi, we performed a Tn-seq experiment. Tn-seq relies on the ability to saturate a bacterial genome by transposon mutagenesis. Cells are grown in a selective environment and individual transposon insertions are mapped using next-generation-sequencing protocols. The frequency of insertions in each gene is used to analyze the importance of each gene under those growth conditions. Those genes that receive few or no insertions are identified as essential (no insertions under any conditions), conditionally essential (no or few insertions under one condition), or conditionally important for fitness (reduced insertion frequency under one particular condition) (see Figure 4).

Figure 4. The design of a Tn-seq experiment. A library of transposon insertion mutants was grown in duplicate in liquid cultures containing either 0.1 mM Pi, 2.0 mM Pi, or 400 mM Pi. Chromosomal DNA was prepared from each sample for deep sequencing to identify the sites of insertion and their frequencies. If an insertion does not affect the growth of a strain, then it is assumed that that gene is not required for growth. Genes with no insertions under any conditions were classified as essential genes. Genes with no or very few insertions under one condition were classified as conditionally essential and those with reduced frequency were classified as important for fitness.

Wild-type E. coli strain MG1655 that harbors a rpsL mutation conferring streptomycin resistance was mutagenized with a mini-Tn5 transposon delivered from a conjugative plasmid that required the lambda Π protein for replication. The donor strain could be counter-selected because it contained a mutation in the dapA gene and required supplementation with diaminopimelic acid. By selecting transconjugants that were kanamycin-resistant and that did not require diaminopimelic acid we were able to obtain a library of about 200,000 independent mutants. Such a library would be predicted to give about 30–50 random insertions per gene. This mutant library was then grown in duplicate in one of three different defined media containing variable Pi concentrations. We used media containing 0.1, 2.0, and 400 mM Pi. Preliminary experiments showed that growth of the wild-type strain in 400 mM Pi was significantly slower than in the other media. This high-Pi medium was also of a significantly higher osmolarity than the other two media. After growing cells until stationary phase, the cultures were harvested and DNA extractions were performed. The chromosomal DNA was then enzymatically fragmented and a polyC tail was then added to these DNA fragments using terminal transferase. Polymerase chain reactions (PCRs) were then performed using transposon-specific and polyG primers to amply DNA where transposons had inserted. A second round of PCR was then used to add primers for Illumina sequencing. The reads were mapped to the published MG1655 genome and the number of reads was normalized to 4 × 106 reads per sample.

any condition in the ppk gene, suggesting that it was an essential gene under these defined growth conditions, as well as in the genes for ATP synthase. Another intriguing class of genes with low frequency of transposon insertions included genes of unknown function, such as ydhP, yodD, yniC, and glcG. We conclude from these results that when placed in very high Pi environments cells need to regulate Pi import and continue to synthesize ATP at high rates for the production of polyP. We expect that there are some previously unknown functions that are necessary to deal with high-Pi stresses that are represented by the "y" genes. These may include other transporters, regulators of transporters, or genes for meta-

Molecular Mechanisms of Phosphate Homeostasis in *Escherichia coli*

http://dx.doi.org/10.5772/67283

347

E. coli inhabits environments with widely ranging Pi concentrations. It is often limiting in environmental conditions and can be quite high in the intestinal lumen of a healthy human [96]. Pi homeostasis is a balancing act of import, export, utilization, and sequestration (see Figure 5). Pi can be imported through the secondary transporters PitA and PitB or through the PstSCAB ABC transporter. The multiple transporters that import Pi have various specificities and expression patterns, which allow them to be used primarily under conditions when they are most needed, but which also permits a considerable amount of redundancy in function. Of primary importance in Pi homeostasis is the ability to increase transcription of genes when environmental Pi levels are low for the high-affinity acquisition of Pi and for the utilization of alternate sources of phosphorous. To monitor extracellular Pi, E. coli utilizes a Pi-signaling complex consisting of the PstSCAB transporter, PhoU and PhoR. In its two states, it can either activate or deactivate the response regulator PhoB. We propose that the signaling complex does not directly sense extracellular Pi, but senses the activity of the Pst transporter by recognizing its alternate conformational states. It is the inward-facing conformation of the Pst transporter that represents Pi-sufficient environments because it is only formed when Pi is actively transported. Once imported, Pi becomes part of an intracellular pool and can be incorporated into ATP through substratelevel phosphorylation or through oxidative phosphorylation. From ATP or its equivalents, the phosphoryl groups are transferred to all other phosphorylated intermediates of the cell. Cellular growth is inhibited when intracellular Pi levels become too elevated, so cells must have mechanisms to control this parameter also. To maintain its intracellular Pi levels near 10 mM, E. coli can either export excess Pi or it can sequester it through the synthesis of PolyP. PitA and PitB are known metal-Pi exporters and rely on high intracellular Pi levels and metals, such as Mg2+, Mn2+, Ca2+, Zn2+, and Co2+ for Pi export [7, 107]. Pi export through the Pit proteins contributes to the generation of a proton-motive force. It has also been suggested that YjbB plays a role in Pi export [12]. This protein is very interesting because it consists of two segments, a hydrophobic N-terminal half with sequence similar-

/Pi transporters and a C-terminal half with sequence similarity to PhoU.

Motomura et al. showed that overexpression of YjbB resulted in lower intracellular polyP levels and that it released significant amounts of Pi into the medium. These results are consistent with YjbB being a Pi exporter. PolyP serves as a Pi buffer to fine-tune intracellu-

4.2. Pi homeostasis model and questions for further research

bolic functions.

ity to Na<sup>+</sup>

lar Pi levels.

To identify genes that are important for growth in high-Pigrowth conditions, we sorted from low to high each of the genes based upon the quotient of the number of hits in high-Pi media divided by the total number of hits in all three media. We were particularly interested in genes with few hits in the high-Pi medium and were able to identify many genes whose functions are important for fitness under these growth conditions. As mentioned above, the high-Pi growth medium that we employed was also high in osmolarity. As an internal control to identify genes that were important for this growth condition, we were able to identify many top hits as occurring in genes that are known to be important in a high osmolarity response, such asompR, envZ, galU, otsB, hupA, cpxR, and hupB [98– 100]. OmpR and EnvZ are two-component regulators that respond to changes in osmolarity. GalU and OtsB are involved in the synthesis of trehalose, a compatible solute, that is produced under high osmolarity growth conditions. hupA encodes for a component of the HU protein, which is a small DNA-binding protein that helps regulate the expression of the osmoresponsive gene proU [101]. CpxR is a response regulator that responds to cell envelope damage and it is known that it participates in the regulation of gene expression in response to osmolarity [99].

We also identified genes that are known to be involved in the control of the Pho regulon, for example, each of the pstSCAB genes was found near the top of the list. Mutations in any of these genes lead to elevated expression of the entire Pho regulon, whose genes are involved in the high-affinity acquisition of Pi and the utilization of alternate Pi sources. It is easy to hypothesize why the expression of these genes would be deleterious when Pi levels are very high. With the Pho regulon fully expressed, Pi may be imported through the phosphonate or other transporters without the requisite expression of genes to accommodate the increased Pi. Another common class of genes that had few transposon insertions under high Pi conditions was genes involved in central metabolism of glucose and most importantly in ATP production (ptsG, pykF, ackA, zwf, pta, and sdhBCD). PstG is the enzyme IIBC component of the phosphotransferase system for glucose uptake [102]. PykF is pyruvate kinase from glycolysis and synthesizes ATP from ADP and phosphoenolpyruvate. Zwf is glucose-6-phosphate-1-dehydrogenase, which catalyzes the first steps in the Enter Doudoroff or oxidative pentose phosphate pathways [103, 104]. AckA and Pta are acetate kinase and phosphotransacetylase, respectively, and are involved in ATP production, acetyl phosphate synthesis, and acetate secretion [105]. SdhBCD are subunits of succinate dehydrogenase, which is part of the TCA cycle. It is interesting to note that these genes are repressed during growth on glucose [106], so it is unclear why mutations in these genes lower the fitness of E. coli grown on glucose high-Pi medium. It is also important to note that there were very few hits under

any condition in the ppk gene, suggesting that it was an essential gene under these defined growth conditions, as well as in the genes for ATP synthase. Another intriguing class of genes with low frequency of transposon insertions included genes of unknown function, such as ydhP, yodD, yniC, and glcG. We conclude from these results that when placed in very high Pi environments cells need to regulate Pi import and continue to synthesize ATP at high rates for the production of polyP. We expect that there are some previously unknown functions that are necessary to deal with high-Pi stresses that are represented by the "y" genes. These may include other transporters, regulators of transporters, or genes for metabolic functions.

### 4.2. Pi homeostasis model and questions for further research

higher osmolarity than the other two media. After growing cells until stationary phase, the cultures were harvested and DNA extractions were performed. The chromosomal DNA was then enzymatically fragmented and a polyC tail was then added to these DNA fragments using terminal transferase. Polymerase chain reactions (PCRs) were then performed using transposon-specific and polyG primers to amply DNA where transposons had inserted. A second round of PCR was then used to add primers for Illumina sequencing. The reads were mapped to the published MG1655 genome and the number of reads was normalized to 4 × 106

346 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

To identify genes that are important for growth in high-Pigrowth conditions, we sorted from low to high each of the genes based upon the quotient of the number of hits in high-Pi media divided by the total number of hits in all three media. We were particularly interested in genes with few hits in the high-Pi medium and were able to identify many genes whose functions are important for fitness under these growth conditions. As mentioned above, the high-Pi growth medium that we employed was also high in osmolarity. As an internal control to identify genes that were important for this growth condition, we were able to identify many top hits as occurring in genes that are known to be important in a high osmolarity response, such asompR, envZ, galU, otsB, hupA, cpxR, and hupB [98– 100]. OmpR and EnvZ are two-component regulators that respond to changes in osmolarity. GalU and OtsB are involved in the synthesis of trehalose, a compatible solute, that is produced under high osmolarity growth conditions. hupA encodes for a component of the HU protein, which is a small DNA-binding protein that helps regulate the expression of the osmoresponsive gene proU [101]. CpxR is a response regulator that responds to cell envelope damage and it is known that it participates in the regulation of gene expression

We also identified genes that are known to be involved in the control of the Pho regulon, for example, each of the pstSCAB genes was found near the top of the list. Mutations in any of these genes lead to elevated expression of the entire Pho regulon, whose genes are involved in the high-affinity acquisition of Pi and the utilization of alternate Pi sources. It is easy to hypothesize why the expression of these genes would be deleterious when Pi levels are very high. With the Pho regulon fully expressed, Pi may be imported through the phosphonate or other transporters without the requisite expression of genes to accommodate the increased Pi. Another common class of genes that had few transposon insertions under high Pi conditions was genes involved in central metabolism of glucose and most importantly in ATP production (ptsG, pykF, ackA, zwf, pta, and sdhBCD). PstG is the enzyme IIBC component of the phosphotransferase system for glucose uptake [102]. PykF is pyruvate kinase from glycolysis and synthesizes ATP from ADP and phosphoenolpyruvate. Zwf is glucose-6-phosphate-1-dehydrogenase, which catalyzes the first steps in the Enter Doudoroff or oxidative pentose phosphate pathways [103, 104]. AckA and Pta are acetate kinase and phosphotransacetylase, respectively, and are involved in ATP production, acetyl phosphate synthesis, and acetate secretion [105]. SdhBCD are subunits of succinate dehydrogenase, which is part of the TCA cycle. It is interesting to note that these genes are repressed during growth on glucose [106], so it is unclear why mutations in these genes lower the fitness of E. coli grown on glucose high-Pi medium. It is also important to note that there were very few hits under

reads per sample.

in response to osmolarity [99].

E. coli inhabits environments with widely ranging Pi concentrations. It is often limiting in environmental conditions and can be quite high in the intestinal lumen of a healthy human [96]. Pi homeostasis is a balancing act of import, export, utilization, and sequestration (see Figure 5). Pi can be imported through the secondary transporters PitA and PitB or through the PstSCAB ABC transporter. The multiple transporters that import Pi have various specificities and expression patterns, which allow them to be used primarily under conditions when they are most needed, but which also permits a considerable amount of redundancy in function. Of primary importance in Pi homeostasis is the ability to increase transcription of genes when environmental Pi levels are low for the high-affinity acquisition of Pi and for the utilization of alternate sources of phosphorous. To monitor extracellular Pi, E. coli utilizes a Pi-signaling complex consisting of the PstSCAB transporter, PhoU and PhoR. In its two states, it can either activate or deactivate the response regulator PhoB. We propose that the signaling complex does not directly sense extracellular Pi, but senses the activity of the Pst transporter by recognizing its alternate conformational states. It is the inward-facing conformation of the Pst transporter that represents Pi-sufficient environments because it is only formed when Pi is actively transported. Once imported, Pi becomes part of an intracellular pool and can be incorporated into ATP through substratelevel phosphorylation or through oxidative phosphorylation. From ATP or its equivalents, the phosphoryl groups are transferred to all other phosphorylated intermediates of the cell. Cellular growth is inhibited when intracellular Pi levels become too elevated, so cells must have mechanisms to control this parameter also. To maintain its intracellular Pi levels near 10 mM, E. coli can either export excess Pi or it can sequester it through the synthesis of PolyP. PitA and PitB are known metal-Pi exporters and rely on high intracellular Pi levels and metals, such as Mg2+, Mn2+, Ca2+, Zn2+, and Co2+ for Pi export [7, 107]. Pi export through the Pit proteins contributes to the generation of a proton-motive force. It has also been suggested that YjbB plays a role in Pi export [12]. This protein is very interesting because it consists of two segments, a hydrophobic N-terminal half with sequence similarity to Na<sup>+</sup> /Pi transporters and a C-terminal half with sequence similarity to PhoU. Motomura et al. showed that overexpression of YjbB resulted in lower intracellular polyP levels and that it released significant amounts of Pi into the medium. These results are consistent with YjbB being a Pi exporter. PolyP serves as a Pi buffer to fine-tune intracellular Pi levels.

5. Conclusion

signal transduction system.

Acknowledgements

Author details

References

10.1126/science.36664

William R. McCleary

Pi homeostasis is essential for life's basic processes. Without the ability to control intracellular levels of Pi within optimal levels, cells would be unable to maintain energy stores, synthesize nucleic acids and phospholipids, or carry out central metabolic pathways. The molecular mechanisms by which E. coli cells maintain intracellular Pi levels include utilizing multiple importers with characteristic patterns of expression, affinities for Pi and rates of Pi import [4]. These cells also employ a highly characterized signal transduction system that monitors extracellular Pi levels through the conformational states of the high-affinity Pi importer to control gene expression for scavenging Pi and utilizing alternate phosphorous sources. In addition, polyphosphate plays an important role in fine-tuning the amounts of free intracellular Pi. Understanding these mechanisms is important because this knowledge can be used to design organisms and pathways for the remediation of phosphate pollution. Moreover, the expression of virulence genes in many organisms is controlled by the PhoBR

Molecular Mechanisms of Phosphate Homeostasis in *Escherichia coli*

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I thank Ramesh Vuppada, a Master's student in our laboratory, who provided the preliminary results about the signaling states of the alternate conformations of PstB and Tanner Robinson, an undergraduate student, for the preliminary Tn-seq results. I also thank the many undergraduate and graduate students who have contributed to the ongoing work and discussions in the laboratory. Work from our laboratory was supported by Public Health Service grant

Microbiology and Molecular Biology Department, Brigham Young University, Provo, UT, USA

[1] Shulman RG, Brown TR, Ugurbil K, Ogawa S, Cohen SM, den Hollander JA. Cellular applications of 31P and 13C nuclear magnetic resonance. Science. 1979;205:160–166. DOI:

[2] Rao NN, Roberts MF, Torriani A, Yashphe J. Effect of glpT and glpD mutations on

expression of the phoA gene in Escherichia coli. J Bacteriol. 1993;175:74–79.

R15GM96222 from the National Institute of General Medical Sciences.

Address all correspondence to: bill\_mccleary@byu.edu

Figure 5. Model for Pi homeostasis in E. coli. Intracellular amounts of Pi are maintained within a modest range around 10 mM. The mechanisms for this homeostatic maintenance include the use of multiple Pi importers with variable affinities and rates of Pi transport. Cells also utilize the sophisticated PhoBR two-component-signaling mechanism that directly controls the expression of genes for high-affinity Pi acquisition and for the use of alternate sources of phosphorous. In addition, when Pi levels become too high, the cells sequester Pi by accumulating polyP, which is produced from ATP by the enzyme Ppk or they export it.

While the general outlines of Pi homeostasis have begun to be filled in, there are still important questions that remain. How do cells sense intracellular levels of Pi to control polyP synthesis/degradation and Pi export? What are the roles of the genes that are repressed by the PhoBR system? What are the functions of the unknown genes that were identified by Tn-seq to be important for fitness in very high levels of environmental Pi? What are the control mechanisms for the expression of PitA and PitB? Why does E. coli retain both the pitA and pitB genes? What are their differential functions? What effects does the stoichiometry of PstSCAB, PhoU, PhoR, and PhoB have on signaling, especially at the level of the single cell? Knowledge gained in studying Pi homeostasis will continue to be important in understanding global regulatory mechanisms, as Pi is involved in so many cellular processes. It will also be important in the engineering of organisms for improved Pi bioremediation.
