Overproduction of YjbB reduces the level of polyphosphate in Escherichia coli : a hypothetical role of YjbB in phosphate export and polyphosphate accumulation

Authors


  • Editor: Wilfrid Mitchell

Correspondence: Akio Kuroda, Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Tel.: +81 82 424 7758; fax: +81 82 424 7047; e-mail: akuroda@hiroshima-u.ac.jp

Abstract

Intracellular phosphate (Pi) is normally maintained at a fairly constant concentration in Escherichia coli, mainly by Pi transport systems and by the ‘phosphate balance’ between Pi and polyphosphate (polyP). We have reported previously that excess uptake of Pi in a phoU mutant results in elevated levels of polyP. Here, we found that the elevated levels of polyP in the mutant could be reduced by the overproduction of YjbB, whose N-terminal half contains Na+/Pi cotransporter domains. The rate of Pi export increased when the YjbB overproducer grew on a medium containing glycerol-3-phosphate. These results strongly suggested that YjbB reduced the elevated levels of polyP in the phoU mutant by exporting intracellular excess Pi.

Introduction

Phosphate (Pi) is essential for all living organisms. It is required for the synthesis of lipids and nucleic acids, and is involved in many biochemical reactions. Intracellular concentrations of Pi are normally maintained at a fairly constant level (10 mM) in Escherichia coli under conditions of aerobic or anaerobic growth on glucose with excess or limiting extracellular Pi (Wanner, 1996). Escherichia coli possesses a number of Pi transporters, including low-affinity Pi transport systems (PitA and PitB) and a high-affinity Pi-specific transport system (PstSCAB) (Rosenberg et al., 1977; Amemura et al., 1985; Surin et al., 1985; Metcalf & Wanner, 1993; Harris et al., 2001). PhnCDE, which is mainly involved in phosphonate metabolism, also functions as a Pi transporter (Metcalf & Wanner, 1993). The PstSCAB system is induced under low external Pi concentrations (<4 μM) as part of the Pho regulon to maintain the intracellular Pi concentration (Amemura et al., 1985; Wanner, 1993). This regulon is controlled by the PhoR/PhoB two-component regulatory system (Amemura et al., 1985; Wanner, 1993). However, because the Pho regulon is only responsive to external Pi, it alone is probably insufficient to maintain the constancy of intracellular Pi concentration.

Escherichia coli contains three kinds of inorganic phosphate: Pi, pyrophosphate, and polyphosphate (polyP). PolyP is a linear polymer of tens to hundreds of Pi residues that is synthesized by polyP kinase (PPK) and degraded to Pi by polyphosphatase (PPX) (Kornberg, 1995). Although the intracellular concentrations of Pi are stable, those of polyP may change drastically. For example, the polyP concentration increases 100-fold, up to 20 mM, under conditions of amino acid starvation (Kuroda et al., 1997; Rao et al., 1998). Because polyP can be converted to Pi by PPX, it also serves as a reservoir for maintaining Pi levels (Kornberg, 1995). Previously, we reported that a mutation in the phoU gene, whose product negatively regulates the Pho regulon, led to polyP accumulation in E. coli (Morohoshi et al., 2002). Constitutive expression of the PstSCAB system and the resulting uptake of excess Pi were responsible for the elevated levels of polyP in the phoU mutant (Morohoshi et al., 2002). Although we did not identify the mechanism controlling the ‘phosphate balance’ between Pi and polyP, the findings confirmed that polyP can serve as a Pi reservoir and that it participates in the maintenance of the intracellular Pi concentration.

Here, we found that the overproduction of YjbB containing both PhoU and Na+/Pi cotransporter domains reduced the elevated levels of polyP in the phoU mutant. It seemed likely that YjbB exports excess Pi in the phoU mutant and thus reduces the levels of polyP. Finally, we discuss the hypothetical role of Pi export and polyP accumulation in maintaining the intracellular Pi concentration.

Materials and methods

Plasmid construction

Plasmids pMWphoU and pMWyjbB were constructed as follows: DNA fragments containing phoU and yjbB genes were amplified from E. coli MG1655 genomic DNA using the primers phoU-fwd/phoU-rev and yjbB-fwd/yjbB-rev, respectively (Supporting Information, Table S1). The PCR fragments were inserted into the HindIII/EcoRI and HindIII/SspI sites of pMW119, respectively (Nippon Gene, Tokyo, Japan).

Mutant construction

A one-step gene disruption method described by Datsenko & Wanner (2000) was used to construct a mutant that lacks all four kinds of Pi transporters (pitA, pitB, pstSCAB, and phnC). For the disruption of pitB, a PCR fragment was generated using primers pitBdel-1 and pitBdel-2 (Table S1) and the pKD4 plasmid (Datsenko & Wanner, 2000) as a template. The amplified fragment was transferred into MG1655 carrying pKD46 (Datsenko & Wanner, 2000) by electroporation. After a kanamycin-resistant strain (MT2001) was selected, the kanamycin resistance gene was eliminated from the chromosomal DNA by expressing FLP recombinase from pCP20 (Datsenko & Wanner, 2000). The resulting strain was designated MT2002. To generate the pitA∷Cmr and phnC∷Kmr strains, primers pitAdel-1/pitAdel-2 and phnCdel-1/phnCdel-2 were used, respectively (Table S1). P1 transduction was used to transfer pitA∷Cmr into MT2002, and the resulting strain was designated MT2003. MT2004 was constructed by transferring phnC∷Kmr to MT2003. Antibiotic resistance genes in MT2004 were then eliminated as described above and the resulting strain was designated MT2005. To disrupt the PstSCAB transporter, a P1 lysate was prepared from BW17335 and then introduced into MT2005. The resulting strain selected for Km resistance lacked all four Pi transporters and was designated MT2006. For the construction of MT1011 (yjbB∷Cmr), a PCR fragment was generated using primers yjbBdel-1 and yjbBdel-2 and the pKD3 plasmid (Datsenko & Wanner, 2000) as a template and then transferred by electroporation into MG1655 carrying pKD46. MT2009 was constructed by P1 transduction of phoA∷Kmr from JW0374 to MT2005. After eliminating the antibiotic resistance gene of MT2009, the genes yjbB∷Cmr from MT1011 and pstSCAB-phoU∷Kmr from BW17335 and the genes yjbB∷Cmr from MT1011 and glpT∷Kmr from JW2234 were transferred into MT2009 by P1 transduction, with the resulting mutants designated MT2013 and MT2014, respectively. After eliminating the antibiotic resistance genes of MT2014, pstSCAB-phoU∷Kmr from BW17335 was transferred into MT2014 by P1 transduction, with the resulting mutant designated MT2016. Disruption of pitA, pitB, phnC, pstSCABphoU, and yjbB was confirmed by PCR using the primer pairs pitA1/pitA2, pitB1/pitB2, phnC1/phnC2, pstX1/pstX2, and yjbB1/yjbB2, respectively. Strains JW0374 (ΔphoA∷Kmr) and JW2234 (ΔglpT∷Kmr) were obtained from the National Institute of Genetics of Japan. All the strains and plasmids used in this study are listed in Table 1.

Table 1.   Bacterial strains and plasmids
Strain or plasmidDescriptionReferences or sources
E. coli strains
 MG1655Wild-type strain; FarcA-1655 fnr-1655Rao et al. (1998)
 MT29MG1655 phoU-A83TMorohoshi et al. (2002)
 BW25113rrnB3ΔlacZ4787 hsdR514Δ(araBAD)567Δ(rhaBAD)568 rph-1Datsenko & Wanner (2000)
 BW17335DE3(lac)X74 Δ(pstSCAB-phoU)560∷KmrSteed & Wanner (1993)
 JW0374BW25113 ΔphoA∷KmrBaba et al. (2006)
 JW2234BW25113 ΔglpT∷KmrBaba et al. (2006)
 MT1011MG1655 ΔyjbB∷CmrThis study
 MT2001MG1655 ΔpitB∷KmrThis study
 MT2002MG1655 ΔpitBfrtThis study
 MT2003MG1655 ΔpitA∷CmrΔpitBfrtThis study
 MT2004MG1655 ΔpitA∷CmrΔpitBfrtΔphnC∷KmrThis study
 MT2005MG1655 ΔpitAfrtΔpitBfrtΔphnCfrtThis study
 MT2006MG1655 ΔpitAfrtΔpitBfrtΔphnCfrtΔ(pstSCAB-phoU) 560∷KmrThis study
 MT2009MG1655 ΔpitAfrtΔpitBfrtΔphnCfrtΔphoA∷KmrThis study
 MT2013MG1655 ΔpitAfrtΔpitBfrtΔphnCfrtΔ(pstSCAB-phoU)560∷KmrΔphoAfrtΔyjbB∷CmrThis study
 MT2014MG1655 ΔpitAfrtΔpitBfrtΔphnCfrtΔphoAfrtΔyjbB∷CmrΔglpT∷KmrThis study
 MT2016MG1655 ΔpitAfrtΔpitBfrtΔphnCfrtΔ(pstSCAB-phoU)560∷KmrΔphoAfrtΔyjbBfrtΔglpTfrtThis study
Plasmids
 pMW119Cloning vector; AmprNippon Gene
 pMWphoUpMW119 containing the E. coli phoU geneThis study
 pMWyjbBpMW119 containing the E. coli yjbB geneThis study
 pTrcPPX1pTrcHisB harboring the PPX1 gene of Saccharomyces cerevisiaeWurst et al. (1995)
 pKD3Template plasmid; Ampr, FRT-flanked chloramphenicol cassetteDatsenko & Wanner (2000)
 pKD4Template plasmid; Ampr, FRT-flanked kanamycin cassetteDatsenko & Wanner (2000)
 pKD46Red recombinase expression plasmid; Ampr, ara-inducible expression, temperature-sensitive replicationDatsenko & Wanner (2000)
 pCP20FLP expression plasmid; Ampr, temperature-sensitive replication and FLP synthesisDatsenko & Wanner (2000)

Growth conditions for polyP accumulation

The accumulation of polyP during amino acid starvation was tested as described below (Kuroda et al., 1997). Escherichia coli MG1655 carrying pMWyjbB was grown to the mid-log phase on a 2 × YT-rich medium (1.6% peptone, 1.0% yeast extract, and 0.5% NaCl) (Sambrook & Russell, 2001) with shaking at 37 °C. The cells were collected by centrifugation, washed once with a morpholinopropane sulfonate (MOPS)-minimal medium [22.2 mM glucose, 40 mM potassium morpholinopropane sulfonate (pH 7.2), 50 mM NaCl, 9.52 mM NH4Cl, 4 mM Tricine, 2 mM K2HPO4, 0.52 mM MgCl2, 0.28 mM K2SO4, 0.01 mM FeSO4, 0.0005 mM CaCl2, and trace metals] (Neidhardt et al., 1974), and resuspended in the same medium. The accumulation of polyP during the cessation of nucleic acid synthesis was tested by adding rifampicin (100 μg mL−1) to mid-log-phase cells (Kuroda & Ohtake, 2000; Kuroda, 2006).

Biochemical assays

Intracellular polyP was extracted using the silica glass method and determined using a two-enzyme assay (Ault-Riche et al., 1998). An E. coli pellet was dissolved in 4 M guanidine isothiocyanate (GITC), and cells were lysed by heat (90 °C), SDS, and sonication. After the addition of ethanol, polyP was precipitated with Glassmilk (MP-Biomedicals, Santa Ana, CA) and was washed with New Wash (MP-Biomedicals). Following DNase and RNase treatment, polyP was readsorbed to the Glassmilk in the presence of GITC and ethanol and was extracted with water. The polyP concentration was measured as an amount of ATP generated by the reaction with PPK and ADP, which is equivalent to the number of Pi residues of polyP. ATP was measured using the ATP Bioluminescence assay kit (Roche, Mannheim, Germany). Alkaline phosphatase activity was measured using the method reported by Freimuth et al. (1990). One unit of alkaline phosphatase activity was defined as the production of 1 nmol p-nitrophenol min−1 per unit of OD600 nm. Protein concentrations were measured using the Bradford method with bovine serum albumin as the standard (Bradford, 1976). Pi concentrations were determined using the molybdenum-blue method (Clesceri et al., 1989).

Pi export assay

Escherichia coli cells grown overnight on the 2 × YT medium with shaking at 37 °C were collected by centrifugation. The pellet was washed twice with Pi-free MOPS medium and resuspended in the same medium containing 2 mM glycerol-3-phosphate to an OD600 nm of 0.2. Samples taken from the cultures were centrifuged, and the supernatants were assayed for Pi.

Results

YjbB contains both PhoU and Na+/Pi cotransporter domains

The Pfam database (Finn et al., 2008) indicated that E. coli YjbB consists of two distinct segments (Fig. 1). The N-terminal half of YjbB contains hydrophobic amino acid residues whose sequence is conserved among eukaryotic type II Na+/Pi cotransporters and is designated the Na+/Pi cotransporter domain (Pfam accession number PF02690). Most Na+/Pi cotransporter proteins consist of two repeats of this domain. In fact, the N-terminal half of YjbB also consists of two repeats of the Na+/Pi cotransporter domain (41% identity over 135 amino acids and 32% identity over 126 amino acids, respectively). The C-terminal half of YjbB contains two repeats of a PhoU domain (Pfam accession number PF01895) (21% identity over 80 amino acids and 15% identity over 60 amino acids, respectively), although the homology was considered insignificant in the database. Similarly, PhoU proteins also consist of two copies of the PhoU domain that form three-helix bundles (Liu et al., 2005; Oganesyan et al., 2005). This analysis suggested that YjbB might be involved both in Pi transport and in the regulation of Pho regulon genes.

Figure 1.

 Hydropathy plot of YjbB. The schematic diagram above the graph indicates the domain structure in the protein family database (Pfam, version 23.0).

Overproduction of YjbB reduced the elevated levels of polyP in a phoU mutant

PhoU negatively regulates the Pho regulon genes (Wanner, 1996). We have reported that a phoU mutant, MT29, accumulated 1000-fold higher levels of polyP than the wild type due to the constitutive expression of pstSCAB (Morohoshi et al., 2002). To test whether the overproduction of YjbB can compensate for the loss of PhoU function, we introduced yjbB on a multicopy plasmid (pMWyjbB) into MT29. MT29 carrying pMWyjbB had significantly lower levels of polyP (Table 2).

Table 2.   Levels of polyP and alkaline phosphatase (AP) activity in the phoU mutant MT29
PlasmidpolyP (nmol mg−1 protein)AP activity (units)
  1. Strains were grown on the 2 × YT medium for 12 h. Concentrations of polyP are given in terms of Pi residues. One unit of alkaline phosphatase activity was defined as the production of 1 nmol p-nitrophenol min−1 per unit of OD600 nm. Results are arithmetic means ± SD of triplicate assays.

pMW119 (control)170 ± 90.35 ± 0.05
pMWphoU0.37 ± 0.03<0.045
pMWyjbB0.35 ± 0.020.79 ± 0.09

One possible explanation for the reduction of polyP by YjbB is that the PhoU domain of YjbB had compensated for the chromosomal phoU mutation as a multicopy suppressor and reduced the expression of the Pho regulon genes. Because the phoA gene (alkaline phosphatase) is also one of the Pho regulon genes, we measured the alkaline phosphatase activity of MT29 carrying pMWyjbB. Unexpectedly, the levels of alkaline phosphatase activity were still high in the transformant, but they were reduced when pMWphoU was introduced (Table 2). It therefore seemed unlikely that YjbB overproduction reduced the expression of the Pho regulon genes. In other words, the reduced levels of polyP may not be due to the suppression of the increased expression of the PstSCAB Pi uptake system.

Escherichia coli accumulates polyP under conditions of amino acid starvation (Kuroda et al., 1997). This polyP accumulation is due to the inhibition of polyP degradation by ppGpp rather than the loss of PhoU function (Kuroda & Ohtake, 2000; Kuroda, 2006). To determine whether YjbB reduces the levels of polyP under conditions of amino acid starvation, we introduced pMWyjbB into the wild-type strain and then subjected the transformant to amino acid starvation. The levels of polyP in the transformant were lower than those of the strain carrying a control vector plasmid (Fig. 2a). Escherichia coli also accumulates polyP when its growth is blocked by antibiotics that inhibit nucleic acid synthesis (Kuroda & Ohtake, 2000; Kuroda, 2006). When treated with rifampicin, the levels of polyP in the transformant were also lower than those in the strain carrying a control vector plasmid (Fig. 2b). These results also supported the hypothesis that the reduction of polyP was not due to the suppression of the expression of Pho regulon genes including pstSCAB.

Figure 2.

 PolyP accumulation in the wild-type strain MG1655 carrying pMWyjbB under amino acid starvation (a) and in the presence of rifampicin (b). Intracellular polyP concentrations were measured in the Escherichia coli wild type carrying pMW119 (open squares) or pMWyjbB (closed squares) at the indicated times after shifting to amino acid-starved conditions or after adding rifampicin.

YjbB increased the rate of Pi export

As noted above, the N-terminal half of YjbB shows homology with Na+/Pi cotransporters, indicating the possible involvement of YjbB in the Pi flux. Escherichia coli possesses four Pi transporters (PitA, PitB, PhnCDE, and PstSCAB). Here, we constructed a mutant strain, MT2006, which lacks all four Pi transporters (Table 1). This mutant lost the ability to grow on a medium containing Pi as the sole source of phosphorus (Pi medium) (Fig. 3a). To test whether YjbB is involved in Pi import, we introduced pMWyjbB into MT2006. However, this transformant still failed to grow on the Pi medium (Fig. 3a). Escherichia coli can utilize glycerol-3-phosphate as the sole source of Pi (Hayashi et al., 1964; Schweizer et al., 1982). The transformant could grow on a medium containing glycerol-3-phosphate as the sole source of phosphorus (GP medium) (Fig. 3b), indicating that YjbB has no or little Pi-uptake activity.

Figure 3.

 Growth of the Pi transporter mutants. Cells were grown in MOPS medium containing 2 mM Pi (a) or 2 mM glycerol-3-phosphate (b) as the sole source of phosphorus at 37°C. Growth was measured as the OD600 nm in the wild type carrying pMW119 (open squares) and pMWyjbB (closed squares), MT2006 carrying pMW119 (open circles) and pMWyjbB (closed circles), and MT2013 carrying pMW119 (open triangles) and pMWyjbB (closed triangles).

On the other hand, the transformant released approximately 1 mM Pi into the supernatant when it grew for 8 h on the GP medium. To exclude the possibility that Pi was due to the degradation of glycerol-3-phosphate by an elevated alkaline phosphatase activity in the phoU mutant, we constructed MT2013 (phoA, yjbB, pitA, pitB, phnC, pstSCAB-phoU). Similar to MT2006, MT2013 and its transformant harboring pMWyjbB lost the ability to grow on Pi medium, but could grow on GP medium (Fig. 3). MT2013 carrying pMWyjbB still released a large amount of Pi into the GP medium, while MT2013 carrying a control vector plasmid only released a small amount of Pi during the lag phase (Fig. 4a and b).

Figure 4.

 Growth, Pi concentrations in the culture supernatant, and intracellular polyP levels of strain MT2013 after shifting to GP medium. Cells grown overnight on 2 × YT medium were washed with the Pi-free MOPS medium and then resuspended in the same medium containing 2 mM glycerol-3-phosphate (GP medium). The growth (a), extracellular Pi (b), and intracellular polyP concentrations (c) in strain MT2013 carrying pMW119 (open circles) or pMWyjbB (closed circles) were measured at the indicated times after shifting to the GP medium. The cells started to grow exponentially after 5 h as shown in Fig. 3b. The concentrations of polyP are given in terms of Pi residues.

Escherichia coli can take up glycerol-3-phosphate via glycerol-3-phosphate transport systems (Ugp and GlpT) (Hayashi et al., 1964; Schweizer et al., 1982). The GlpT transport system can function in the exchange mode, so that glycerol-3-phosphate is taken up in exchange with internal Pi, while the Ugp system does not release Pi. MT2016 (glpT, phoA, yjbB, pitA, pitB, phnC, pstSCAB-phoU) carrying pMWyjbB still released the same amount of Pi as MT2013 (pMWyjbB) when it was shifted to GP medium (data not shown), indicating that the Pi was not released in exchange for glycerol-3-phosphate by the GlpT system. Because Ugp belongs to the Pho regulon, it is upregulated in a phoU mutant such as MT2013. Therefore, in this mutant, G3P would be taken up mainly by the Ugp system. It has been shown that intracellular Pi is generated following the assimilation of glycerol-3-phosphate (Xavier et al., 1995). Because we could not observe any growth defects of MT2013 carrying pMWyjbB, the Pi (up to 1 mM) in the supernatant may be ascribed to the specific export of Pi rather than the nonspecific leaking of intracellular materials from cells.

YjbB reduced the elevated levels of polyP in a phoU mutant by exporting excess Pi

The assimilation of glycerol-3-phosphate would generate excess Pi that could either be released or converted to polyP. MT2013 carrying pMW119 accumulated a large amount of polyP when it was shifted to GP medium, while MT2013 carrying pMWyjbB did not (Fig. 4c). MT2013 carrying pMW119 did not release Pi and therefore might have an increased intracellular Pi concentration, leading to polyP accumulation. This is similar to the polyP accumulation resulting from excess Pi uptake in a phoU mutant (Morohoshi et al., 2002).

We found that the overproduction of YjbB caused two phenotypes: the reduction of the intracellular polyP level and the increase in the rate of Pi export. To exclude the possibility that YjbB reduces the intracellular polyP level, resulting in the increase in the rate of Pi export, we introduced a PPX-overproducing plasmid (pTrcPPX1) (Wurst et al., 1995) instead of pMWyjbB. Overproduction of PPX reduces the level of polyP by degrading polyP. If Pi release is simply a consequence of polyP degradation or inhibition of polyP synthesis, the transformant would be expected to release a large amount of Pi. MT2013 carrying pTrcPPX1 did not accumulate polyP. However, the amount of Pi in the supernatant was approximately 250 μM at 4 h after shifting to the GP medium, which was almost the same amount as that of MT2013 carrying a control vector plasmid. Therefore, the reduced polyP levels in the YjbB overproducer are probably a consequence of the increase in Pi export.

Discussion

The excess uptake of Pi in a phoU mutant results in elevated levels of polyP (Morohoshi et al., 2002). Because overproduction of YjbB containing two PhoU domains reduced the elevated levels of polyP in the phoU mutant, we had expected that YjbB may act as a multicopy suppressor compensating for the loss of PhoU function. However, our results indicated that YjbB reduced the level of polyP independent of PhoU function. Furthermore, the overproduction of YjbB increased the rate of Pi export during growth on GP medium. Therefore, we proposed that YjbB reduced the elevated levels of polyP in the phoU mutant by exporting excess Pi. This is consistent with the fact that the overproduction of YjbB also reduced the accumulation of polyP under conditions of amino acid starvation or in the presence of rifampicin by a mechanism that does not depend on the loss of PhoU function. In Saccharomyces cerevisiae, high concentrations of polyP accumulate in the vacuole during growth. Pho91 serves as a vacuolar Pi transporter that exports Pi from the vacuolar lumen to the cytosol and negatively regulates polyP accumulation (Hurlimann et al., 2007). Although we have not yet obtained direct evidence that YjbB has a Pi-export activity, we propose that YjbB, whose N-terminal half contains Na+/Pi cotransporter domains, also functions as a Pi exporter and thus reduces polyP accumulation. However, it remains a question of considerable interest as to what factors control the direction of Pi transport.

We cannot exclude the possibility that the PhoU domains of YjbB play an important role in Pi export. Some transporters and channels possess regulatory domains in addition to the transmembrane domains. For example, many bacterial K+ transporters and channels, such as the K+ efflux channel KefC, are controlled by a Ktn domain (Roosild et al., 2002). In S. cerevisiae, the SPX domain of the low-affinity Pi transporter regulates transport activity through a physical interaction with the regulatory protein (Hurlimann et al., 2009). Although the exact mechanism is poorly understood, PhoU homologs play an important and conserved role in Pi signaling and metabolism. Indeed, a recent study showed that PhoU modulates the activity of the Pst transporter (Rice et al., 2009). The PhoU domains of YjbB might also be involved in the sensing of the intracellular Pi concentration and the regulation of exporting activity.

The ‘phosphate balance’ between Pi and polyP plays an important role in the maintenance of the intracellular Pi concentration. Cells must use energy to convert Pi to ATP for the synthesis of polyP. PolyP is degraded and Pi can be fully reused when needed. On the other hand, the export of excess Pi by YjbB would not require energy input because intracellular Pi concentrations normally far exceed extracellular ones. However, exported Pi would occasionally be lost. Pi export-based control would thus appear more prompt, but less flexible in the case of fluctuating Pi availability than polyP-based control.

Because the levels of polyP were lower in the YjbB overproducer, we expected that the polyP levels would be higher in a chromosomal yjbB mutant. However, we did not observe such an increase in MT1011, whose polyP levels were less than 1 nmol (as Pi residues) mg−1 protein when it grew on 2 × YT medium. Furthermore, we did not detect promoter activity in the yjbB upstream fragment under Pi-rich or Pi-limited conditions when the fragment was inserted into a promoter-probe vector (data not shown). We hypothesized that the Pi export-based control may have been largely replaced by a polyP-based one in E. coli during the course of evolution. The phn genes, mainly involved in phosphonate metabolism, are known to be cryptic due to duplication or, in the E. coli K strain, insertion of an 8-bp sequence (Makino et al., 1991). Interestingly, their activation occurs after prolonged incubation on media containing methylphosphonate as the sole source of phosphorus (Makino et al., 1991). This phenomenon suggested that E. coli might utilize a Pi export-based method for maintaining the intracellular Pi concentration in response to some environmental stimuli. Further experiments are needed to understand the mechanism of YjbB activation and its relationship with the ‘phosphate balance’ between Pi and polyP.

Acknowledgements

This work was supported by a Grant-in-Aid for JSPS Fellows from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We are grateful to the National BioResource Project (National Institute of Genetics, Japan) for the E. coli strains from the KEIO collection.

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