The nature of polyphosphate

Inorganic polyphosphate (polyP) is a linear, unbranched polymer of orthophosphate residues linked by phosphoanhydride bonds (Figure 1.1).  PolyP ranges in size from three to over one thousand orthophosphate residues (Kornberg et al., 1999).  PolyP is widespread in bacteria and yeasts and has been found in plant and animal cells (polyP is also formed by dehydration and condensation of phosphate at the elevated temperatures of benthic and volcanic vents (Kornberg et al., 1999).

Linear structure of polyphosphate
PolyP was first found in yeast cells by Liebermann (1888). Further work by Wiame (1947), Kornberg (1956) and others through the 1940s and 1950s established the role of polyP, or 'volutin' as it was then known in the accumulation of phosphate and in energy storage by microorganisms. PolyP was observed in many microorganisms as metachromatic particles and was historically used as a diagnostic tool for certain pathogens such as Corynebacterium diphtheriae (Robinson & Wood, 1986). PolyP, like other anions, shifts the absorption of basic dyes such as toluidine blue, to a shorter wavelength (630 to 530nm) therefore giving rise to a metachromatic effect. When viewed by electron microscopy intracellular polyP appears as dark, electron dense granules. The presence of polyP in cells may also be detected by other techniques such as 31P-NMR analysis (Glonek et al., 1971) and by fluorescence of 4-6-diamidino-2-phenylindole (DAPI) (Allan & Miller, 1980).

 Under certain conditions, such as nutrient limitation, during stationary phase or osmotic stress some microorganisms have been shown to accumulate relatively large amounts of polyP (Tzeng & Kornberg, 1998).  In Acinetobacter johnsonii polyP may account for up to 30% of the dry biomass (Deinema et al., 1985).  Such levels of polyP are well in excess of the normal metabolic requirements for phosphate and indicate an important role of polyP in response to changes in nutritional status or environmental conditions (Kornberg et al., 1999).

Phosphate transport systems in prokaryotes

Before polyP synthesis can take place, it is necessary for uptake of inorganic phosphate and its transport across the cytoplasmic membrane.  Phosphate transport has been extensively studied in a number of microorganismsIn bacteria two major phosphate transport systems are involved; the low affinity Pit (phosphate inorganic transport) system, and the high affinity Pst (phosphate specific transport) system (van Veen, 1997).

Phosphate inorganic transport (Pit)  

Pit is a constitutively expressed secondary transporter consisting of a single trans-membrane protein (van Veen, 1997).  Uptake of phosphate, in the form of a neutral metal phosphate complex is in symport with a proton (Figure 1.2).  Transport of phosphate is achieved by binding and dissociation of the neutral metal phosphate complex and H+ on the outer and inner surface of the trans-membrane protein carrier protein (van Veen, 1997).  Transport of phosphate by Pit is mediated by the proton motive force across the cell membrane (van Veen et al., 1994; van Veen, 1997).  Phosphate inorganic transport (Pit)Pit is reversible and therefore allows for both influx and efflux of divalent ions and phosphate.   







 Pit has a relatively low specificity for both phosphate and arsenate, (toxic analogue of phosphate) with a Km of 25μM.  The optimum pH for this system is 6.0.  For optimal functioning of Pit it is necessary to maintain an alkaline internal pH to facilitate dissociation of a proton from the trans-membrane carrier protein (van Veen, 1997)

Phosphate specific transport (Pst). 

Pst is a periplasmic protein-dependent transporter that operates as a primary transport mechanism i.e. unidirectional phosphate transport is coupled to a chemical reaction (van Veen, 1997).  Pst consists of four subunits, a phosphate-binding protein located in the periplasmic space, two cytoplasmic associated proteins that contain six membrane spanning helices and a dimeric ATP binding protein (Figure 1.3) (Wanner, 1993; van Veen, 1997).  Phosphate is transported by the Pst system in the form of H2PO4- and HPO4 2-; the predominant phosphate species over the pH range 5.0 to 9.0: the proportion of H2PO4- increases with decreasing pH (van Veen et al., 1994) (van Veen, 1997).  Pst has a relatively high substrate affinity, the Km for phosphate is less than 1.0μM.  Pst is part of the PHO regulon in Escherichia coli and is therefore phosphate-starvation inducible (van Veen, 1997).  During phosphate limitation alkaline phosphatase production and phosphate uptake by Pst enable effective scavenging of phosphate under adverse conditions (Bonting et al., 1992).  The Pst system may be differentiated from the Pit system by the sensitivity of the former to cold osmotic shock and H+-ATPase inhibitors such as N,N’-dicycolhexylcarbodiimide (DCCD) (van Veen et al., 1993).  In contrast to Pst, Pit is relatively insensitive to either cold osmotic shock or DCCD but sensitive to disruption of the proton motive force by agents such as valinomycin-nigericin or carbonyl cynanide 3-chlorophenyl hydrazone  (van Veen, 1997).

  Phosphate inorganic transport (Pit)

 Other phosphate transport systems.

Besides the main specific phosphate transport systems phosphate may also enter the cell in the form of esters such as sn-glycerol-3-phosphate, glucose-6-phosphate or mannose-6-phosphate (van Veen, 1997).  Other organic phosphate compounds may diffuse through the outer membrane before hydrolysis in the periplasm by phosphatases - allowing transport of Pi into the cytoplasm.  Pi linked antiport systems of sn-glycerol-3-phosphate (GlpT) and glucose-6-phosphate (UhpT) mediate the pH dependent translocation of organo-phosphate compounds across the cell membrane (van Veen, 1997).  Phosphate is also accepted as an analogue of organo-phosphate by these exchange systems; the affinity for phosphate is 10 fold lower than for the organo-phosphate.  Regulation of GlpT and UhpT is specifically by extracellular glucose-6-phosphate and sn-glycerol-3-phosphate respectively, as opposed to phosphate (van Veen, 1997).  PhoE pores are formed in E. coli cell membranes during phosphate limitation and have a preference for anions such as phosphate and phosphate-containing nutrients, facilitating the unspecific entry of phosphate into the cytoplasm by diffusion (van Veen, 1997). 

The enzymes involved in polyphosphate metabolism

In order to study polyP accumulation an understanding of the enzymes involved and their regulation is required. The main enzymes involved in prokaryotic polyphosphate metabolism are polyphosphate kinase (PPK), polyphosphate glucose-6-transferase, polyphosphate adenosine monophosphate phosphotransferase, adenylate kinase and exopolyphosphatase.

Enzymes of polyphosphate synthesis

Polyphosphate kinase

The biosynthesis of polyP in prokaryotes is primarily mediated by the enzyme polyphosphate kinase (polyphosphate; ADP phosphotransferase; PPK; EC (Kornberg, et al., 1956)Now known as PPK 1, PPK has been studied most extensively in E. coli (Li & Brown, 1973) (Ahn & Kornberg, 1990) and to a lesser degree in Acinetobacter spp., (Neisseria meningitidis , Pseudomonas aeruginosa,, Propionibacterium shermanii and Vibrio cholera.

The PPK of E. coli is a membrane bound homotetramer of molecular mass 80kDa (Akiyama et al., 1992).  Formation of polyP is achieved through the reversible transfer of the gamma phosphate of ATP to a growing polyP chain (Equation 1.1) (Akiyama et al., 1992).  An intermediate, in the form of an N-linked phosphoenzyme, has been demonstrated in E. coli and N. meningitidis (van Dien et al., 1997). Phosphate has been shown to have allosteric effects on PPK activity in some bacteria, indicating a role in phosphate regulation (Robinson et al., 1987).  Divalent cations, especially Mg2+ are required for the activation of PPK (Murata et al., 1988).  The PPK of E. coli may operate in the reverse direction; synthesising ATP from polyP and ADP (Ahn & Kornberg, 1990). 

 Equation 1.1

PolyPn + ATP = polyPn+1 + ADP

A second PPK enzyme that has polyP synthesising ability has been reported: polyP dependent nucleoside diphosphate kinase activity, also known as PPK2. This differs from PPK1 in that it has a preference for GTP over ATP, Mn2+ as opposed to Mg2+. and favours the reverse reaction to synthesise GTP at the expense of polyP. This enzyme appears during stationary phase and is required for alginate synthesis (Ishige et al., 2002).

Other polyP synthesising enzymes

Although polyP is mainly synthesised by PPK alternative pathways are known to exist.  Reduced levels of polyP are present in ppk mutants of both P. aeruginosa 8830 (Zago et al., 1999) and Acinetobacter sp. Strain ADP1 (Trelstad et al., 1999); 2-10% of the wild type levels of polyP are also present in ppk mutants of N. meningitidis BNCV (Tinsley & Gotschlich, 1995).  Poly-β-hydroxybutrate-calcium-polyP membrane complexes are present in ppk mutants of E. coli (Castuma et al., 1995). This activity is likely to be that of PPK2 which was not known at the time. PolyP is accumulated to very high levels in yeast cells but without demonstrable PPK activity (Kulaev & Kulakovskaya, 2000).

A number of possible pathways have been proposed for polyP synthesis by mechanisms other than via PPK.  One possible alternative pathway for synthesis of polyP in prokaryotes is by the enzyme 1, 3 diphosphoglycerate-polyphosphate-phosphotransferase EC that catalyses the formation of polyP from 1, 3 diphosphoglycerate (Kulaev & Kulakovskaya, 2000).  It has also been proposed that polyP degrading enzymes such as exopolyphosphatase and endopolyphosphatase may form polyphosphate by operating in reverse in association with other enzymes (Kulaev & Kulakovskaya, 2000).  Binding of enzymes to membrane bound polyphosphatases that are present at higher levels in eukaryotes than prokaryotes could alter their activity sufficiently to favour polyP synthesis. 

 Enzymes of polyphosphate utilisation

PolyP is a substrate for a number of specific phosphotransferases and hydrolases and as mentioned earlier, reverse activity of PPK can hydrolyse polyP to form ATP.


Exopolyphosphatase (PPX; EC has been found in many bacteria and catalyses the hydrolysis of terminal phosphate residues from long chain polyP to form orthophosphate (Equation 1.2) (Akiyama et al., 1993).  The PPX of E. coli is a cytoplasmic membrane bound dimer with a subunit mass of 58KDa.  This enzyme has a high affinity for high molecular weight polyP (Km = 9nM for polyP500) and a high requirement for K+ (Akiyama et al., 1993).

 Equation 1.2

PolyPn + H2O = PolyPn-1 + Pi              

 Polyphosphate AMP phosphotransferase and adenylate kinase

Polyphosphate AMP phosphotransferase (Bonting et al., 1991) (Kornberg et al., 1999) and adenylate kinase (Kornberg et al., 1999) are used to regenerate ATP.  Phosphate is transferred from polyP to AMP by polyphosphate AMP phosphotransferase producing ADP (Equation 1.3).  This ADP can be utilised by adenylate kinase to form 1 ATP and AMP molecule from 2 ADP molecules (Equation 1.4). 

Equation 1.3

PolyPn + AMP = PolyPn-1 + ADP                                     

Equation 1.4

2ADP = ATP + AMP  

 ATP can be formed by this process in the absence of a carbon source  (van Groenestijn et al., 1989).  Recently it has been suggested that the polyphosphate AMP phosphotransferase activity detected in cell extracts of E. coli is the result of an enzyme complex involving PPK, adenylate kinase and polyP (Ishige & Noguchi, 2000. Adenylate kinase is thought to be unable to use polyP directly as a phosphate donor for phosphorylation of AMP and obtains a donor phosphate from an autophosphorylated PPK, a potent phosphodonor (Ishige & Noguchi, 2000).

 Polyphosphate glucokinase

Polyphosphate: glucose phosphotransferase (polyphosphate glucokinase; EC (Szymona & Ostrowski, 1964)  catalyses the phosphorylation of glucose and glucosamine to glucose-6-phosphate and glucosamine-6- phosphate respectively (Equation 1.5).  A glucokinase activity has been identified in a number of bacteria including: Mycobacterium tuberculosis (Hsieh et al., 1996) and P. shermanii (Phillips et al., 1993).  The glucokinases of the more phylogenetically ancient species show a higher preference for polyP as opposed to ATP than more recent species; indicating a more significant role for polyP in primitive prokaryotes (Phillips et al., 1993).

 Equation 1.5

PolyPn + glucose ® PolyPn-1 + glucose-6-phosphate 

Other polyP utilising enzymes

Polyphosphate: ADP phosphotransferaseis a similar, yet distinct activity to reverse action of PPK that has recently been reported in P. aeruginosa.  This enzyme requires short chain polyP and has a higher preference for GDP than ADP (Ishige & Noguchi, 2001).  This activity appears to be involved in exopolysaccharide alginate synthesis that requires GTP.  Other polyP degrading enzymes that have been reported are: tripolyphosphatase (EC in Methanobacterium thermautotrophicum (van Alebeek et al., 1994), endopolyphosphatase activity in S. cerevisae (Kulaev & Kulakovskaya, 2000) (this activity has not been reported in prokaryotes), polyP-dependent NAD kinase (Wood & Clark, 1988), as well as a number of other bacterial polyP phosphotransferase activities.

Regulation of enzyme expression associated with polyP metabolism

Relatively little is known about the regulation of enzyme activities involved in polyP metabolism.  The genetics of PPK have been studied in E. coli, N. meningitidis, P. aeruginosa and Acinetobacter sp.  Interestingly, ppk has been found to be highly conserved in the genomes of those bacteria studied (Kornberg & Fraley, 2000).  However regulation of ppk expression and the activity of PPK and PPX by these bacteria and the involvement of the PHO regulon are inconsistent (Gavigan et al., 1999).

The PHO regulon is central to assimilation of phosphate and regulation of phosphate metabolism.  The PHO regulon includes a number of phosphate starvation-inducible genes, the products of which include the environmental phosphate sensor (PhoR), the PHO regulon activator (PhoB), alkaline phosphatase, polyanionic specific outer membrane porin and the high and low affinity phosphate transport systems: phosphate specific transport (Pst) and phosphate inorganic transport (Pit) respectively (Metcalf and Wanner, 1991).  During phosphate limitation, genes under control of the PHO regulon may be induced by over 100 fold.  These genes are under control of the 2 component sensor-regulator complex that is comprised of PhoR and PhoB (Wanner, 1993).  The PhoB DNA binding protein functions as an activator of transcription; PhoB is activated by phosphorylation by PhoR during phosphate limitation  (Wanner, 1993).  The action of PhoR is in turn regulated by Pst, which acts as a cell surface receptor complex and an accessory protein PhoU (Wanner, 1993).

The involvement of the PHO regulon in the regulation of PPK and PPX is unclear.  In E. coli and Enterobacter aerogenes the ppk and ppx genes are located on the same operon (Figure 1.4) (Akiyama et al., 1993). PHO components do appear to be involved in some way: the genes encoding PPK and PPX are under control of two promoters, both of which contain putative PhoB boxes that indicate potential activation by PhoB, a functional PhoB is required for polyP accumulation by E. coli (Rao et al., 1998), a phoU mutant strain of E. coli was shown to accumulate 5 fold more polyP than the wild type (Zuckier & Torriani, 1981).  But accumulation of inorganic polyphosphate has also been shown in another study in phoU mutants of E. coli and Synechocystis sp. (Morohoshi et al., 2002).  In addition the induction of ppk in E. coli requires amino acid starvation in addition to phosphate limitation alone (Kornberg et al., 1999).  Therefore it is more likely that the ppk-ppx operon of E. coli is not under control by the PHO regulon.

The organisation of ppk and ppx genes in E. coli The organisation of other bacterial ppk and ppx differs from that of E. coli and the closely related E. aerogenes.  In all other bacteria studied, ppk is not followed by a ppx gene (Geißdörfer et al., 1998).  Therefore the regulation of PPK and PPX activities is likely to be different to that of E. coli and enable such bacteria to accumulate polyP to a much greater degree. Phosphate starvation has been demonstrated to induce ppk transcription in Acinetobacter sp. ADP1 (Trelstad et al., 1999) and Acinetobacter baumannii 252 (Gavigan et al., 1999).  Putative phoB sequences have not been found in Acinetobacter strain ADP1 indicating that the phoB is different to that of E. coli or another regulator without homology to PhoB is involved (Geißdörfer et al., 1998).

Accumulation of polyP by PPK can also be regulated at the enzymatic level (Rao et al., 1998).  PPX may be selectively inhibited by the stress response nucleotides guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp); often referred to as the stringent response factors (Kuroda et al., 1997) (Kuroda & Ohtake, 2000).  The levels of ppGpp and pppGpp increase in response to nutritional stress (Kuroda et al., 1997).  In response to amino acid starvation polyP levels of E. coli may increase by up to 1000 fold yet PPK and PPX activities remain unchanged.  E. coli mutants that are unable to produce pppGpp exhibit reduced synthesis of polyP (Ahn & Kornberg, 1990).  Zago et al. (1999) have also described the selective inhibition of PPX in P. aeruginosa mucoid strain 8830 by pppGpp leading to polyP accumulation. During exposure to other conditions, such as osmotic stress, PPX and PPK may be inhibited by stress- induced proteins such as the sigma factor RpoS (Ault-Riche et al., 1998).  RpoS and other regulatory signals may either inhibit PPX or stimulate PPK resulting in a decrease or increase in polyP synthesis respectively (Ault-Riche et al., 1998). 

Roles and functions of polyphosphate.

Since the early studies of Wiame (1947) and others, it has become increasingly apparent that polyphosphate has functions other than simply acting as a phosphate storage polymer (Kornberg et al., 1999).  New assays have shown the involvement of polyP in many diverse roles.  This molecule often referred to in the past as a ‘molecular fossil’ is now better described as a multifunctional biopolymer (Kulaev et al., 1999).

Phosphate reserve.  Maintenance of intracellular phosphate concentration is essential for cellular metabolism and growth.  In periods of phosphate surplus, phosphate can be channelled into a polyP reserve (Kornberg & Fraley, 2000).  Reserves of polyP are present as an osmotically inert aggregate associated with multivalent cations.  Conversely, polyP reserves can be readily hydrolysed to provide phosphate in periods of phosphate limitation by a number of polyP degrading enzymes (Kornberg et al., 1999). Phosphate has also been reported to allosterically activate PPK in a number of bacteria suggesting a role in the maintenance of the intracellular phosphate concentration (Tinsley et al., 1993; Mullan et al., 2002).

Alternative to ATP.  PolyP is readily degraded by the action of a number of enzymes.  PolyP may subsitute for ATP in various kinase reactions with, AMP, ADP and glucose and for other sugars, nucleosides, and proteins (Kornberg et al., 1999) (Kulaev & Kulakovskaya, 2000). Although present at a much higher concentration than ATP, intracellular polyP reserves could only directly subsitute ATP for a matter or seconds as the turnover of ATP is rapid (Kornberg et al., 1999) (Kornberg & Fraley, 2000).  Its potential as an direct energy source for cell metabolism is, therefore, limited. However, phosphate generated from the degradation of polyP by PPX may be transported out of the cell by the Pit system and generate a transmembrane proton gradient, thereby acting as an energy conservation mechanism (van Veen et al., 1994). This phosphate efflux is often observed during stationary phase of polyP rich cells (Mullan, PhD thesis 2003).

Chelator of metal ions.  The polyanionic properties of polyP facilitate the chelation of metal ions from the environment and intracellularly.  Intracellular polyP reserves participate in heavy metal detoxification (Kulaev & Kulakovskaya, 2000).  Release of phosphate from polyP by exopolyphosphatase activity enables heavy metal-phosphate complexes to be transported out of the cells (Keasling & Hupf, 1996).  Cadmium tolerance of E. coli cells depends on polyP metabolism (Keasling & Hupf, 1996).  PolyP rich cells of Anacystis nidulans also show greater tolerance to cadmium than polyP deficient cells (Keyhani et al., 1996).  

Intracellular pH regulation.  PolyP is utilised as a pH-stat mechanism in some microorganisms.  In the halotolerant green alga Dunaliella and S. cerevisiae polyP is hydrolysed to maintain internal cytoplasmic pH during alkaline stress (Pick et al., 1990) (Pick et al., 1991) (Castro et al., 1995).  Hydrolysis of polyP liberates H+ ions that restore the pH of the cytosol (Pick et al., 1990) (Castro et al., 1995).  Similarily a decrease in internal pH may be prevented by capture of H+ ions by polyP synthesis. Fluorescent staining with the pH indictor BCECF ( 2',7'-bis-(2-carboxyethyl) -5-(and-6)-carboxyfluorescein) has also revealed polyP inclusions to be a region of lower pH than the surrounding cytoplasm (Mullan, unpublished results). PolyP has been demonstrated in the acidified calcium compartments with H+ pumps known as acidocalcisomes (Seufferheld et al., 2004).

Component of cell capsule.  The bacterial cell capsule may be comprised of large amounts of polyP.  The cell capsule has been associated with microbial pathogenicity (Tinsley et al., 1993) (Tinsley & Gotschlich, 1995).  In Neisseria species, half of the total cellular polyP is loosely attached to the surface membrane (Tinsley et al., 1993).  Mutants of Neisseria, with reduced ability to synthesise polyP have lower pathogenicity, possibly due to reduced resistance to either complement fixation or phagocytosis (Tinsley & Gotschlich, 1995).  PolyP in the cell envelope of fungi is important in maintaining the negative charge of the cell surface and preventing cytoplasmic damage by ionic compounds (Kulaev & Kulakovskaya, 2000). 

Cell membrane channel complex.  PolyP has been found complexed with polyhydroxybutyrate and Ca2+ in the membranes of competent E. coli cells by Reusch & Sadoff 1988.  These complexes are associated with structural changes in the cell membranes that facilitate DNA transport into the cell, by an as yet unknown mechanism.

Other functions  

PolyP has recently been associated with a range of virulence factors of bacterial pathogens such as Helicobacter pylori (Kornberg & Fraley, 2000), P. aeruginosa (Rashid et al., 2000) (Rashid et al., 2000), and V. cholera (Ogawa et al. 2001).  H. pylori accumulates polyP during its infection stage (Rashid et al., 2000).  Accumulation of polyP may therefore be necessary for successful colonisation by H. pylori.  PolyP is required by P. aeruginosa for quorum sensing and biofilm formation (Rashid et al., 2000) and for proper functioning of flagella (Rashid & Kornberg, 2000.  Mutants of V. cholera unable to accumulate polyP also show reduced motility and attachment to abiotic surfaces.  PolyP accumulation would appear to be essential for the establishment of infection in these pathogens.  Kornberg & Fraley (2000) suggest that antimicrobial drugs could be developed to inhibit polyP synthesis by PPK and therefore reduce the virulence of microbial pathogens. 

Polyphosphate accumulation in response to environmental stress

An important role of polyP is that of cellular regulation in response to changes in nutritional status or environmental conditions.  Under certain conditions microbial cells accumulate extensive polyP reserves; accumulation may be induced by different conditions in different microorganisms.

 In E. coli polyP accumulation has been detected in response to osmotic stress, amino acid starvation and under nitrogen and phosphate limitation (Ault-Riche et al., 1998).  Under other conditions such as carbon limitation, changes in pH or temperature or oxidative stress, polyP accumulation was not induced (Ault-Riche et al., 1998).  P. aeruginosa mucoid strain 8830 accumulates polyP during stationary phase, amino acid starvation and phosphate limitation (Tzeng & Kornberg, 1998).  PolyP is also accumulated by a range of microorganisms in response to the conditions imposed by EBPR. Relatively high levels of polyP may be accumulated following a shift from a defined medium, lacking phosphate to one with an excess of phosphate (20mM) (Ogawa et al., 2001).  This is known as the ‘polyP overplus’ phenomenon and has been reported for many microorganisms including E. aerogenes (Harold, 1966), V. cholerae (Ogawa et al., 2001), Acinetobacter spp. ADP1 (Geißdörfer et al., 1998) and in yeast (Kulaev & Vagabov, 1983).

Interactions of polyP with DNA, RNA and polymerases as part of the RNA degradosome during stress conditions have been reported (Blum et al., 1997).  PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyP and regenerating ATP consumed by RNA helicase (Blum et al., 1997).  PolyP is therefore considered to be involved in a regulatory capacity during changing conditions, such as entry into stationary phase (Kornberg et al., 1999).  PolyP appears to have an interdependent regulatory system with the alternate RNA polymerase factor, RpoS in E. coli (Ault-Riche et al., 1998).  RpoS is essential for the expression of over 50 genes induced in stationary phase that are involved in stress resistance and long-term survival (Loewen & Hengge-Aronis, 1994).  RpoS may be regulated by the stringent response factor ppGpp.  It has been reported that polyP-depleted cells are unable to induce the transcription of rpoS and katE (an rpoS dependent gene) (Shiba et al., 2000) and also that rpoS mutants fail to accumulate polyP (Ault-Riche et al., 1998).  PolyP deficient cells of E. coli exhibit lower percentage survival to heat, oxidants and osmotic challenge compared to cells with long-chain polyP (Crooke et al., 1994) (Rao & Kornberg, 1996).

A model for stress-induced polyP accumulation in E. coli has been proposed by Ault-Riche et al. (1998) (Figure 1.5).  In this model polyP accumulation is dependent on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB.  Requirement for RpoS implies another unknown factor(s) designated “X” may be involved (Ault-Riche et al., 1998) (Hengge-Aronis et al., 1991).  Accumulation of polyP may be induced during nitrogen limitation by the nitrogen regulation signal cascade involving Utase, UR, NtrB, NtrC, RpoS and PhoB.  NtrC may act as a response regulator for RpoS to activate factor(s) “X” which could in turn activate polyP synthesis by stimulation of PPK or by inhibition of PPX. During nutrient limitation ppGpp accumulates by the activity of RelA and SpoT.  ppGpp (or ppGppp) may selectively inhibit PPX activity resulting in polyP accumulation.  PolyP accumulation during osmotic stress does not result from the osmotic sensor EnvZ.  It is thought that secondary effects such as nutrient limitation may trigger polyP synthesis.  Under some conditions in which the stringent response factor ppGpp and RpoS accumulate such as acid or alkaline pH, temperature or oxidative stress polyP accumulation did not occur in E. coli indicating that other, as yet unknown factors may be involved.

 A model for stress-induced polyP accumulation in E. coli


Figure 1.5. A model for stress-induced polyP accumulation in E. coli. Adapted from (Ault-Riche et al., 1998).

Accumulation of polyphosphate by microorganisms in response to low pH.
Accumulation of polyP due to the acidification of poorly buffered cultures was first reported by Dugiud et al. (1954) in E. aerogenes (then Bacterium aerogenes).  Cells grown in a medium in which the pH dropped to 4.5 were observed to contain granules presumed to contain ‘metaphosphate’.  The granules were present in the largest amount at between pH 4.2 and 5.2.  Granules were not observed in cells grown in buffered cultures at pH 6.0 or above.  Although research on polyP metabolism under such conditions as phosphate limitation continued, the findings of Duguid et al. were essentially forgotten until recently.

PolyP accumulation at low pH was reported in the environmental yeast isolate Candida humicola G-1 (McGrath & Quinn, 2000).  During growth at pH 5.5, phosphate removal was found to be 4.5 fold higher than cells grown at pH 7.5.  This increase in phosphate removal was associated with an increase in intracellular polyP.

A further study of activated sludge isolates revealed that a significant number of isolates exhibited a higher phosphate uptake during growth at a reduced pH (McGrath et al., 2001).  Some 34% of the isolates were capable of enhanced phosphate uptake at pH 5.5.  A further 44% of the isolates had no difference in phosphate removal at pH 5.5 and 7.5.  The remaining isolates either grew poorly or demonstrated lower phosphate removal at pH 5.5.  Most of these isolates that removed increased levels of phosphate at low pH appeared to be bacterial. Increased polyP accumulation at low pH has been described in the ectomycorrhizal fungus Suillus bovinus growing in association with the roots of Scots Pine (Gerlitz & Gerlitz, 1994).  PolyP accumulation was reported to be optimal at pH 5.5, some 35% greater than at pH 7.5. 

Further research within our laboratory has centered around Burkholderia cepacia AM19 to investigate the underlying mechanisms involved in polyP accumulation in acidic pH environments. This has resulted in improved methods for polyP extraction and quantification (Mullan et al., 2002) and a novel method for assay of PPK activity (Mullan et al., 2002). In a pilot biological activated sludge plant it was also shown that P removal from wastewater could be improved 2 fold by operation at reduced pH (Mullan et al., 2005). This has since led to full scale trials.

The phosphate transport systems have now been characterised using a high sensitivity colorimetric approach. RT-PCR has been used to show the effect of pH and P status on regulation of PPK transcription. Sequenced isolates have also been characterised using these new methods and 'in silico' techniques used to compare genes involved in phosphate metabolism.

PolyP accumulation is an important feature for survivial during growth in low pH environments and key roles in areas such as solubilsation of phosphate from soils and pathogenesis have been identified.

How to cite this article

Mullan, Alan (2005). [On-line]. Available from: . Accessed: 14 April, 2024.