Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

The biological process for phosphate (P_i) removal is based onthe use of bacteria capable of accumulating inorganic polyphosphate(polyP). We obtained Escherichia coli mutants which accumulatea large amount of polyP. The polyP accumulation in these mutantswas ascribed to a mutation of the phoU gene that encodes a negativeregulator of the P_i regulon. Insertional inactivation of thephoU gene also elevated the intracellular level of polyP inSynechocystis sp. strain PCC6803. The mutant could remove fourfoldmore P_i from the medium than the wild-type strain removed.

INTRODUCTION

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Introduction

Inorganic phosphate (P_i) is recognized as one of the major nutrientscausing eutrophication of lakes, bays, and waterways (18). Considerableattention has been paid to effective P_i removal from wastewater(6). Many bacteria are capable of accumulating excess P_i inthe form of inorganic polyphosphate (polyP), which is a linearpolymer of hundreds of P_i residues linked by high-energy phosphoanhydridebonds (7, 9, 10). Improvement of the ability to accumulate polyPcontributes to increased P_i removal from wastewater (18).

Previously, we have demonstrated genetic improvement of polyPaccumulation in Escherichia coli (8). High levels of accumulatedpolyP were achieved by increasing the dosage of the E. coligenes encoding polyP kinase (ppk) and the P_i-specific transportsystem (pstSCAB). The E. coli recombinant accumulated approximately16% of its dry weight as phosphorus (P) (49% as P_i) (8). Over60% of cellular P was stored in the form of polyP in the geneticallyengineered E. coli strain. However, growth of the E. coli recombinantwas severely limited in minimal medium (8). In addition, thisrecombinant released polyP back into the medium when it accumulatedhigh levels of polyP. In this paper, we report that a mutationin the phoU gene, which encodes a negative regulator of theP_i regulon (3, 21), led to high levels of accumulated polyPin E. coli. phoU mutants could be easily screened on agar platescontaining 5-bromo-4-chloro-3-indolylphosphate (XP) after N-methyl-N'-nitro-N-nitrosoguanidinemutagenesis. Therefore, isolating phoU mutants seems to be auseful way to improve the ability of bacteria to accumulatepolyP. To show whether this method is effective in another bacterium,we performed insertional inactivation of the phoU gene in Synechocystissp. strain PCC6803 and showed that the intracellular level ofpolyP increased in the mutant.

Isolation of E. coli mutants.

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Isolation of e. coli...

The levels of polyP in E. coli MG1655 were very low (less than1 nmol of P_i residues/mg of protein) when the organism was grownon a rich medium (11). PolyP was recovered with silicate glassfrom cells lysed with guanidine isothiocyanate, and the polyPcontent was determined by a two-enzyme assay (4). PolyP wasfirst converted to ATP by polyphosphate kinase, and then theamount of ATP was measured by a bioluminescence assay. We firstselected alkaline phosphatase constitutive mutants, which couldform blue colonies on agar plates containing XP (50 mg/liter)under P_i-sufficient conditions after N-methyl-N'-nitro-N-nitrosoguanidinemutagenesis (17). One of 150 mutants, designated MT4, accumulateda large amount of polyP (approximately 100 nmol of P_i residues/mgof protein). The levels of polyP were at least 100-fold higherthan the levels in the wild-type strain, MG1655.

To assess P_i transport (14), the E. coli mutants were grownovernight in MOPS (morpholinepropanesulfonic acid) medium (12)containing either 0.1 mM P_i (P_i limiting) or 2 mM P_i (P_i sufficient).Cells were harvested by centrifugation at 8,000 x g for 5 minand washed with HEPES buffer (13). Assays were started by adding³²P_i to a final concentration of 2.5 µM, and the amountof ³²P_i incorporated into the cells was determined by usinga scintillation counter (Packard). Only MT4 exhibited P_i-specifictransport even when the organisms were grown with excess P_i(Fig. 1).

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FIG. 1. P_i transport as assessed by using P_i-sufficient MG1655 ( ${blacksquare}$ ), P_i-limited MG1655 (•), P_i-sufficient MT4 ( ${triangleup}$ ), and P_i-limited MT4 ( ${circ}$ ) cultures. Cells were grown in MOPS medium containing either 2 mM P_i (P_i sufficient) or 0.1 mM P_i (P_i limited). OD, optical density at 600 nm.

Analysis of the mutation of MT4.

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Analysis of the mutation...

An E. coli DNA library based on the SuperCos plasmid (Stratagene)was introduced into MT4 by transformation (17). Approximately200 transformants were examined for the ability to accumulatepolyP in 2xYT medium (17). One transformant reduced the levelsof polyP to the level in the wild-type strain. This transformantcarried a recombinant plasmid containing a 30-kb DNA fragmentof the E. coli chromosome. Nucleotide sequence analysis of the30-kb insert revealed that this fragment contained the pst andbgl operons which were located at 84 min on the E. coli linkagemap (5). Subcloning and complementation analysis revealed thata 3.0-kb EcoRI fragment, carrying the entire phoU gene, couldcomplement the mutation of MT4. The chromosomal phoU gene ofMT4 was amplified by PCR with primers EU1 (5'-ATTGGGATTTGTCTGGTGAA-3')and EU2 (5'-AGAAGACTACATCACCGGTC-3') and cloned into the pGEM-Tvector (Promega). Nucleotide sequence analysis showed that the29th codon of the phoU gene was changed from a glycine codonto an aspartic acid codon in MT4. We again selected a polyP-accumulatingmutant from the mutants producing alkaline phosphatase constitutively.In this mutant, the 83rd codon of the phoU gene was changedfrom an alanine codon to a threonine codon. These results indicatedthat mutation of the phoU gene resulted in elevated intracellularlevels of polyP in E. coli.

To further confirm that phoU mutants could accumulate high levelsof polyP, the chromosomal phoU gene of MG1655 was disruptedby inserting a kanamycin resistance (Km^r) gene cassette intothe wild-type gene. A 0.8-kb DNA fragment containing the entirephoU gene was amplified by PCR with the EU1 and EU2 primersand inserted into the pGEM-T vector. The resultant plasmid wasdigested with ClaI and ligated with the Km^r gene cassette ofpUC4K (Amersham-Pharmacia), and an EcoRI fragment containinga disrupted phoU gene was inserted into an EcoRI site of pGP704containing the sacB gene (16). This plasmid was introduced intoE. coli S17-1 and then transferred into MG1655 by transconjugation(22). Transconjugants were selected on agar plates containing5% sucrose and kanamycin (50 mg/liter). Disruption of the chromosomalphoU gene was confirmed by Southern hybridization. As expected,the insertional phoU mutant accumulated high levels of polyP(approximately 400 nmol of P_i residues/mg of protein).

One might assume that ppk is a member of the P_i regulon in E.coli and that derepressed expression of this gene in the phoUmutants results in polyP accumulation. However, no significantincrease in the level of polyphosphate kinase was observed underP_i-limited conditions in E. coli (Morohoshi and Kuroda, unpublisheddata). Furthermore, expression of a single-copy ppk-lacZ transcriptionalfusion did not increase under P_i limitation conditions (L. Zhouand B. L. Wanner, personal communication). P_i transport is ratelimiting for polyP accumulation in E. coli (8). A phoU mutantexhibited P_i-specific transport even under P_i-excess conditions(Fig. 1). We transferred a ${Delta}$ (pstSCAB-phoU)::km mutation fromBW17335 (20) to the wild-type strain, MG1655, by using P1 phage.This mutant failed to accumulate polyP (0.5 nmol/mg of protein).Consequently, it is likely that constitutive expression of P_i-specifictransport (PstSCAB) is responsible for the elevated levels ofpolyP in the phoU mutants.

Although MT4 grew relatively slowly, it removed twofold moreP_i from the medium than the wild type removed (Fig. 2). Introductionof multicopy plasmid pBC29, which contains the E. coli ppk gene(1), into the wild-type strain, MG1655, did not increase P_iremoval significantly (Fig. 2). By contrast, MT4(pBC29) removedabout twofold more P_i than MT4 removed (Fig. 2). The P contentof MT4(pBC29) reached approximately 9% on a dry weight basis(27% as P_i) and was about sixfold greater than that of the wild-typestrain, MG1655. PolyP granules were detected in MT4(pBC29) whenthe cells were observed by fluorescent microscopy after theywere stained with 4',6'-diamidino-2-phenylindole (DAPI) (2)(Fig. 3).

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FIG. 2. Growth (A) and P_i uptake (B) of E. coli MG1655, MG1655(pBC29), MT4, and MT4(pBC29). Strains MG1655 ( ${blacksquare}$ ), MG1655(pBC29) (•), MT4 ( ${triangleup}$ ), and MT4(pBC29) ( ${circ}$ ) were grown in MOPS medium containing 2 mM P_i at 28°C. OD₆₀₀, optical density at 600 nm.

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FIG. 3. PolyP granules in MT4(pBC29) detected by DAPI fluorescence. Strain MT4(pBC29) was grown overnight on 2xYT medium, stained with DAPI at a final concentration of 50 µg/ml, and observed with a fluorescence microscope (Olympus BX-40).

phoU mutant of Synechocystis sp. strain PCC6803.

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phoU mutant of Synechocystis...

A chromosomal phoU mutant of Synechocystis sp. strain PCC6803was also constructed by inserting a Km^r gene cassette into thewild-type gene. A 2.0-kb DNA fragment, which carried the entirephoU gene, was amplified by PCR with primers SU1 (5'-GGTACCATCAACCTGATCGCCTAT-3')and SU2 (5'-GCTACTGCTCCAGTCGACCCGAGT-3') and cloned into pUC119.The resultant plasmid was digested with BglII, ligated to theKm^r gene cassette of pUC4K, and introduced into Synechocystissp. strain PCC6803 by electroporation (17). Disruption of thechromosomal phoU gene was confirmed by Southern hybridization(data not shown). The Synechocystis phoU mutant, designatedSPU101, accumulated about 15% as much polyP (on a dry weightbasis) as P_i. As in E. coli MT4(pBC29), polyP particles weredetected in SPU101 by DAPI fluorescence (Fig. 4). The totalP content of the mutant strain reached a maximum of 6% on adry weight basis. P_i uptake experiments were performed withgrowing cultures of PCC6803 and SPU101 (Fig. 5). Cultures weregrown in BG-11 medium (19) with 1% CO₂ at 30°C under light(5,000 lx). Strain SPU101 removed about fourfold more P_i fromthe medium than the parental strain removed. The growth of SPU101was almost equivalent to that of PCC6803.

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FIG. 4. PolyP granules in Synechocystis sp. strain PCC6803 and phoU mutant SPU101 detected by DAPI fluorescence. Strains PCC6803 and SPU101 were grown in BG-11 medium at 30°C under light (5,000 lx) with 1% CO₂ for 3 days and stained with DAPI. Cells were observed with either visible light (A and B) or UV light (C and D).

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FIG. 5. Growth (A) and P_i uptake (B) of Synechocystis sp. strain PCC6803 and phoU mutant SPU101. Strains PCC6803 (•) and SPU101 ( ${circ}$ ) were grown in BG-11 medium at 30°C under light (5,000 lx) with 1% CO₂. OD₇₅₀, optical density at 750 nm.

We showed that a mutation of the phoU gene, which encodes anegative regulator of the P_i regulon, led to high levels ofaccumulated polyP in both E. coli and Synechocystis sp. Raoet al. previously reported that a phoU mutation had no effecton polyP accumulation (15). The phoU mutant of these authorsprobably had a secondary mutation in the Pst system, as describedpreviously (20).

In enhanced biological P removal, sludge microorganisms accumulate3 to 5% of their dry weight as P (18). Similar levels of accumulatedpolyP were observed with the phoU mutant of Synechocystis sp.strain PCC6803. In general, the levels of carbon sources inwastewater are relatively low. This is likely to limit P_i removalfrom wastewater by sludge microorganisms (18). Use of the Synechocystismutant, which is able to accumulate polyP if light is present,may improve biological removal of P_i from wastewater.

ACKNOWLEDGMENTS

We thank B. L. Wanner for a gift of the phoU and pstSCAB mutantand M. Ikeuchi for a gift of strain PCC6803.

This work was supported in part by the New Energy and IndustrialTechnology Development Organization (NEDO) and by the ResearchInstitute of Innovative Technology for the Earth (RITE).

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Phone: 81-824-24-7758. Fax: 81-824-24-7047. E-mail: akuroda{at}hiroshima-u.ac.jp.

REFERENCES

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