Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830 - PubMed (original) (raw)

Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830

A Zago et al. Appl Environ Microbiol. 1999 May.

Abstract

Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P. aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of P. aeruginosa 8830. Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that ppk is transcribed from a sigmaE-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.

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Figures

FIG. 1

FIG. 1

(A) Organization of ppk and ppx genes. (B) Alignment of P. aeruginosa 8830 PPX with E. coli PPX (accession number L06129) and E. coli pppGpp-5′-phosphohydrolase (GppA) (accession number M83316) (CLUSTAL X). Sequences were retrieved from GenBank. ∗, amino acid identity; “:” and “.”, limited conserved substitutions.

FIG. 2

FIG. 2

Southern blot analysis of ppk knockout mutant genomic DNA, determined by using the 1.7-kb PCR probe. Lanes: 1, P. aeruginosa 8830 chromosomal DNA digested with _Sph_I; 2, ppk::Tc genomic DNA digested with _Sph_I; 3, P. aeruginosa 8830 chromosomal DNA digested with _Kpn_I; 4, ppk::Tc genomic DNA digested with _Kpn_I. The different band patterns for the parental and mutant strains are consistent with the insertion of the Tc cassette of 1,720 bp having one _Sph_I restriction site and no _Kpn_I sites.

FIG. 3

FIG. 3

TLC analysis of PPK enzymatic activity product. Lanes: 1, P. aeruginosa PPK reaction mixture; 2, polyP purified with Glassmilk from the reaction mixture shown in lane 1; 3, P. aeruginosa polyP digested with rPPX1; 4, purified polyP synthesized by E. coli PPK; 5, E. coli polyP digested with rPPX1; 6, reaction mixture in lane 1 digested with an M. bovis ATPase; 7, E. coli polyP digested with M. bovis ATPase; 8, P. aeruginosa polyP digested with M. bovis ATPase.

FIG. 4

FIG. 4

TLC analysis of P. aeruginosa PPX enzymatic activity. [32P]polyP (267 μM) was incubated for various time periods as specified with 200 ng of PPX protein in 20 μl of total reaction mixture. Samples (1 μl) were taken periodically and spotted onto a polyethyleneimine plate.

FIG. 5

FIG. 5

(A) Primer extension analysis of the transcription start sites of the ppk gene. P. aeruginosa 8830 total RNA was annealed to oligonucleotide P1 and extended with murine reverse transcriptase. Lanes G, A, T, and C represent the dideoxy sequencing ladder carried out with the same primer. Lane 1, primer extension products. (B) Comparison of −35 and −10 motifs identified upstream of the transcription start site (the C residue that gave the most intense band) with ςE consensus sequences.

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