• Journal of Life Science
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• v.10 no.4
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• pp.397-402
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• 2000

Polyphosphate kinase catalyzes the formation of polyphosphate from ATP. To understand the mechanism of phosphate accumulation, the Serratia marcescens gene encoding ppk was cloned from the genomic library by the method of Southern hybridization. The hybridization positive DNA fragment region from pDH3 was subcloned into the expression vector. The ppk gene product, a polypeptide of 75 kDa, was confirmed by SDS-PAGE. Expression of the Serratia marcescens ppk is regulated by the catabolite repression system. The enzyme activity polyphosphate kinase was increased in the E. coli strain harboring plasmid pMH4 with ppk gene.

• Journal of Microbiology and Biotechnology
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• v.13 no.1
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• pp.139-145
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• 2003

The gene encoding Thermus caldophilus GK24 polyphosphate kinase (Tca PPK) was cloned and sequenced. The gene contains an open reading frame encoding 608 amino acids with a calculated molecular mass of 69,850 Da. The deduced amino acid sequence of Tca PPK showed a 40% homology to Escherichia coli PPK, and $39\%$ to Klebsiella aerogenes PPK. The Tca ppk gene was expressed under the control of the T7lac promoter on pET-22b(+) in E. coli and its enzyme was purified about 70-fold with $36\%$ yield, following heating and HiTrap chelating HP column chromatography. The native enzyme was found to have an approximate molecular mass of 580,000 Da and consisted of eight subunits. The optimum pH and temperature of the enzyme were 5.5 and $70^{\circ}C$, respectively. A divalent cation was required for the enzyme activity, with $Mg^2+$ being the most effective.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1061-1065
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• 2001

The antibacterial effects of water extracts of Scutellariate Radix (a dried root of Scutellaria baicalensis GEORGI) and its major flavonoid components, Baicalin and Baicalein, on Salmonella typhimurium, a representative enteric pathogen, were studied. Through a Kriby-Bauer disc analysis, the growth-inhibition activity of Scutellariae Radix against. S. typhimurium was found to be compatible with commercial antibiotics, such as ampicillin, chloramphenicol, and streptomycin. In contrast, the growth of a nonpathogenic E. coli strain was unaffercted by Scutellariae Radix. To examine the effect of polyphosphate kinase (ppk), a putative virulence factor, on the antibacterial activity of Scutellariae Radix, the growth profile of a ppk mutant of S. typhimurium was investigated in a tryptic soy broth containing different concentrations of water extracts of Scutellariae Radix. The ppk mutant was able to grow in 6 mg/ml of water extracts of Scutellariae Radix, whereas in 6 mg/ml of water extracts of Scutellariae Radix, whereas the wild-type could not, implying that the inactivation of ppk made S. typhimurium more resistant to the antibacterial activity of Scutellariae Radix. No enhanced resistance was observed in a ppk mutant of S. typhimurium complemented with a ppk expression vector. The attenuation of the virulence by ppk inactivation was also observed in a virulence assay using BLAB/c mice. Neither Baicalin nor Baicalein exhibited any growth-inhibition activity against S. typhimurium. The water extracts of Scutellariae Radix stimulated the transcription of ppk, especially in the early growth-stage of S. typhimurium.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1527-1535
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• 2009

Polyphosphate (polyP) plays diverse physiological functions in prokaryotes and eukaryotes, but most of their detailed mechanisms are still obscure. Here, we show that deletion of polyphosphate kinase (PPK), the principal enzyme responsible for synthesis of polyP, resulted in augmented expression of cAMP receptor protein (CRP) and rpoS and lowered $H_2O_2$ sensitivity in Salmonella Typhimurium ATCC14028. The binding of cAMP-CRP complex to rpoS promoter and further stimulation of its transcription were proved through electrophoretic mobility shift assay, lacZ fusion, and exogenous cAMP addition, respectively. The rpoS expression increased in cpdA (cAMP phosphodiesterase coding gene) mutant, further suggesting that cAMP-CRP upregulated rpoS expression. These results demonstrate that PPK affects oxidative stress response by modulating crp and rpoS expression in S. Typhimurium.

• Proceedings of the Korean Society of Life Science Conference
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• 2000.09a
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• pp.77-77
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• 2000

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.114-120
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• 2002

A 1,989-bp genomic region encoding nickel resistance genes was isolated from Legionella pneumophila, a pathogen for legionellosis. From a sequencing and computer analysis, the region was found to harbor two structural genes, a nreB-like protein gene (1,149 bp) and a nreA-like protein gene (270 bp), in a row. Both genes exhibited a significant degree of similarity to the corresponding genes from Synechocystis sp. PCC6803 ($54\%$ amino acid sequence identity) and Achromobacter xylosoxidans 31A ($76\%$). The gene was successfully expressed in E. coli MG1655 and conferred a nickel resistance of up to 5 mM in an LB medium and 3 mM in a TMS medium including gluconate as the sole carbon source. E. coli harboring the nickel resistance gene also exhibited a substantial resistance to cobalt, yet no resistance to cadmium or zinc. Since the extracellular concentration of nickel remained constant during the whole period of cultivation, it was confirmed that the nickel resistance was provided by an efflux system like the $Ni^2＋$permease (nrsD) of Synechocystis sp. strain PCC6803. Since polyphosphate (poly-P) is known as a global regulator for gene expression as well as a potential virulence factor in E. coli, the nickel resistance of a ppk mutant of E. coli MG 1655 harboring the nickel resistance gene from L. pneumophila was compared with that of its parental strain. The nickel resistance was significantly attenuated by ppk inactivation, which was more pronounced in an LB medium than in a TMS medium.

• Korean Journal of Veterinary Research
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• v.53 no.4
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• pp.211-216
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• 2013

This study was focusing on evaluating the protection of polyphosphate kinase (ppk) deleted and/or temperature-sensitive (ts) Salmonella Enteritidis (SE) as an attenuated vaccine in chickens. We constructed SEppk, SEts and SEppk::ts mutants and screened those mutants by growth capability in vitro, protection study in mice model and antibody response in chickens. Among the mutants, SEppk::ts-3 was selected because it showed higher growth capability, good protection against highly virulent SE in mice model, and good antibody response in chickens. SEppk::ts-3 also showed good protection against highly virulent SE isolate because it decreased colonization of virulent SE challenge strain in spleen, liver and cecum compared with the non-vaccinated control. The SEppk::ts-3 mutant showed cross-protection against S. Gallinarum (SG) challenge although the its cross-protection rate was a little lower than that of SG9R, a commercial vaccine against SG infection. To use for live attenuated vaccine in chickens, it should further be characterized.