• Journal of Microbiology and Biotechnology
• /
• 제13권1호
• /
• pp.139-145
• /
• 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.

• 생명과학회지
• /
• 제10권4호
• /
• pp.397-402
• /
• 2000

본 연구는 인산 축적능이 뛰어난 균주를 분자 육종하여 생물학적 폐수처리 및 토양의 인산 집적을 해결시키는 산업적 유용한 재료로 이용하기 위한 기초연구를 목표로 하고 있다. Polyphosphate kinase의 ATP의 phosphate를 단리하여 한분자씩 결합시키는 형태로 polyphosphate의 합성반응을 촉매한다. 인산 축적에 관한 대사과정의 분자적 이해를 위하여 Serratia marcescens균주로부터 Southern hybridization방법으로 ppk를 암호하는 유전자를 찾아내어 새조합시킨 pDH3를 구축하였다. pDH3으로부터 ppk를 암호하는 유전자 영역의 4.0 kb 단편을 가진 subclone을 작성하였다.Serratia marcescens의 polyphosphate kinase의 활성은 catabolite repression에 의한 조절을 받았다. 발현멕타에 삽입시킨 재조합 플라스미드를 대장균에 도입시킨 결과, polyphosphate kinase의 효소활성이 크게 증가됨을 확인 하였다. 또한 대량 발현시킨 결과를 SDS-PAGE를 통하여 75 KDa의 발현산물을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제19권12호
• /
• pp.1527-1535
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• 제12권1호
• /
• pp.114-120
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• 제28권11호
• /
• pp.1846-1849
• /
• 2018

Recent human gut microbiome studies have supported that the genus Bifidobacterium is one of the most beneficial bacteria for human intestinal health. To develop a new probiotic strain for functional food applications, fourteen fecal samples were collected from healthy Koreans and the strain BCBR-583 was newly selected and isolated from a 25-year-old Korean woman's fecal sample using the selective medium for Bifidobacterium. Subsequent fructose-6-phosphate phosphoketolase (F6PPK) test and 16S rRNA gene sequencing analysis of the strain BCBR-583 confirmed that it belongs to B. longum subsp. longum. The stress resistance tests showed that it has oxygen and heat tolerance activities (5- and 3.9-fold increase for 24 h at 60 and 120 rpm, respectively; $78.61{\pm}6.67%$ survival rate at $45^{\circ}C$ for 24 h). In addition, gut environment adaptation tests revealed that this strain may be well-adapted in the gut habitat, with gastric acid/bile salt resistance ($85.79{\pm}1.53%$, survival rate under 6 h treatments of gastric acid and bile salt) and mucin adhesion ($73.72{\pm}7.36%$). Furthermore, additional tests including cholesterol lowering assay showed that it can reduce $86.31{\pm}1.85%$ of cholesterol. Based on these results, B. longum BCBR-583 has various stress resistance for survival during food processing and environmental adaptation activities for dominant survival in the gut, suggesting that it could be a good candidate for fermented food applications as a new probiotic strain.