摘要
采用双亲本接合法对从文山湖富营养化水体中分离得到肺炎克雷伯氏菌进行Tn5转座子插入诱变,在含四环素和卡那霉素的LB培养基上获得接合子,利用饥饿冷冻高渗透压法对接合子进行细菌VBNC状态转化突变株的筛选和特性的研究。结果表明,带有卡那霉素基因的Tn5成功地插入到了肺炎克雷伯氏菌的染色体中,在双抗性培养基上获得约2.3×104cell/mL的接合子,转座效率为6.58×10-4。对多个接合子和对照肺炎克雷伯氏菌的诱导、筛选及比较,发现KPQT-7是最快进入VBNC状态的突变株,该突变株在胁迫诱导9d后超过30%的细胞都进入了VBNC状态,而对照的肺炎克雷伯氏菌至少要在诱导21d后才大部分进入VBNC状态。在此基础上,对VBNC转化突变株KPQT-7作进一步的遗传学分析将会为细菌VBNC状态分子机理的研究奠定坚实的基础。
VBNC （viable but non-culturable state） bacteria are considered to represent a sub-population of cells which are not able to grow in the usual culture media but yet remain metabolically active, and can be resuscitated when conditions become favorable. So far,the molecular basis for entry into and resuscitation from the VBNC state are still poorly known,and all these obscurations must make clear, because VBNC bacteria is concerned about public health, evaluating water quality, epidemic prevention. To better understand the molecular mechanism of bacterial VBNC state, this study used transposon Tn5 to induce the gram negative bacteria-Klebsiella peneumoniae by parental conjugation method. Exconjugations were iso- lated from culture medium with 500μg/mL km and 15μg/mL Tc and detected by PCR using primers IS1 and IS2 which had been designed using Tn5 as the template. PCR products were visualized on a 1% agarose gel. After that, exconjugations were incubated in sterilized distilled water with 3% sodium chloride at 4℃ for screening mutants which was the fastest and the easiest entered the VBNC state. The results showed that transposon Tn5 were inserted in chromosome of K. peneumoniae successfully, about 2.3 ×10^4 cell/mL exconjugations were obtained, and its transposon frequency was high at 7.6 ×10^-5. Exconjugations could be detected by PCR using primers IS1 and IS2. Comparing with the other stains, a mutant KPQT-7 was found much faster and easier to enter VBNC state, throughout the incubation, total direct cell counts remained near the original inoculum level of 10^6 to 10^7 cell/mL,and the plate cell counts decreased day by day,more than 30% of KPQT-7 population had been into VBNC state after 9d stress treatment,while the control stain entered the VBNC state after 21d stress treatment. It can be concluded that K. peneumoniae entered the VBNC state might deal with the sequence flanking the Tn5 insertion site. The genetic analysis on the mutant KPQT-7 are ongoing,this experiment has laid a solid foundation for the further research on molecular mechanism of the VBNC transformation in aquatic bacteria.
出处
《水生生物学报》
CAS
CSCD
北大核心
2009年第1期28-33,共6页
Acta Hydrobiologica Sinica
基金
国家自然科学基金(30470281
30770340)
国家重点基础研究发展计划课题(2002CB412309)资助
