Identification and characterization of KvgAS,a two-component system in Klebsiella pneumoniae CG43

A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions.     

Continuous culture studies on the synthesis of capsular polysaccharide by Klebsiella pneumoniae K1

The synthesis of capsular polysaccharide by Klebsiella pneumoniae K1 was investigated in a minimal salts medium by continuous culture. The organism produced larger amounts of polysaccharide under nitrogen-limited conditions than under carbon-limited conditions. The synthesis of polysaccharide was dependent not only on the availability of excess carbon, but also on growth rate. The rate of polysaccharide synthesis was greatest at low dilutions, low temperature (30°C) and at neutral pH. Prolonged growth in nitrogen-limited culture resulted in the development of non-mucoid variants, possibly due to a selective growth advantage over mucoid cells. The non-mucoid isolate was more susceptible to some bacteriophages, possibly due to the reduction or absence of capsular polysaccharide.     

Regulation of the homologous two-component systems KvgAS and KvhAS in Klebsiella pneumoniae CG43

In Klebsiella pneumoniae CG43, deletion of the sensor gene kvgS reduced the kvgAS expression in M9 medium with 0.2 mM paraquat, 0.2 mM 2,2-dihydropyridyl, or 300 mM NaCl. This result shows an autoregulatory role of KvgS and a stress-responsive expression of the two-component system (2CS). The kvgS deletion also appeared to decrease the expression of kvhAS, paralogous genes of kvgAS. Additionally, measurements of the promoter activity in kvgA(-) mutant revealed that KvgA is probably an activator for the expression of kvgAS and kvhAS. The subsequent electrophoretic mobility shift assay, indicating a specific binding of the recombinant KvgA to the putative promoters P(kvgAS) and P(kvhAS), also supported an interacting regulation between the 2CSs. In P(kvgAS) and P(kvhAS), the presence of RpoS binding elements suggested an RpoS-dependent regulation. Nevertheless, the rpoS deletion reduced the expression of kvgAS but increased that of kvhAS. Moreover, the kvgA deletion reduced the expression of katG and sodC. The overexpression of KvhA altered the susceptibility to fosfomycin and an increasing activity of UDP-N-acetylglucosamine enolpyruvyl transferase, the target protein of fosfomycin, which suggesting a regulation by KvhA. Taken together, these indicated that the two 2CSs probably belong to different regulatory circuits of the RpoS regulon.     

合成肺炎链球菌荚膜多糖基因的研究进展

肺炎链球菌是导致婴幼儿和老年人罹患肺炎、脑膜炎、中耳炎等疾病的主要病原体之一,其致病力与位于细菌表面的荚膜多糖密切相关,而荚膜多糖层的薄厚和多糖结构是影响致病力的主要因素。在分子水平探索参与荚膜多糖合成的相关基因,不仅有助于进一步理解肺炎链球菌的致病机理,而且可从基因水平选育高表达荚膜多糖的肺炎链球菌菌株用于多糖疫苗的研发。鉴于此,现就合成肺炎链球菌荚膜多糖基因的作用机制和研究方法作一综述。     

Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.     

Application of flow cytometry to the study of antiphagocytic properties of Klebsiella pneumoniae capsular polysaccharide

Abstract We used flow cytometry to compare the effects of whole cells and capsular polysaccharides of Klebsiella pneumoniae on the phagocytic ability ot polymorphonuclear leukocytes. Our results showed a light descrease in phagocytic activity in the presence of capsular polysaccharides, but a marked decrease with whole cells. Our findings suggest that the resistance to phagocytosis in these microorganisms is not due exclusively to their capsule, as claimed by other authors.     

肺炎克雷伯氏菌荚膜多糖表达量评价指标的建立及发酵条件优化

建立了特异性强的肺炎克雷伯氏菌荚膜多糖全菌ELISA检测方法,检测结果与多糖表达量相关性好;以全菌ELISA值结合菌数为评价指标,对影响荚膜多糖表达的培养基组成及发酵条件进行了优化,优化后的摇瓶培养条件下发酵液活性和生物量分别比优化前提高72.7和33倍,并经7L罐放大实验,绘制发酵动力学曲线,为肺炎克雷伯氏菌荚膜多糖进一步开发打下基础。     

The influence of the O-antigen and the capsular polysaccharide on the establishment of PlCmts lysogeny in Klebsiella pneumoniae

The O-antigen of the lipopolysaccharide in Klebsiella pneumoniae caused a significant reduction in the frequency of establishment of PlCmts lysogeny, while the capsular polysaccharide showed no effect on this frequency. The bacterial receptor for PlCmts are the lipopolysaccharide-core oligosaccharides, the results suggest that K. pneumoniae strains with an O-antigen in their lipopolysaccharide have a poorly accessible lipopolysaccharide-core (the PlCmts bacterial receptor), while K. pneumoniae strains lacking the O-antigen have a highly accessible lipopolysaccharide-core. The accessibility of the receptor is independent of the K antigen (capsular polysaccharide).     

一种溶菌酶样蛋白对肺炎链球菌毒力及荚膜多糖合成的影响

摘要：【目的】为了研究肺炎链球菌（Streptococcus pneumoniae, S.pn）的一种假想的溶菌酶样蛋白在细菌生物学性状及其致病中的作用。【方法】利用长臂同源PCR对该基因进行敲出,并同时构建带有拯救质粒的缺失菌株,观察D39野生菌、缺失菌与带有拯救质粒的缺失菌株在相关生物学性状及其致病力改变,从而鉴定这种假想溶菌酶样蛋白的功能。【结果】缺失菌与野生菌相比,细菌生长减缓,毒力下降,荚膜多糖合成明显减少。而将拯救质粒转入缺失菌株后,该溶菌酶样蛋白的mRNA表达水平较野生菌高,其毒力及荚膜合成     

Epitopes in the capsular polysaccharide and the porin OmpK36 receptors are required for bacteriophage infection of Klebsiella pneumoniae

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