肺炎克氏杆菌NifA对巴西固氮螺菌nifH启动子的转录激活作用

用PR方法克隆了巴西固氮螺菌Yu62 nifH的启动子片段,DNA序列分析表明菌株Yu62与标准菌株sp7之间的DNA序列差异很小。利用启动子探针质粒载体pcBl82,构建了3个不同的nifH：：lacz转录融合质粒,在大肠杆菌中分别测定肺炎克氏杆菌NifA对它们的转录激活作用。结果表明巴西固氮螺菌nifH启动子的转录是依赖于NifA的,缺失了上游激活序列的启动子不能被NifA激活转录,肺炎克氏杆菌NifA对其自身nifH及巴西固氮螺菌nifH启动子的转录激活作用并无很大差异。     

Electron transport to nitrogenase in Klebsiella pneumoniae: purification and properties of the nifJ protein

In Klebsiella pneumoniae, the physiological electron flow to nitrogenase involves specifically, in addition to nitrogenase reductase, the products of the nifF and nifJ genes. The J protein was purified to homogeneity and was found to be an iron-sulfur protein devoid of molybdenum. In its native state, the J protein is a dimer of Mr about 245 000, made up of two subunits of the same molecular weight. It contains about 30 mol iron and 24 mol labile sulfur/mol protein. The addition of J protein to crude extracts of a nifJ mutant reestablishes pyruvate-supported acetylene-reducing activity. This activity is further enhanced by addition of pure nitrogenase (Kp1). Based on its physical properties, the J protein is probably an oxidoreductase whose physiological role might be to transfer electrons from a metabolic donor to the F protein. In addition, another protein whose activity is also dependent on the nifJ gene seems to be required for the formation of a fully active Kp1.     

Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae.

Crude extracts of the wild-type Klebsiella pneumoniae reduced C2H2 with either pyruvate or formate as reductant (specific activity, 3 nmol min-1 mg of protein-1), whereas crude extracts of nifF mutant were almost inactive (specific activity, 0.05). However, activity in the latter extracts was stimulated by adding Azotobacter chroococcum flavodoxin (specific activity, 10). Thus, nifF mutants may lack an electron transport factor. Crude extracts of nifJ mutants had about 20% of the wild-type level of active MoFe protein, and thus nifJ has a presumptive role in maintaining active MoFe protein. Studies on pyruvate or formate as reductants for nitrogenase in extracts of the nifJ mutants suggest in addition a role in electron input to nitrogenase for the following reasons. (i) Nitrogenase activity with these reductants was very low (specific activity, 0.06) and was not stimulated by extra MoFe protein or the flavodoxin. (ii) Activity was increased by adding a crude extract of a mutant lacking the structural nif genes (specific activity, 1) or a crude extract of the nifF mutant (specific activity, 4).     

Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae.

Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae has been demonstrated. Studies on the oxygen regulation of nifB-lacZ and nifH-lacZ fusions in the presence of the nifLA operon, which contains either an intact or a deleted nifL gene, indicate that possibly both the nifL promoter and the nifL product are responsible for nif repression by oxygen.     

MALDI-TOF MS用于肺炎克雷伯菌同源性分析的初步研究

目的评价基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight mass spectrometry,MALDI-TOF MS)对肺炎克雷伯菌(Klebsiella pneumoniae,KPN)同源性分析的能力。方法对2013年2-4月青岛大学附属医院重症监护病房(ICU)分离的7株KPN和其他科室分离的13株KPN进行溯源性回顾分析,采用方法为脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)、多位点序列分型(multilocus sequence typing,MLST)和MALDI-TOF MS。结果 PFGE和MLST结果一致,MALDI-TOF MS同源性分析中将20株菌株分为2类,Ⅰ类为来自ICU的7株菌,Ⅱ类为其他科室的13株菌,与前2种方法得到的结果基本一致。结论 MALDI-TOF MS技术能够准确鉴定肺炎克雷伯菌且可对其进行同源性分析,较其他同源性分析方法快捷、方便,可满足临床对院感工作的需求。     

肺炎克雷伯菌基因组可移动遗传元件的研究进展

肺炎克雷伯菌是目前临床上最主要的耐药致病菌之一,对人类健康造成了很大威胁.近年来,细菌耐药成为治疗肺炎克雷伯菌感染的主要难题,尤其是高毒力、高耐药性肺炎克雷伯菌的出现对临床工作造成了巨大挑战,而研究表明其耐药基因和毒力基因主要由可移动遗传元件携带而传播.因此,为了更好地认识及防控肺炎克雷伯菌感染,本文对肺炎克雷伯菌基因...     

肺炎克雷伯菌的分布特点耐药性分析

目的分析2010年至2012年房县人民医院患者肺炎克雷伯菌的耐药特点,为临床合理用药提供依据。方法对2010年至2012年该院各类感染患者标本中分离获得的肺炎克雷伯菌,采用纸片扩散法（K—B法）检测抗菌药物的敏感性,产超广谱β-内酰胺酶（ESBLs）检测采用ESBLs表型筛选与确证试验,并用WHONET5．3软件对药敏结果进行统计分析。结果3年分离的肺炎克雷伯菌共计248株,分离出产ESBLs菌株102株,其对亚胺培南、美洛培南无耐药,对头孢哌酮／舒巴坦、哌拉西林／三唑巴坦、阿米卡星和头孢替坦的耐药率较低,对其他抗菌药物的耐药率均较高。结论肺炎克雷伯菌对多种抗菌药物均具有较高的耐药性,临床上治疗该菌感染时应根据药敏与ESBLs检测结果选择抗菌药物。     

Metabolism of acrylonitrile by Klebsiella pneumoniae

A gram-negative rod-shaped bacterium capable of utilizing acrylonitrile as the sole source of nitrogen was isolated from industrial sewage and identified as Klebsiella pneumoniae. The isolate was capable of utilizing aliphatic nitriles containing 1 to 5 carbon atoms or benzonitrile as the sole source of nitrogen and either acetamide or propionamide as the sole source of both carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae was capable of hydrolyzing 6.15 mmol of acrylonitrile to 5.15 mmol of acrylamide within 24 h. The acrylamide was hydrolyzed to 1.0 mmol of acrylic acid within 72 h. Another metabolite of acrylonitrile metabolism was ammonia, which reached a maximum concentration of 3.69 mM within 48 h. Nitrile hydratase and amidase, the two hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds, were induced by acrylonitrile. The optimum temperature for nitrile hydratase activity was 55°C and that for amidase was 40°C; both enzymes had pH optima of 8.0.Abbreviations PBM phosphate buffered medium - GC gas chromatography - GC/MS gas chromatography/mass spectrometry