Transformation of Gram-positive microorganisms with the Gram-negative broad-host-range cosmid vector pJRD215

Abstract In an in vitro direct assay with tissue-type plasminogen activator (tPA), plasminogen and the chromogenic substrate S-2251, the ability of Mycoplasma fermentans KL4 to stimulate tPA-mediated activation of plasminogen to plasmin was studied. Mycoplasma cells markedly enhanced the activation of plasminogen by tPA in a concentration-, temperature- and pH-dependent manner. Nonidet P-40 (0.01%), sonication, and freezing and thawing of the cells substantially increased the stimulatory effect of mycoplasma on tPA activity. In contrast, the activation of plasminogen by urokinase was refractory to mycoplasma cells. The mycoplasma-mediated stimulation of tPA activity was prevented by ϵ-aminocaproic acid (EACA), a lysine analogue known to block lysine-binding sites (LBS) in plasminogen and tPA. Among several Mycoplasma fermentans strains tested, incognitus strain demonstrated the highest stimulation activity. These results suggest that mycoplasma cells interact with LBS in tPA and plasminogen to enhance plasminogen activation.     

2008-2009年南京地区儿童肺炎支原体感染情况分析

目的了解2008年至2009年南京地区儿童呼吸道肺炎支原体（MP）的感染情况。方法应用MP快速培养法对南京地区2008年1月至2009年12月980例急性呼吸道感染患儿的咽拭子标本进行MP培养检测。结果 980例急性呼吸道感染患儿的咽拭子标本中MP培养阳性384例,总阳性率为39.2%。其中〈3岁、35岁、〉5岁各年龄组患儿中MP阳性率分别是36.9%,38.5%和43.3%;不同季度MP感染的检出率分别是：春季（13月）45.7%,夏季（46月）24.8%,秋季（79月）20.4%,冬季（1012月）50.3%,其中冬、春两季MP感染的检出率明显高于其他两季（P〈0.01）。结论 MP为南京地区儿童急性呼吸道感染的重要病原体,各年龄儿童普遍易感;冬、春两季高发。     

Characterization of a Mycoplasma hominis gene encoding lysyl-tRNA synthetase (LysRS)

Abstract The gene encoding lysyl-tRNA synthetase ( lysS ) in Mycoplasma hominis was cloned and sequenced. The gene was found to have an open reading frame of 1466 bp encoding a polypeptide with a predicted molecular mass of 57 kDa. The amino acid sequence showed 44.3% and 43.7% identity to the Escherichia coli lysyl-tRNA synthetases, encoded by lysS and lysU . Only one lysyl-tRNA synthetase encoding gene was found in M. hominis . The G+C content of the gene was found to be 28.6%, which is significantly lower than in other prokaryotes. The gene was located 4 kb upstream of the M. hominis PG21 rRNA B operon.     

Cloning and sequence analysis of the aminopeptidase My gene from Mycoplasma salivarium

Abstract The complete nucleotide sequence of a major component of aminopeptidase My purified from Mycoplasma salivarium was determined. The protein gene encoded a protein consisting of 520 amino acids with a molecular mass of 58079 Da. The protein contained two tryptophan residues, one of which was encoded by UGA. A computer-aided homology search suggested that aminopeptidase My had properties similar to those of leucine aminopeptidase (EC 3.4.11.1).     

表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌对小鼠的免疫原性

【目的】构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,对重组菌株的生物学特性以及对小鼠的免疫原性进行研究。【方法】分别将猪肺炎支原体的免疫原性基因p36、p46、p65和p97R1-Nrdf克隆到pYA3493,得到重组质粒pYA-36、pYA-46、pYA-65和pYA-97R1-Nrdf。重组质粒和空质粒pYA3493分别电转asd基因缺失株C500ˉ,获得重组菌株C36(pYA-36)、C46(pYA-46)、C65(pYA-65)、C97R1-Nrdf(pYA-97R1-Nrdf)和空质粒菌株CpYA(pYA3493)。研究重组菌株的生物学特性,并以小鼠为动物模型评价重组菌株在口服、肌注两种不同免疫途径下的免疫原性。【结果】成功构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,重组菌株能表达外源蛋白,生化和生长特性未发生改变,插入的外源基因亦稳定存在。小鼠的免疫原性结果显示:口服C36+C46+C65+C97R1-Nrdf组的猪肺炎支原体抗体极显著高于口服C36+C46+C65组和肌注商品疫苗组(P<0.01),但与肌注C36+C46+C65组无显著性差异(P>0.05);IFN-γ为肌注C36+C46+C65组显著高于肌注商品疫苗组(P<0.05),而与口服C36+C46+C65或C36+C46+C65+C97R1-Nrdf组差异均不显著(P>0.05);IL-4水平为口服C36+C46+C65组>口服C36+C46+C65+C97R1-Nrdf组>肌注商品疫苗组>肌注C36+C46+C65组,但各组之间差异均不显著(P>0.05)。对照组的猪肺炎支原体抗体、IFN-γ以及IL-4均与试验组差异极显著(P<0.01)。【结论】构建的表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,采用肌注免疫时具有较好的免疫原性,有望发展为猪肺炎支原体的基因工程疫苗。     

Culture-independent multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae

A PCR approach for multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae directly from respiratory samples was developed and evaluated on 54 specimens from M. pneumoniae-positive pneumonia patients. The method resulted in a clearly identifiable MLVA type in all samples tested and can be used for MLVA without laborious isolation of the pathogen.     

Quantification of mRNA and protein and integration with protein turnover in a bacterium

Biological function and cellular responses to environmental perturbations are regulated by a complex interplay of DNA, RNA, proteins and metabolites inside cells. To understand these central processes in living systems at the molecular level, we integrated experimentally determined abundance data for mRNA, proteins, as well as individual protein half‐lives from the genome‐reduced bacterium Mycoplasma pneumoniae. We provide a fine‐grained, quantitative analysis of basic intracellular processes under various external conditions. Proteome composition changes in response to cellular perturbations reveal specific stress response strategies. The regulation of gene expression is largely decoupled from protein dynamics and translation efficiency has a higher regulatory impact on protein abundance than protein turnover. Stochastic simulations using in vivo data show how low translation efficiency and long protein half‐lives effectively reduce biological noise in gene expression. Protein abundances are regulated in functional units, such as complexes or pathways, and reflect cellular lifestyles. Our study provides a detailed integrative analysis of average cellular protein abundances and the dynamic interplay of mRNA and proteins, the central biomolecules of a cell.     

滑液支原体NADH氧化酶的酶学活性及亚细胞定位研究

【背景】许多研究表明,支原体的NADH氧化酶(NADH oxidase,NOX)不仅在胞浆中发挥生物酶学功能,也存在于细胞膜上发挥黏附宿主细胞功能。【目的】对滑液支原体(Mycoplasma synoviae,MS)的NOX进行酶学活性及亚细胞定位研究,分析其在MS致病过程中的潜在作用。【方法】对MS的NOX蛋白进行原核表达、纯化,然后对重组MSNOX (rMSNOX)的酶学活性及影响酶活的条件进行研究,测定其酶比活力、米氏常数及最大反应速率,接着用MS阳性血清及制备的rMSNOX兔多克隆抗体,分别与rMSNOX蛋白及MS全菌、膜蛋白和胞浆蛋白进行Western blotting反应,鉴定rMSNOX的免疫原性及其在MS中的分布情况。【结果】在大肠杆菌BL21(DE3)中成功表达rMSNOX蛋白,相对分子质量约为53 kD,并获得纯化的rMSNOX蛋白;酶活测定显示rMSNOX蛋白的酶比活力为14.17IU/mg,最适酶促温度为37℃,最适pH为7.5,双倒数法求得rMSNOX的最大反应速率V_(max)为21.8μmol/(L·min),米氏常数K_m(NADH)为244.0μmol/L;rMSNOX蛋白与MS阳性血清特异性结合,证明具有良好的免疫原性;亚细胞定位研究表明NOX蛋白主要存在于MS的胞浆中,细胞膜上有少量的分布。【结论】首次证实MS的NOX不仅具有NADH氧化酶活性,也是MS具有免疫原性的膜蛋白,为进一步探索NOX在MS致病过程中的作用提供了分子基础。