Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis

Promoter-probe and terminator-probe plasmid vectors make possible to rapidly examine whether particular sequences function as promoter or terminator signals in various genetic backgrounds and under diverse environmental stimuli. At present, such plasmid-based genetic tools are very scarce in the Gram-positive pathogenic bacteria Streptococcus pneumoniae and Enterococcus faecalis. Hence, we developed novel promoter-probe and terminator-probe vectors based on the Streptococcus agalactiae pMV158 plasmid, which replicates autonomously in numerous Gram-positive bacteria. As reporter gene, a gfp allele encoding a variant of the green fluorescent protein was used. These genetic tools were shown to be suitable to assess the activity of promoters and terminators (both homologous and heterologous) in S. pneumoniae and E. faecalis. In addition, the promoter-probe vector was shown to be a valuable tool for the analysis of regulated promoters in vivo, such as the promoter of the pneumococcal fuculose kinase gene. These new plasmid vectors will be very useful for the experimental verification of predicted promoter and terminator sequences, as well as for the construction of new inducible-expression vectors. Given the promiscuity exhibited by the pMV158 replicon, these vectors could be used in a variety of Gram-positive bacteria.     

Isolation of a temperature-sensitive dnaA mutant of Staphylococcus aureus

Of 750 temperature-sensitive mutants of Gram-positive Staphylococcus aureus, one was complemented by the dnaA gene. This mutant had a single base transition in the dnaA gene causing the amino-acid substitution mutation, Ala40Thr. Phage transduction experiments showed that this temperature-sensitive phenotype was linked with a drug-resistant marker inserted near the dnaA gene, suggesting the dnaA mutation is responsible for the phenotype. Flow cytometric analysis revealed that the dnaA mutant was unable to initiate DNA replication at a restrictive temperature and exhibited asynchrony in the replication initiation at a permissive temperature. This is the first report of a temperature-sensitive dnaA mutant in S. aureus, and the results show that DnaA is required for the initiation of chromosomal replication and for the regulation of synchrony in the bacterial cells.     

Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.Communicated by H. Ikeda     

Plasmid maintenance in Bacillus stearothermophilus is strain-dependent

We studied the segregational stability of plasmids based on pTB913, a 4.5-kb rolling-circle plasmid derived from the thermophilic Bacillus plasmid pTB19. In Bacillus stearothermophilus the stability of pTB913 derivatives appeared to be strain-dependent. In strain CU21 large amounts of single-stranded pTB913 DNA were found and the plasmid was highly unstable at 57 degrees C. In strain NUB3621, however, very low amounts of single-stranded plasmid DNA were formed and pTB913-based replicons were only slightly unstable at 57 degrees C. The NUB3621/pTB913 host-vector system seems appropriate for molecular cloning. A RepA-based replicon, also derived from pTB19 but replicating by a theta mechanism, was highly unstable in B. stearothermophilus NUB3621.     

pNK289衍生质粒在牙孢杆菌中的分离稳定性

报道关于一系列pNK289衍生质粒分离稳定性研究结果。这些起源相同的质粒在Bacillus,subtilis AS1.1176中的分离稳定性存在差异,这种差异与质粒的大小和复制方式无关,而与质粒的考贝数有一定的关系。由于不稳定质粒pNK219在B.subtilis BD224宿主中能稳定遗传,所以推测宿主的遗传背景可能影响质粒的分离稳定性,这些研究不仅为进一步寻找与pNK289衍生质粒稳定性相关的基因奠定了基础,而且为在芽孢杆中构建稳定的重组质粒提供了理论依据。     

重庆地区儿童2000年至2004年首位革兰阴性细菌和阳性细菌的耐药性监测

目的了解重庆地区儿童感染的分离至临床标本的首位革兰阴性细菌和阳性细菌对常用抗生素的耐药趋势,指导临床合理使用抗生素。方法常规方法分离、培养细菌,应用美国德灵公司WalkAway-40细菌鉴定仪对2000年至2004年我院细菌室分离至临床标本的首位革兰阴性细菌和阳性细菌共2854株进行细菌鉴定及药敏试验。结果2000年至2004年检出的首位革兰阴性细菌和阳性细菌分别为大肠埃希菌和金黄色葡萄球菌。2000年至2004年前5位革兰阴性菌5777株,革兰阳性菌1565株,其中大肠埃希菌2090株,金黄色葡萄球菌764株,分别占36.2%和48.8%;5年间大肠埃希菌对氨苄西林、头孢吡肟、头孢西丁、庆大霉素、亚胺培南、环丙沙星、头孢噻肟、头孢他啶的总耐药率分别为80.9%、37.5%、15.4%、54.0%、0.8%、34.0%、46.6%、46.2%;金黄色葡萄球菌对青霉素、红霉素、复方新诺明、万古霉素、阿莫西林/克拉维酸的总耐药率分别为95.6%、63.4%、5.8%、0%、11.0%。结论通过细菌耐药监测发现：大肠埃希菌对常用抗生素的总耐药率变化不大,金黄色葡萄球菌对常用抗生素的总耐药率有下降趋势,应引起临床医生重视。     

Gut immune stimulation by non pathogenic Gram(+) and Gram(-) bacteria. Comparison with a probiotic strain

We analyzed the gut immune stimulation induced by Gram-positive bacteria: non probiotic Lactobacillus acidophilus CRL 1462 and Lactobacillus acidophilus A9; two potentially probiotic strains: L. acidophilus CRL 924 and Lactobacillus delbrueckii subsp. bulgaricus CRL 423; comparatively with a probiotic strain: Lactobacillus casei CRL 431. We also studied Gram-negative bacteria: Escherichia coli 129 and E. coli 13-7 in BALB/c mice. All the strains increased the number of IgA+ cells. We analyzed the cytokines IFNγ, TNFα, IL-17, IL-12, IL-6 and MIP-1α. The Gram(+) strains increased the number of IL-10+ cells. Gram(−) strains did not increase IL-10+ cells, but they increased the number of IL-12+ cells. The probiotic strain increased mainly IFNγ and TNFα. In the study of the receptors TLR-2, TLR-4 and CD-206, we demonstrated that only the probiotic strain increased the number of CD-206+ cells. All the Gram(+) strains increased the number of TLR-2+ cells and the Gram(−) strains of the TLR-4+ cells. The probiotic strain induced the release of IL-6 by a preparation enriched in intestinal epithelial cells (IEC). Gram(+) and Gram(−) bacteria activated different immune receptors and induced a different cytokine profile. The probiotic strain showed a great activity on the immune cells and the enriched population in IEC, activating mainly cells of the innate immune system.     

革兰氏阳性菌蛋白结构域特征分析

结构域是蛋白质序列中具有独特功能的区域,这些区域影响着蛋白质的功能,因此研究结构域的特征对于了解蛋白质功能很有帮助。构建革兰氏阳性菌蛋白质4个亚细胞位置数据集,对该数据集中的蛋白质进行结构域的搜索和功能分析,找到了革兰氏阳性菌的细胞壁、细胞质、细胞膜和细胞外四个蛋白质区域的结构域。分析这四个位置结构域的功能并在PDBsum数据库中找到了这些结构域的二级结构和三级结构图,利用这些特征信息可以更深入的了解革兰氏阳性菌蛋白质的结构和功能。     

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences. Received: 13 January 1998 / Accepted: 10 June 1998     

Gene arrangements and branching orders of gram-positive bacteria

The availability of complete genomic sequence data allows one to develop new methods of reconstructing phylogenetic trees. A simple method of reconstructing branching orders based on gene transposition (or lateral transfer) is presented. It is argued that specific gene arrangements on four different genomes could determine a branching order. A computer search for such gene arrangements was carried out against gene order data of completely sequenced Gram-positive bacteria. Gene arrangements around ribosomal protein S4 gene, murC (UDP-N-acetylmuramate:alanine ligase) gene and dnaE (DNA polymerase III alpha chain) gene each suggest a branching order in which actinobacteria with a high genomic G+C content first branched off from other Gram-positives with a low G+C content and then a split occurred between Mycoplasma species and a group closely related to Bacillus subtilis. A recently sequenced thermophilic bacterium Thermoanaerobacter tengcongensis is suggested to have branched off from the lineage leading to the low G+C Gram-positives prior to the split between the Mycoplasma and Bacillus groups. By contrast to the indel analysis in which a single evolutionary event of insertion or deletion of a signature sequence is assumed, the present method does not necessarily require such a parsimonious assumption of gene transposition.     

Water-soluble phosphate prodrugs of pleuromutilin analogues with potent in vivo antibacterial activity against Gram-positive pathogens

A phosphate prodrug strategy was investigated to address the problem of poor aqueous solubility of pleuromutilin analogues. Water-soluble phosphate prodrugs 6a, 6b and 6c of pleuromutilin analogues were designed and synthesized. Three compounds all exhibited excellent aqueous solubility (>50 mg/mL) at near-neutral pH and sufficient stability in buffer solution. In particular, the phenol pleuromutilin prodrug 6c displayed favourable pharmacokinetic profiles and comparable potency with vancomycin against MSSA and MRSA strains in vivo.     

Construction and use of a new vector/transposon, pLBT::mim-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium

Abstract The previously described pLOFKm transposon delivery plasmid (J. Bacteriol. (1990) 172, 6557–6567) was engineered such that a promoterless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.     

上海某三甲医院2014—2021年血流感染病原菌的分布及耐药性分析

本研究旨在分析血流感染病原菌种类分布及其耐药率,为临床合理使用抗菌药物提供参考。对2014—2021年同济大学附属东方医院血培养标本分离的主要病原菌及其耐药性进行统计分析,结果显示剔除重复菌株后,本院8年血培养标本共检出病原菌1 396株,以革兰氏阴性杆菌为主,占56.3%,其次为革兰氏阳性球菌(38.4%)和真菌(5.3%)。居前8位的菌株为大肠埃希菌(21.5%)、凝固酶阴性葡萄球菌(coagulase negative Staphylococcus, CNS) (17.4%),肺炎克雷伯菌(14.9%)、金黄色葡萄球菌(7.2%),鲍曼不动杆菌(5.4%),粪肠球菌(4.2%),屎肠球菌(3.7%)和铜绿假单胞菌(3.5%)。大肠埃希菌对第3代头孢菌素的耐药率超过50%,对碳青霉烯类药物的耐药率低至1%;肺炎克雷伯菌对碳青霉烯类药物的耐药率高达46.3%;鲍曼不动杆菌对头孢类、氨基糖苷类和碳青霉烯类的累计耐药率均大于45%。耐甲氧西林金黄色葡萄球菌和耐甲氧西林凝固酶阴性葡萄球菌(methicillin-resistant coagulase-negative Staphylococci, MRCNS)分别为44.6%和74.5%的占比。     

Physical evidence for plasmids in autotrophic,especially hydrogen-oxidizing bacteria

Agarose gel electrophoresis of crude lysates from 23 species of autotrophic bacteria revealed plasmids of various sizes in 12 species. The plasmid pattern varied considerably. While the majority of the plasmid-bearing species harbored one or two plasmids, one species, Alcaligenes latus, exhibited more than six ccc-DNA bands. With one exception the molecular masses of the plasmids were 50×10⁶ or higher. In Achromobacter carboxydus, Alcaligenes latus, Derxia gummosa and three strains of Paracoccus denitrificans large plasmids of molecular masses higher than 300×10⁶ were resolved. The examination of Thiobacillus A2 resulted in the discovery of two plasmids while Pseudomonas oxalaticus was apparently free of resident plasmid DNA. So far these plasmids can only be characterized as cryptic. Future studies may allow to correlate them with specific metabolic activities of their hosts such as the ability to grow on carbon monoxide or thiosulfate, to fix molecular nitrogen and to form soluble NAD-reducing and/or membrane-bound hydrogenases.     

SecretP: Identifying bacterial secreted proteins by fusing new features into Chou’s pseudo-amino acid composition

Protein secretion plays an important role in bacterial lifestyles. Secreted proteins are crucial for bacterial pathogenesis by making bacteria interact with their environments, particularly delivering pathogenic and symbiotic bacteria into their eukaryotic hosts. Therefore, identification of bacterial secreted proteins becomes an important process for the study of various diseases and the corresponding drugs. In this paper, fusing several new features into Chou’s pseudo-amino acid composition (PseAAC), two support vector machine (SVM)-based ternary classifiers are developed to predict secreted proteins of Gram-negative and Gram-positive bacteria. For the two types of bacteria, the high accuracy of 94.03% and 94.36% are obtained in distinguishing classically secreted, non-classically secreted and non-secreted proteins by our method. In order to compare the practical ability of our method in identifying bacterial secreted proteins with those of six published methods, proteins in Escherichia coli and Bacillus subtilis are collected to construct the test sets of Gram-negative and Gram-positive bacteria, and the prediction results of our method are comparable to those of existing methods. When performed on two public independent data sets for predicting NCSPs, it also yields satisfactory results for Gram-negative bacterial proteins. The prediction server SecretP can be accessed at http://cic.scu.edu.cn/bioinformatics/secretPV2/index.htm.