Evidence for binding of at least two factors, including T-rich strand-binding factor(s) to the single-stranded ARS1 sequence in Saccharomyces cerevisiae.

Summary To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors interacting with the ARS1 region located near the centromere of chromosome IV in Saccharomyces cerevisiae. Using the gel shift assay, we found protein factor(s) which specifically bound to the T-rich strand of the region containing the core consensus and its flanking sequences in ARS1, but not to the opposite strand. We designated this factor ATS (ARS1, T-rich strand-binding factor(s)). Similar specific complexes were also detected with oligonucleotide probes specific for the H4 or C2G1 ARS. As we have previously identified another binding factor, we conclude that at least two factors bind to the single-stranded ARS1 sequence.     

DNA vaccines based on genetically detoxified derivatives of pneumolysin fail to protect mice against challenge with Streptococcus pneumoniae

The 7-valent polysaccharide conjugate vaccine currently administered against Streptococcus pneumoniae has been shown to be highly effective in high risk-groups, but its use in developing countries will probably not be possible due to high costs. The use of conserved protein antigens using the genetic vaccination strategy is an interesting alternative for the development of a cost-effective vaccine. We have analyzed the potential of DNA vaccines expressing genetically detoxified derivatives of pneumolysin (pneumolysoids) against pneumococcal infections, and compared this with immunization using recombinant protein. The purified recombinant pneumolysoid with the highest residual cytolytic activity was able to confer partial protection against a lethal intraperitoneal challenge, with the induction of high antibody levels. Immunization with DNA vaccines expressing pneumolysoids, on the other hand, induced a significantly lower antibody response and no protection was observed.     

Protective efficacy of PspA (pneumococcal surface protein A)-based DNA vaccines: contribution of both humoral and cellular immune responses

Streptococcus pneumoniae is a major public health problem and new strategies for the development of cost-effective alternative vaccines are important. The use of protein antigens such as PspA (pneumococcal surface protein A) is a promising approach to increase coverage at reduced costs. We have previously described the induction of a strong antibody response by a DNA vaccine expressing a C-terminal fragment of PspA. Fusion of this fragment with the cytoplasmic variant of SV40 large T-antigen (CT-Ag) caused reduction in specific interferon-gamma produced by stimulated spleen cells. In this work we show that the DNA vaccine expressing the C-terminal region of PspA elicits significant protection in mice against intraperitoneal challenge with a virulent strain of S. pneumoniae. Furthermore, fusion with CT-Ag completely abrogated the protection elicited by DNA immunization with this fragment. In this case, protection did not correlate with total anti-PspA antibody production nor with total IgG2a levels. The anti-PspA sera obtained from both constructs showed equivalent opsonic activity of pneumococci, indicating that the antibodies produced were functional. We could, though, observe a correlation between a lower IgG1:IgG2a ratio, which is indicative of a stronger bias towards Th1 responses, and protection. We also show that a vector expressing the most variable N-terminal alpha-helical region induces higher antibody formation, with increased protection of mice against intraperitoneal challenge with a more virulent strain of S. pneumoniae. As a whole, these results indicate that antibodies elicited against PspA would not be solely responsible for the protection induced by DNA vaccination and that cell-mediated immune responses could also be involved in protection against pneumococcal sepsis.     

High efficiency transduction of single strand plasmid DNA into enteric bacteria

Summary This report demonstrates high efficiency transduction of enteric bacteria using single strand plasmids packaged in M13 phage capsids. Transformation by plasmid DNA is usually a very inefficient process in many enteric bacteria other than Escherichia coli K12. Plasmids carrying an M13 origin of replication can be replicated and packaged when cells carrying such plasmids are infected with M13 or a derivative helper phage. By introducing an F plasmid into E. coli, Serratia marcescens, Citrobacter freundii, and Enterobacter aerogenes, these species can now be infected at high efficiency with M13 phage and with packaged single strand plasmids, yielding an efficient method to introduce cloned DNA fragments into these bacteria. The titer of colony forming units in a lysate was essentially equivalent in all the bacteria, demonstrating an equal efficiency of transduction of these other enteric bacteria compared to E. coli.     

肺炎链球菌假想的溶菌酶样蛋白的活性分析和鉴定

目的 探索肺炎链球菌中一种假想的溶菌酶样蛋白的活性.方法 生物信息学分析该基因的功能结构域;利用长臂同源PCR对该基因进行敲除,观察D39野生菌和缺陷菌在生物学性状的改变;利用底物PNP-（GIcNAc）和溶壁微球菌,构建过表达载体,绘制生长曲线,对截断和全长蛋白的溶菌酶活性进行鉴定.结果 生物信息学分析结果显示该基因的编码产物为β-1,4-N-乙酰胞壁酸糖苷酶,属于糖基水解酶25家族;野生菌为长链生长,缺陷菌呈短链状;溶菌酶和假想的溶菌酶样蛋白均可使底物释放出游离的对硝基苯酚,A405nm吸光度值分别为1.166和0.792;同时也可使得溶壁微球菌发生溶解;含过表达质粒的肺炎链球菌较之野生菌,溶解较快.结论 假想的溶菌酶样蛋白具有溶菌酶活性,是一种新的溶菌酶.     

Replication of ColE2 and ColE3 plasmids In vitro replication dependent on plasmid-coded proteins

Summary We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.     

以gfp为报告基因的肺炎链球菌启动子诱捕文库的构建与分析

首先构建一个以gfp(green fluorescence protein)为报告基因的自杀质粒pEVP3-SDGFP,将肺炎链球菌基因组DNA的随机酶切片段(200bp～800 bp)克隆到该质粒gfp基因上游的多克隆位点,得到约58000个含有肺炎链球茵基因组DNA随机酶切片段的重组子,提取质粒即为质粒库,该库大约覆盖肺炎链球菌基因组全长的5倍,插入率达到90%以上,且有较强的随机性,质量较高.将该质粒库转化入肺炎链球菌TIGR4菌株,带有随机片段的报告质粒通过同源重组的方式将gfp基因融合于细菌染色体上该随机片段之后,利用质粒的抗生素抗性基因筛选出重组菌株,从而构建出相应的菌株库,共获得包含约500000个肺炎链球菌转化子的菌株库,经体内、外实验表明,其包含插入了S.pn体内、外表达基因片段的细菌,可以报告特定条件下的基因表达,并可通过流式细胞仪识别、分选.该文库的构建为进一步利用差异荧光诱导技术筛选肺炎链球菌体内诱导基因奠定了基础.     

Probing single-stranded DNA and its biomolecular interactions through direct catalytic activation of factor XII, a protease of the blood coagulation cascade

Triggered self-activation of factor XII, a blood coagulation protease, was utilized for the amplified visual detection of ss-DNA targets in a non-sequence specific way. Factor XII holds potential as a low-affinity and therefore non-interfering probe for DNA secondary structure and for the screening of protein binding to ss-DNA. The observation that ss-DNA also accelerates coagulation of human blood plasma is relevant to the emerging field of aptamer therapeutics.     

Characterization of the Replication Region of the Lactobacillus reuteri Plasmid pTC82 Potentially Used in the Construction of Cloning Vector

A 3.2 kb DNA fragment containing the replication region (RR) from pTC82 was cloned, sequenced, and found to contain elements typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR), including double-strand origin (DSO), replication protein gene (rep), and single-strand origin (SSO). The DSO of pTC82 contains two domains showing 55.5% and 84.6% similarities in nucleotide (nt) sequence to the conserved functional elements bind and nic, respectively, which are required for the initiation of the leading strand typical of the pC194-RCR family. Although the predicted rep gene product of pTC82 (Rep82) shares little identity (less than 24%) with other known Reps, a region containing three motifs, characteristic of the pC194-family Reps, was identified, indicating the Rep82 as a novel Rep protein of this family. Downstream of the rep82 gene, strong similarity to the typical palT type-SSO could be detected. This is the first palT type-SSO to be identified from Lactobacillus. Through a series of deletion studies, the minimal replicon of the cloned RR was found to be 2.66 kb in size including the DSO region and rep gene. This RR was further identified as being highly stable in L. reuteri and also bearing a very narrow host-range property, suggesting it to be a good replicon potentially useful in vector construction for developing L. reuteri as a vaccine carrier.     

Murine DNA cytosine C(5)-methyltransferase: in vitro studies of de novo methylation spreading

The preference of murine DNA (cytosine-5)-methyltransferase (Dnmt1) for single stranded DNA substrates is increased up to 50-fold by the presence of a proximal 5-methyl cytosine (5(me)C). This modulation is distance-dependent and is due to an enhanced binding affinity and minor changes in catalytic efficiency. No modulation was observed with double stranded DNA. Modulation requires that the 5(me)C moiety be attached to the DNA strand containing the CpG methylation target. Our results support a model in which 5(me)C binding by the enzyme occurs to at least one site outside the region involved in CpG recognition. No modulation in response to 5(me)C is observed with the bacterial enzyme M.SssI, which lacks the large N-terminal regulatory domain found in Dnmt1. We suggest that this allosteric modulation involves the N-terminal domain of Dnmt1.     

Rapid identification of Streptococcus pneumoniae by PCR amplification of ribosomal DNA spacer region

Abstract Streptococcus pneumoniae is one of the important human pathogens in clinical microbiology. A polymerase chain reaction assay was designed to detect and identify S. pneumoniae through amplification of the ribosomal DNA spacer regions between the pneumococcal 16S-23S ribosomal RNA genes. Thirty-two Streptococcus and non- Streptococcus strains were tested to verify the specificity of the assay, and only S. pneumoniae strains gave a positive reaction. This method is a powerful technique for the rapid identification of S. pneumoniae .     

儿童感染肺炎链球菌的青霉素结合蛋白基因突变分析

目的 分析儿童感染肺炎链球菌的青霉素结合蛋白基因突变与青霉素耐药水平之间的关系。方法 自2012年1月至2014年12月期间分离的1 317株肺炎链球菌中随机抽取出青霉素MIC=2.0 µg/mL、4.0 µg/mL、≥8.0 µg/mL各20株共60株作为实验菌株,采用PCR方法对实验菌株进行青霉素结合蛋白PBP1a、PBP1b、PBP2a、PBP2b、PBP2x、PBP3的基因扩增,扩增产物进一步纯化和测序,测序结果与青霉素敏感肺炎链球菌R6就国际上公认的PBPs保守序列进行比对分析。结果 60株肺炎链球菌的PBP2b、PBP1a、PBP2x、PBP2a基因的保守区或保守区附件均发现氨基酸突变,未发现PBP3与PBP1b突变。中介与耐药菌株基因突变位点存在重合,主要出现在单一的PBP1a序列的370STMK模体元件内Thr371Ser置换突变或伴有PBP2b序列的Thr451Ala/Ser和Ala624Gly置换突变,同时PBP2a序列的465SLN模体元件前置位发生Ser461Ala的置换突变。结论 肺炎链球菌对青霉素中、高水平耐药菌株绝大部分合并有不同PBP序列中4~6个氨基酸的置换突变,但合并多个氨基酸置换突变并非必然引起耐药水平相应升高。中、高水平耐药与PBP1a、PBP2b、PBP2a的变异关系密切,其中PBP1a的STMK保守区域Thr371Ser置换是引起耐药的主要因素之一。