Molecular modeling and dynamics studies of purine nucleoside phosphorylase from Bacteroides fragilis

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the purine base. This work describes for the first time a structural model of PNP from Bacteroides fragilis (Bf). We modeled the complexes of BfPNP with six different ligands in order to determine the structural basis for specificity of these ligands against BfPNP. Comparative analysis of the model of BfPNP and the structure of HsPNP allowed identification of structural features responsible for differences in the computationally determined ligand affinities. The molecular dynamics (MD) simulation was assessed to evaluate the overall stability of the BfPNP model. The superposition of the final onto the initial minimized structure shows that there are no major conformational changes from the initial model, which is consistent with the relatively low root mean square deviation (RMSD). The results indicate that the structure of the model was stable during MD, and does not exhibit loosely structured loop regions or domain terminals.     

新疆哈萨克族正常血压人群和高血压人群肠道菌群中拟杆菌属、梭菌属结构特征分析

目的分析新疆哈萨克族正常血压人群和高血压人群肠道菌群中拟杆菌属、梭菌属的结构特征,探讨两人群肠道两菌属的差异。方法使用16S DNA-PCR-DGGE技术比较哈萨克族正常血压人群和高血压人群肠道菌群中两菌属结构的差异,将差异条带克隆、测序,与GenBank数据库提供的序列进行比对,确定细菌种类。结果哈萨克族高血压人群肠道梭菌属的16S DNA V3区DGGE图谱中出现一条优势带,其在2组人群中出现的频率经分析后差异具有统计学意义(P0.05)。基因序列在GenBank数据库上用Blast程序进行比对,确定为Uncul-tured bacterium clone nbw1009b01c1。结论这种细菌可能会影响哈萨克族高血压的发生和发展。     

16S rRNA PCR鉴定脆弱类杆菌

目的:应用16SrRNA序列设计PCR引物鉴别脆弱类杆菌。方法:通过脆弱类杆菌16SrRNA序列特异性位点设计引物,对4株脆弱类杆菌及大肠杆菌、乳酸杆菌、嗜热链球菌等进行PCR扩增。应用琼脂糖电泳法对PCR扩增产物进行特异性检测。结果:脆弱类杆菌在176bp左右出现特异性条带,而其他细菌均未出现特异性条带。结论:通过16SrRNA序列中特异位点设计引物进行PCR,可特异性鉴定脆弱类杆菌。     

Immunization with outer-membrane protein(s) of Bacteroides fragilis: an experimental study in mice

A crude outer-membrane protein (OMP) preparation from a strain of Bacteroides fragilis, grown in supplemented brain-heart infusion broth, was tested for its protective effect against subcutaneous infection in mice. Immunization with six doses, each of 100, 150 or 200 g OMP preparation, gave some protection: abscesses completely disappeared 15 to 22 days after immunization. In non-immunized animals and animals immunized with doses of 10, 20, 40 or 80 g each, well demarcated abscesses were seen beyond day 22 post-immunization. Although crude OMP elicited good antibody response, with maximum titres on day 4 post-immunization, high titres could not be associated with healing of the abscesses.     

Isolation and characterization of naturally occurring hairpin structures in single-stranded DNA of coliphage M13

With precise conditions of digestion with single-strand-specific nucleases, namely, endonuclease S1 of $\text{Aspergillus oryzae}$ and exonuclease I of $\text{Escherichia coli}$, nuclease-resistant DNA cores can be obtained reproducibly from single-stranded M13 DNA. The DNA cores are composed almost exclusively of two sizes (60 and 44 nucleotides long). These have high (G + C)-contents relative to that of intact M13 DNA, and arise from restricted regions of the M13 genome. The resistance of these fragments to single-strand-specific nucleases and their nondenaturability strongly suggest the presence of double-stranded segments in these core pieces. That the core pieces are only partially double-stranded is shown by their lack of complete base complementarity and their pattern of elution from hydroxyapatite.     

Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis

The Bacteroides fragilis enterotoxin is an extracellular zinc metalloprotease that has been implicated in diarrheal disease of humans and animals. This toxin causes fluid accumulation in intestinal loops and is cytotoxic for HT-29 cells, an intestinal carcinoma cell line. Here we report the cloning and sequencing of the toxin gene (bftP). bftP is 1191 nucleotides coding for a 397 amino acid protein of 44.4 kDa. The toxin has a signal peptide of 18 amino acids that is typical of many lipoproteins followed by a 379 amino acid protoxin. The portion of the protoxin found in culture filtrates and stools begins at amino acid 212. An additional open reading frame located immediately upstream shows some sequence identity with cobra cytotoxins. If expressed, the ORF protein product could also play a role in the virulence of B. fragilis.     

Isolation and characterization of enterotoxigenic Escherichia coli mutants that produce abnormal heat-labile enterotoxins

Abstract Bacteroides fragilis populations were separated according to the size of surface structure. Subculture of the separated populations produced cultures enriched for 3 different structures; a large capsule, a small capsule and an electron-dense layer (EDL). The ability of these subpopulations to haemagglutinate (HA) erythrocytes from a number of species was examined. Populations which produced either a large or s small capsule did not have HA activity, whereas those with an extracellular EDL did. By mixing populations with EDL and those with either the large or small capsule, the degree of HA could be altered. HA was dependent on the proportion of EDL-bearing bacteria present. Fimbriae were not observed on electron microscopy.     

Several regions of the repeat domain of the Staphylococcus caprae autolysin,AtlC, are involved in fibronectin binding

The chromosome of Bacteroides fragilis strain YCH46 was shown to be a single circular DNA molecule of about 5.3 Mb having 16 NotI, seven AscI, and six I-CeuI sites. A physical map of the chromosome was constructed by four independent experimental approaches: linking clone analysis, cross-Southern hybridization, partial restriction digestion, and two-dimensional pulsed-field gel electrophoresis. Six rRNA operons and 10 known genes were localized on the physical map.     

Characterisation of Bacteroides from sheep periodontal disease by SDS-PAGE of outer membrane proteins

Isolates of Bacteroides species obtained from a longitudinal study of developing periodontal disease in sheep were analysed by SDS-PAGE. Protein profiles of Sarkosyl-insoluble outer membrane extracts were compared within groups of isolates which had already been defined by conventional biochemical techniques. Heterogeneity was exhibited within most groups. Isolates of B. gingivalis and B. asaccharolyticus shown to be similar to human isolates by conventional biochemical tests, gave different protein profiles from the respective type cultures. The sheep B. gingivalis-like isolates were however homogeneous, while the B. asaccharolyticus-like organisms could be divided into 3 subgroups. SDS-PAGE appears to be a useful tool for the examination of bacterial flora and recognition of subgroups of subspecies.