Glycylprolyl dipeptidase activity of Bacteroides gingivalis W50 and the avirulent variant W50/BEI

Glycylprolyl dipeptidase activity was measured in cells, extracellular vesicles (ECV) and the soluble extracellular protein fraction (EP) of batch cultures of strains W50 and W50/BEI. Total culture enzyme activity of W50 dropped with age whilst that of W50/BEI remained constant. Activity was highest in the cellular fraction, greater for W50/BEI than W50 and rose with culture age. Both strains showed similar ECV activities but these declined with culture age. The EP glycylprolyl dipeptidase activity of W50/BEI in older cultures rose to a level 13-fold greater than W50. The majority of extracellular activity was represented by the ECV for strain W50 but by EP for W50/BEI. Variable but incomplete attenuation of activity was achieved by dithiothreitol. ECV and EP activities were associated with a high molecular mass fraction, but a smaller fraction (molecular mass 30,000) was detected in W50/BEI EP.     

Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.     

Polysaccharide utilization loci in Bacteroides determine population fitness and community-level interactions

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Use of a Bacteroides thetaiotaomicron-specific α-1-6, mannanase quantitative PCR to detect human faecal pollution in water

Aims: The aims of this work were to develop a quantitative test, based on Bacteroides thetaiotaomicron, for human faecal pollution in water and to evaluate test performance. Methods and Results: qPCR primers, based on the complete genomic sequence of B. thetaiotaomicron VPI 5482, were designed and tested. The single-copy putative mannanase homologue, α-1-6 mannanase, was selected as the particular target and sequences within this gene chosen as the qPCR primers by Blast search for specificity to B. thetaiotaomicron. The average concentration of B. thetaiotaomicron in human faeces was 1·39 × 10⁸ cells per gram faeces and the detection limit was 9·3 B. thetaiotaomicron copies per qPCR procedure. Comparison of B. thetaiotaomicron content in sewage vs pooled nonhuman faecal samples indicated that the current assay is specific for sewage. Conclusion: The subject assay is potentially useful for quantification of sewage pollution in water. Significance and Impact of the Study: Bacteroides-associated markers, proposed for faecal source tracking, have exclusively been based on gene sequences related to generally classified and uncultured bacteria. However, genes associated with host-microbe interaction have been suggested as more specific markers. The present assay targets such a gene of B. thetaiotaomicron which is considered to be a symbiont in the human gut.     

A silent carbapenemase gene in strains of Bacteroides fragilis can be expressed after a one-step mutation

High-level carbapenem-resistant (CpmR) mutants, with MICs for imipenem and carbapenem of greater than 128 micrograms/ml, were selected in vitro from four carbapenem-susceptible (CpmS) clinical isolates of Bacteroides fragilis. The CpmS strains produced very low levels of beta-lactamase activity, which was increased approx. 50- to 100-fold in the CpmR mutants. Isoelectric focussing and enzyme kinetic analysis (Km and Vrel) of the 'carbapenemases' from the CpmR mutants and similarly resistant clinical isolates suggested a close relatedness of the enzymes. A probe covering most of the cfiA gene encoding such an enzyme (Thompson, J.S. and Malamy, M.H. (1990) J. Bacteriol. 172, 2584-2593) hybridized with DNA from the CpmR mutants, their CpmS parental strains as well as clinical CpmR isolates, but not from randomly chosen carbapenem-susceptible strains. The possibility is considered that mutations leading to expression of the silent carbapenemase gene, and thereby to clinically relevant carbapenem resistance, may also occur in the clinical setting.     

Cloning and expression of the Bacteroides fragilis YCH46 neuraminidase gene in Eschirichia coli and Bacteroides uniformis

Abstract A neuraminidase-encoding gene nanH of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The nanH gene was subcloned from the cosmid and was located within a 2.2-kb Xho I- Kpn I fragment. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial chromosome. Neuraminidase activity expressed in the initial Escherichia coli clone was approximately 3600-fold lower than that expressed in B. fragilis YCH46. However, when nanH was transferred from E. coli to B. uniformis by mobilization of a shuttle plasmid, the transconjugant expressed 1100-fold higher activity than the E. coli donor did. These results suggest that modes of nanH expression in E. coli and Bacteroides are heterologous.     

Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces

Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source-tracking studies for identifying and quantifying sources of nonpoint fecal contamination. We present results using standard and real-time PCR to show cross-amplification of Bacteroides 16S rRNA gene molecular assays targeting human fecal pollution with fecal DNA from freshwater fish species. All except one of the presumptively human-specific assays amplified fecal DNA from at least one fish species, and one real-time PCR assay amplified DNA from all fish species tested. Sequencing of PCR amplicons generated from fish fecal DNA using primers from the real-time assay revealed no mismatches to the human-specific probe sequences, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria may be different strains. Our results strongly demonstrate the potential for cross-amplification of human-specific PCR assays with fish feces, and may call into question the results of studies in which these Bacteroides- specific molecular markers are used to quantify human fecal contamination in waters where fish contribute to fecal inputs.     

A Ribose-Scavenging System Confers Colonization Fitness on the Human Gut Symbiont Bacteroides thetaiotaomicron in a Diet-Specific Manner

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厌氧产黑色素杆菌群超微结构观察

厌氧产黑色素杆菌群在人口腔内的各处感染中起着重要作用，本文利用电子显微镜对八种产黑色素杆菌做了动态的超微结构观察，以进一步揭示菌体结构与其毒性的关系，为口腔感染疾患的细菌病因学机制提供超微形态学的理论依据。     

Genome Analysis of Bacteroides by Pulsed-field Gel Electrophoresis: Chromosome Sizes and Restriction Patterns

The chromosomal DNAs of nine strains of seven Bacteroides speciesincluding B. fragilis, the type species of the genus Bacteroides,were digested with rare-cutting restriction enzymes I-Ceu I,Not I, and Asc I and analysed by pulsed-field gel electrophoresis.The genome sizes of B. fragilis, B. distasonis, B. eggerthii,B. ovatus, B. thetaiotaomicron, B. uniformis, and B. vulgatuswere determined to be 5.3, 4.8, 4.4, 6.9, 4.8, 4.6, and 5.1Mbp, respectively. B. distasonis and B. vulgatus, and also B.uniformis and B. eggerthii, showed simillar I-Ceu I restrictionprofiles. I-Ceu I cut B. uniformis and B. eggerthii genomesinto four, B. ovatus into five, B. fragilis and B. thetaiotaomicroninto six, and B. distasonis and B. vulgatus into seven fragments.On the basis of genome size, restriction profile, and I-CeuI fragment number, a phylogenetic tree of the Bacteroides specieswas proposed. This was in overall agreement with the previousphylogenetic tree obtained by 16S rRNAdata, with the exceptionsof B. distasonis and B. ovatus.