Group and type antigens of Capnocytophaga.
(25/1602)
Group-specific and type-specific antigens have been identified and purified from sonic extracts of Capnocytophaga. The group-specific antigen, which was purified by affinity chromatography, was found to be identical to an antigen present in all 26 strains tested. The antigen is sensitive to trypsin, sodium dodecyl sulfate and heat labile, and composed predominantly (55%) of protein. The type-specific antigen, which was obtained by preparative immunoelectrophoresis, was found to be present in only 3 of the 26 strains tested. This antigen was resistant to heat, trypsin, and sodium dodecyl sulfate and was primarily composed of carbohydrate (47% phenol-sulfuric acid-positive material, 8% amino sugar). Agglutination and fluorescent antibody data suggest that both the group- and type-specific antigens reside on the cell surface. (+info)
Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1.
(26/1602)
NBU1 is an integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision-required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision. The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRS(HIII)) inhibited excision when provided in trans on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized. (+info)
Colonic bacteria express an ulcerative colitis pANCA-related protein epitope.
(27/1602)
Bacteria are a suspected pathogenic factor in inflammatory bowel disease, but the identity of the relevant microbial species remains unresolved. The pANCA autoantibody is associated with most cases of ulcerative colitis (UC) and hence reflects an immune response associated with the disease process. This study addresses the hypothesis that pANCA identifies an antigen(s) expressed by bacteria resident in the human colonic mucosa. Libraries of colonic bacteria were generated using aerobic and anaerobic microbiologic culture conditions, and bacterial pools and clonal isolates were evaluated for cross-reactive antigens by immunoblot analysis using the pANCA monoclonal antibody Fab 5-3. Two major species of proteins immunoreactive to pANCA monoclonal antibodies were detected in bacteria from the anaerobic libraries. Colony isolates of the expressing bacteria were identified as Bacteroides caccae and Escherichia coli. Isolation and partial sequencing of the B. caccae antigen identified a 100-kDa protein without database homologous sequences. The E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC. Enzyme-linked immunosorbent assay with human sera demonstrated elevated immunoglobulin G anti-OmpC in UC patients compared to healthy controls. These findings demonstrate that a pANCA monoclonal antibody detects a recurrent protein epitope expressed by colonic bacteria and implicates colonic bacterial proteins as a target of the disease-associated immune response. (+info)
Increase in bacterial community diversity in subsurface aquifers receiving livestock wastewater input.
(28/1602)
Despite intensive studies of microbial-community diversity, the questions of which kinds of microbial populations are associated with changes in community diversity have not yet been fully solved by molecular approaches. In this study, to investigate the impact of livestock wastewater on changes in the bacterial communities in groundwater, bacterial communities in subsurface aquifers were analyzed by characterizing their 16S rDNA sequences. The similarity coefficients of restriction fragment length polymorphism (RFLP) patterns of the cloned 16S ribosomal DNAs showed that the bacterial communities in livestock wastewater samples were more closely related to those in contaminated aquifer samples. In addition, calculations of community diversity clearly showed that bacterial communities in the livestock wastewater and the contaminated aquifer were much more diverse than those in the uncontaminated aquifer. Thus, the increase in bacterial-community diversity in the contaminated aquifer was assumed to be due to the infiltration of livestock wastewater, containing high concentrations of diverse microbial flora, into the aquifer. Phylogenetic analysis of the sequences from a subset of the RFLP patterns showed that the Cytophaga-Flexibacter-Bacteroides and low-G+C gram-positive groups originating from livestock wastewater were responsible for the change in the bacterial community in groundwater. This was evidenced by the occurrence of rumen-related sequences not only in the livestock wastewater samples but also in the contaminated-groundwater samples. Rumen-related sequences, therefore, can be used as indicator sequences for fecal contamination of groundwater, particularly from livestock. (+info)
Degradation of pectins with different degrees of esterification by Bacteroides thetaiotaomicron isolated from human gut flora.
(29/1602)
A complete human fecal flora and cultures of defined species obtained from fecal flora were investigated in vitro to determine their ability to ferment the dietary fiber pectin. Bacteroides thetaiotaomicron was tested as a pectin-degrading microorganism alone and in coculture with Escherichia coli. Macromolecular pectins with different degrees of esterification were used as substrates in microbial degradation studies. The levels of oligogalacturonic acids formed in batch cultures were estimated during a 24- or 48-h incubation period by using high-performance thin-layer chromatography and high-performance anion-exchange chromatography. The spectrum and the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the culture media during incubation with the complete fecal flora. After 24 h, no oligogalacturonic acids were detected. The pectin-degrading activities of pure cultures of B. thetaiotaomicron were lower than the pectin-degrading activity of a complete fecal flora. Cocultures of B. thetaiotaomicron and E. coli exhibited intermediate levels of degradation activity. In pure cultures of E. coli no pectin-degrading activity was found. Additionally, the rate of pectin degradation was affected by the degree of esterification of the substrate. Saturated oligogalacturonic acids were not found during pectin fermentation. The disappearance of oligogalacturonic acids in the later stages of fermentation with both the complete fecal flora and B. thetaiotaomicron was accompanied by increased formation of short-chain fatty acids. (+info)
Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes.
(30/1602)
We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 x 10(-5) to 2.8 x 10(-7) g (dry weight) of feces/liter and 6.8 x 10(-7) g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments. (+info)
PCR assay for species-specific identification of Bacteroides thetaiotaomicron.
(31/1602)
Bacteroides thetaiotaomicron is the second most frequently encountered species of the anaerobes isolated from clinical specimens. We developed a PCR-based assay for the rapid identification of B. thetaiotaomicron. Specific primers were based on shared amplicons of about 1.2 kb generated from B. thetaiotaomicron by randomly amplified polymorphic DNA. This 1.2-kb fragment was sequenced and then used to design a set of PCR amplification primers. This PCR generated an amplification product of 721 bp, which was unique to all 65 isolates of B. thetaiotaomicron tested. There was no amplification with isolates of other bacterial species. Restriction enzyme digestion of the amplification product and dot blot hybridization further verified the specificity of the assay. These results suggest that this PCR assay targets a nucleotide sequence that is strongly conserved in B. thetaiotaomicron. This simple and rapid PCR assay provides a rapid and accurate method for identification of B. thetaiotaomicron and shows promise for the detection of B. thetaiotaomicron in clinical samples. (+info)
Dyadobacter fermentans gen. nov., sp. nov., a novel gram-negative bacterium isolated from surface-sterilized Zea mays stems.
(32/1602)
A Gram-negative bacterium, designated NS114T, was isolated from duplicate treatments of surface-sterilized Zea mays stems. The plants were grown in synthetic soil under greenhouse conditions and watered with fertilizer containing no nitrogen. Strain NS114T could not be isolated from plants watered with the standard level or 20% (w/v) of the standard level of nitrogen. Cells occurred as pairs in young cultures that attached to form angled arrangements in R2A broth and occasionally formed rounded, horseshoe arrangements in YM broth. Cell variation resulted in flocculent chains of coccoid cells in old cultures. Strain NS114T fermented glucose and sucrose. The G + C content was 48 mol%. Phylogenetic analysis of the 16S rRNA gene showed that the strain was a member of the domain Bacteria and branched from a point equidistant from an aquatic organism, Runella slithyformis and a marine isolate, 'Microscilla furvescens'. Phenotypic and genotypic analyses indicated that strain NS114T could not be assigned to any recognized genus; therefore a new genus and species, Dyadobacter fermentans gen. nov., sp. nov., is proposed, for which NS114T is the type strain. (+info)