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Transglutaminase-Mediated Cross-Linking of GerQ in the Coats of Bacillus subtilis Spores. Katerina Ragkousi, 2004.The spores of Bacillus subtilis show remarkable resistance to many environmental stresses, due in part to the presence of an outer proteinaceous structure known as the spore coat . GerQ is a spore coat protein essential for the presence of CwlJ, an enzyme involved in the hydrolysis of the cortex during spore germination, in the spore coat . Here we show that GerQ is cross-linked into higher-molecular-mass forms due in large part to a transglutaminase . GerQ is the only substrate for this transglutaminase identified to date . In addition, we show that cross-linking of GerQ into high-molecular-mass forms occurs only very late in sporulation, after mother cell lysis . These findings, as well as studies of GerQ cross-linking in mutant strains where spore coat assembly is perturbed, lead us to suggest that coat proteins must assemble first and that their cross-linking follows as a final step in the spore coat formation pathway . Enhanced Anaerobic Biodegradation of Benzene-Toluene-Ethylbenzene-Xylene-Ethanol Mixtures in Bioaugmented Aquifer Columns. Marcio L. B. Da Silva, 2004.Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline . Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula . Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation . Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation . Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions . Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential . bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential . Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility . This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions . Genomic and Functional Analyses of SXT, an Integrating Antibiotic Resistance Gene Transfer Element Derived from Vibrio cholerae. John W. Beaber, 2002.SXT is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes . In recent years, SXT-related conjugative, self-transmissible integrating elements have become widespread in Asian Vibrio cholerae . We have determined the 100-kb DNA sequence of SXT . This element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid- and phage-related genes, as well as to many genes from unknown sources . We constructed a nearly comprehensive set of deletions through the use of the one-step chromosomal gene inactivation technique to identify SXT genes involved in conjugative transfer and chromosomal excision . SXT, unlike other conjugative transposons, utilizes a conjugation system related to that encoded by the F plasmid . More than half of the SXT genome, including the composite transposon-like structure that contains its antibiotic resistance genes, was not required for its mobility . Two SXT loci, designated setC and setD, whose predicted amino acid sequences were similar to those of the flagellar regulators FlhC and FlhD, were found to encode regulators that activate the transcription of genes required for SXT excision and transfer . Another locus, designated setR, whose gene product bears similarity to lambdoid phage CI repressors, also appears to regulate SXT gene expression . Sizing the Holin Lesion with an Endolysin-ß-Galactosidase Fusion. Ing-Nang Wang, 2003.Double-stranded DNA phages require two proteins for efficient host lysis: the endolysin, a muralytic enzyme, and the holin, a small membrane protein . In an event that defines the end of the vegetative cycle, the  holin S acts suddenly to permeabilize the membrane . This permeabilization enables the R endolysin to attack the cell wall, after which cell lysis occurs within seconds . A C-terminal fusion of the R endolysin with full-length ß-galactosidase (ß-Gal) was tested for lytic competence in the context of the late-gene expression system of an induced
lysogen . Under these conditions, the hybrid R-ß-Gal product, an active tetrameric ß-Gal greater than 480 kDa in mass, was fully functional in lysis mediated by the S holin . Western blot analysis demonstrated that the lytic competence was not due to the proteolytic release of the endolysin domain of the R-ß-Gal fusion protein . The ability of this massive complex to be released by the S holin suggests that S causes a generalized membrane disruption rather than a regular oligomeric membrane pore . Similar results were obtained with an early lysis variant of the S holin and also in parallel experiments with the T4 holin, T, in an identical lambda context . However, premature holin lesions triggered by depolarization of the membrane were nonpermissive for the hybrid endolysin, indicating that these premature lesions constituted less-profound damage to the membrane . Finally, a truncated T holin functional in lysis with the endolysin is completely incompetent for lysis with the hybrid endolysin . A model for the formation of the membrane lesion within homo-oligomeric rafts of holin proteins is discussed .
The Spoilage Yeast Zygosaccharomyces bailii Forms Mitotic Spores: a Screening Method for Haploidization. Fernando Rodrigues, 2003.Zygosaccharomyces bailii ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content . Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants . Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of Z . bailii . This new set of Z . bailii strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies . Formate-Dependent Growth and Homoacetogenic Fermentation by a Bacterium from Human Feces: Description of Bryantella formatexigens gen . nov., sp . nov.. Meyer J. Wolin, 2003.Formate stimulates growth of a new bacterium from human feces . With high formate, it ferments glucose to acetate via the Wood-Ljungdahl pathway . The original isolate fermented vegetable cellulose and carboxymethylcellulose, but it lost this ability after storage at -76°C . 16S rRNA gene sequencing identifies it as a distinct line within the Clostridium coccoides supra-generic rRNA grouping . We propose naming it Bryantella formatexigens gen . nov., sp . nov .
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