|
|
|
|
|
|
Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene. Melanie Duhamel, 2004.A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described . The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) x 108 16S rRNA gene copies/µmol of Cl– when grown on VC and hydrogen . Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) x 108 16S rRNA gene copies/µmol of Cl– . The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well . PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1 . A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H2 enrichment culture . This second Dehalococcoides sequence was identical to that of BAV1 . As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group . Identification and Functional Characterization of flgM, a Gene Encoding the Anti-Sigma 28 Factor in Pseudomonas aeruginosa. A. Frisk, 2002.We describe here the functional characterization of the putative flgM gene of Pseudomonas aeruginosa . FlgM of P . aeruginosa is most similar to FlgM of Vibrio parahaemolyticus . A conserved region is present in the C-terminal half of the FlgM of P . aeruginosa and in FlgM homologues of other organisms that includes the  28 binding domain . A role for the flgM gene of P . aeruginosa in motility was demonstrated by its inactivation . The ß-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA, the sigma factor that regulates fliC, was increased . Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant of P . aeruginosa strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA . Direct interaction of the P . aeruginosa FlgM with the alternative sigma factor
28 was demonstrated by utilizing the yeast two-hybrid system . Three putative consensus
54 recognition sites and one
28 site were found in the flgM upstream region . However, analysis of the transcriptional fusion of the flgM promoter to lacZ in different mutant backgrounds showed that the flgM promoter was not entirely dependent on either
28 or
54 . A transcript was detected by primer extension that was 8 bp downstream of the consensus
28-binding site . Thus, a system for the control of flagellin synthesis by FlgM exists in P . aeruginosa that is different from that in the enteric bacteria and seems to be most similar to that of V . cholerae where both
28-dependent and -independent mechanisms of transcription exist .
A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture. Andrew D. Sails, 2003.A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture . The specificity of the assay for C . jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera . The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents . The assay was used to detect C . jejuni in both naturally and artificially contaminated food samples . Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37°C for 24 h, followed by 42°C for 24 h . Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods . DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay . A total of 66 samples were positive for C . jejuni by either method, with 57 samples positive for C . jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay . The results of both methods were concordant for 84 of the samples . The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar . This assay significantly reduces the total time taken for the detection of C . jejuni in foods and is an important model for other food-borne pathogens .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
