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Inhibiting Cell Division in Escherichia coli Has Little If Any Effect on Gene Expression. S. J. Ryan Arends, 2004.DNA microarrays were used to compare gene expression in dividing and nondividing (filamentous) cultures of Escherichia coli . Although cells from these cultures differed profoundly in morphology, their gene expression profiles were nearly identical . These results extend previous evidence that there is no division checkpoint in E . coli, and progression through the cell cycle is not regulated by the transcription of different genes during different parts of the cell cycle . Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization. Jan Vinjé, 2004.In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters . Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST) . Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses . We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages . These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses . The RT-PCR assays were validated by using 190 field and prototype strains . Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described . Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified . We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters . Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qß, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples . In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies . Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea. Matthias Labrenz, 2004.We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example . Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances . The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T . denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content . It allowed quantification of T . denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T . denitrificans-like cell . Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization . These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract . Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification . Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude . Quantification of enrichments with or without a preamplification step yielded comparable results . T . denitrificans-like 16S rRNA molecules ranged from 7.1 x 103 to 4.4 x 109 copies ml–1 or 0.002 to 49.7% relative abundance . T . denitrificans-like 16S rRNA genes ranged from 9.0 x 101 to 2.2 x106 copies ml–1 or 0.01 to 49.7% relative abundance . Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml–1 . The number of ribosomes per T . denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments . The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment . CDP-2,3-Di-O-Geranylgeranyl-sn-Glycerol:L-Serine O-Archaetidyltransferase (Archaetidylserine Synthase) in the Methanogenic Archaeon Methanothermobacter thermautotrophicus. Hiroyuki Morii, 2003.CDP-2,3-di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of Methanothermobacter thermautotrophicus cells was characterized . The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and L-serine . The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation . The enzyme showed maximal activity in the presence of 10 mM Mn2+ and 1% Triton X-100 . Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase . The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate . The activity of D-serine with the enzyme was 30% of that observed for L-serine . A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment . A gene (MT1027) in M . thermautotrophicus genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to Bacillus subtilis pssA but not to Escherichia coli pssA. The substrate specificity of phosphatidylserine synthase from B . subtilis was quite similar to that observed for the M . thermautotrophicus archaetidylserine synthase, while the E . coli enzyme had a strong preference for CDP-1,2-diacyl-sn-glycerol . It was concluded that M . thermautotrophicus archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (B . subtilis type) on the basis of not only homology but also substrate specificity and some enzymatic properties . The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed . Molecular Analyses of Salmonella enterica Isolates from Fish Feed Factories and Fish Feed Ingredients. Live L. Nesse, 2003.Isolates of the most commonly observed salmonella serovars in Norwegian fish feed factories from 1998 to 2000 (Salmonella enterica serovar Agona, S . enterica serovar Montevideo, S . enterica serovar Senftenberg, and S . enterica serovar Kentucky) were studied by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis and compared to isolates of the same serovars from fish feed ingredients, humans, and other sources (a total of 112 isolates) . Within each serovar, a variety of distinct PFGE types (with similarity levels less than 90%) were observed in the feed ingredients and other sources, while only two distinct types of each serovar were identified in the factories . The combined results of PFGE and plasmid analyses showed that each factory harbored only a few S . enterica clones . Some of these clones persisted for at least 3 years in the factories, indicating that there was long-lasting contamination probably due to inadequate decontamination procedures .
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