Utilization of tmRNA sequences for bacterial identification

In recent years, molecular approaches based on nucleotide sequences of ribosomal RNA (rRNA) have become widely used tools for identification of bacteria [1-4]. The high degree of evolutionary conservation makes 16S and 23S rRNA molecules very suitable for phylogenetic studies above the species level [3-5]. More than 16,000 sequences of 16S rRNA are presently available in public databases [4,6]. The 16S rRNA sequences are commonly used to design fluorescently labeled oligonucleotide probes. Fluorescence in situ hybridization (FISH) with these probes followed by observation with epifluorescence microscopy allows the identification of a specific microorganism in a mixture with other bacteria [2-4]. By shifting probe target sites from conservative to increasingly variable regions of rRNA, it is possible to adjust the probe specificity from kingdom to species level. Nevertheless, 16S rRNA sequences of closely related strains, subspecies, or even of different species are often identical and therefore can not be used as differentiating markers [3]. Another restriction concerns the accessibility of target sites to the probe in FISH experiments. The presence of secondary structures, or protection of rRNA segments by ribosomal proteins in fixed cells can limit the choice of variable regions as in situ targets for oligonucleotide probes [7,8]. One way to overcome the limitations of in situ identification of bacteria is to use molecules other than rRNA for phylogenetic identification of bacteria, for which nucleotide sequences would be sufficiently divergent to design species specific probes, and which would be more accessible to oligonucleotide probes. For this purpose we investigated the possibility of using tmRNA (also known as 10Sa RNA; [9-11]). This molecule was discovered in E. coli and described as small stable RNA, present at ~1,000 copies per cell [9,11]. The high copy number is an important prerequisite for FISH, which works best with naturally amplified target molecules. In E. coli, tmRNA is encoded by the ssrA gene, is 363 nucleotides long and has properties of tRNA and mRNA [12,13]. tmRNA was shown to be involved in the degradation of truncated proteins: the tmRNA associates with ribosomes stalled on mRNAs lacking stop codons, finally resulting in the addition of a C-terminal peptide tag to the truncated protein. The peptide tag directs the abnormal protein to proteolysis [14,15]. 165 tmRNA sequences have so far (August 2001; The tmRNA Website: http://www.indiana.edu/~tmrna/) been determined [16,17]. The tmRNA is likely to be present in all bacteria and has also been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana[10,17,18]

Influence of bioturbation by the polychaete Nereis diversicolor on the structure of bacterial communities in oil contaminated coastal sediments

Patterns of change in the structure of bacterial communities monitored by ribosomal intergenic spacer analysis (RISA) in oil contaminated sediments inhabited or not by the marine polychaete Nereis diversicolor were studied during 45 days under laboratory conditions. Results supported by principal component analysis showed a marked response of the bacterial communities to the oil contamination and to the presence of N. diversicolor. Phylogenetic affiliation of specific RISA bands showed that, in the contaminated sediments, the presence of the marine polychaetes favoured the development of bacteria which may play an active role in natural bioremediation processes of oil polluted environments

Molecular biological methods for studying the gut microbiota : the EU human gut flora project

Seven European laboratories co-operated in a joint project (FAIR CT97-3035) to develop, refine and apply molecular methods towards facilitating elucidation of the complex composition of the human intestinal microflora and to devise robust methodologies for monitoring the gut flora in response to diet. An extensive database of 16S rRNA sequences for tracking intestinal bacteria was generated by sequencing the 16S rRNA genes of new faecal isolates and of clones obtained by amplification with polymerase chain reaction (PCR) on faecal DNA from subjects belonging to different age groups. The analyses indicated that the number of different species (diversity) present in the human gut increased with age. The sequence information generated, provided the basis for design of 16S rRNA-directed oligonucleotide probes to specifically detect bacteria at various levels of phylogenetic hierarchy. The probes were tested for their specificity and used in whole-cell and dot-blot hybridisations. The applicability of the developed methods was demonstrated in several studies and the major outcomes are described

Emerg Infect Dis

12604007PMC2901952610

Identification of archaeal rDNA from subgingival dental plaque by PCR amplification and sequence analysis

A PCR assay for the amplification of small subunit ribosomal DNA (SSU rDNA) of Euryarchaea was developed and used to detect archaeal rDNA in 37 (77%) out of 48 pooled subgingival plaque samples from 48 patients suffering from periodontal disease. One major group of cloned periodontal sequences was identical to Methanobrevibacter oralis and a second minor group to Methanobrevibacter smithii. These two groups and a third novel group were found to be more than 98% similar to each other over an 0.65-kb segment of the 16S rRNA gene sequenced. M. oralis was found to be the predominant archaeon in the subgingival dental plaque. Phylogenetic analysis of partial SSU rDNA sequences revealed evidence for a distinct cluster for human and animal Methanobrevibacter sp. within the Methanobacteriaceae famil

<i>Vibrio rotiferianus</i> sp. nov., isolated from cultures of the rotifer <i>Brachionus plicatilis</i>

Five Gram-negative bacterial strains, oxidase-positive, motile by means of more than one polar flagella, facultative anaerobe, arginine dihydrolase-negative, lysine- and ornithine decarboxylase-positive, sensitive to the vibriostatic agent O/129, were isolated from a flow-through rotifer culture system in Gent, Belgium, and previously characterized by fluorescent amplified fragment length polymorphism. Comparison of the 16S rDNA sequence of strain LMG 21460T indicated close relationships (~99 % similarity) to Vibrio campbellii, Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus. However, DNA hybridization experiments revealed similarity values below 70 % with its closest species V. campbellii and V. harveyi. Additionally, the analysed strains differ from related Vibrio species by the utilization of melibiose and production of acid from L-arabinose and amygdalin. Among the strains analysed, differences were observed in some phenotypic characters, particularly susceptibility to ampicillin, polymyxin B and amikacin, and urease activity. The major fatty acids identified were 16:0, 18:1 ?7c, 14:0, 12:0 3-OH and 18:0. Vibrio rotiferianus sp. nov. is proposed, with type strain LMG 21460T (=CAIM 577T); it has a DNA G+C content of 44·5 ± 0·01 mol%

Isolation and description of keratinase producing marine actinobacteria from South Indian Coastal Region

A unique standard starch casein medium has been implemented for the isolation of actinobacteria from the south Indian marine sediments. A combination of techniques, morphological, physiological and biochemical tests provided the evidence for the isolated actinobacteria. All the 56 isolates were inoculated on milk agar and soya meal agar plates for the primary proteolytic screening and the proportional study was made by ANOVA. Among the 56 isolates, nine showed proteolytic activity in terms of making clear zone around their colony on the plates. Then, nine isolates were subjected to the secondary screening on feather broth where three isolates (IS -1, 2 and 18) showed degradation of feather between seven and ten days. The keratinolytic characters of crude enzymes were scrutinized by feather keratin as substrate and the protein concentration was determined. Then, the isolates were identified at molecular level by 16S rRNA gene amplification technique.Key words: Actinobacteria, keratinase, milk agar, soya meal agar, 16S rRNA gene amplification