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Direct Glutaminyl-tRNA Biosynthesis and Indirect Asparaginyl-tRNA Biosynthesis in Pseudomonas aeruginosa PAO1. Pierre-Marie Akochy, 2004.The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA . We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS) . The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P . aeruginosa PAO1, with the homologous tRNA as the substrate . The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P . aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNAGln, that AspRS is nondiscriminating, and that its Asp-tRNAAsn product is transamidated by AdT . On the other hand, tRNAGln is directly glutaminylated by GlnRS . These results show that P . aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway . The essential role of AdT in the formation of Asn-tRNA in P . aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen . Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria . Characterization of the groEL and groES Loci in Bifidobacterium breve UCC 2003: Genetic, Transcriptional, and Phylogenetic Analyses. Marco Ventura, 2004.The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes, including the GroEL and GroES proteins . The groES and groEL genes are highly conserved among eubacteria and are typically arranged as an operon . Genome analysis of Bifidobacterium breve UCC 2003 revealed that the groES and groEL genes are located in different chromosomal regions . The heat inducibility of the groEL and groES genes of B . breve UCC 2003 was verified by slot blot analysis . Northern blot analyses showed that the cspA gene is cotranscribed with the groEL gene, while the groES gene is transcribed as a monocistronic unit . The transcription initiation sites of these two mRNAs were determined by primer extension . Sequence and transcriptional analyses of the region flanking the groEL and groES genes of various bifidobacteria revealed similar groEL-cspA and groES gene units, suggesting a novel genetic organization of these chaperones . Phylogenetic analysis of the available bifidobacterial groES and groEL genes suggested that these genes evolved differently . Discrepancies in the phylogenetic positioning of groES-based trees make this gene an unreliable molecular marker . On the other hand, the bifidobacterial groEL gene sequences can be used as an alternative to current methods for tracing Bifidobacterium species, particularly because they allow a high level of discrimination between closely related species of this genus . Genotyping and Toxigenic Potential of Bacillus subtilis and Bacillus pumilus Strains Occurring in Industrial and Artisanal Cured Sausages. Alessandra Matarante, 2004.Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products . Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP) . The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species . Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B . subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes . Moreover, fAFLP analysis made it possible to distinguish B . subtilis from B . pumilus strains . The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques . Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies . However, some isolates were found to be positive for hemolytic and lecithinase activity . The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B . cereus cells . Anaerobic Transformation of Alkanes to Fatty Acids by a Sulfate-Reducing Bacterium, Strain Hxd3. Chi Ming So, 2003.Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al . (F . Aeckersberg, F . Bak, and F . Widdel, Arch . Microbiol . 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and 13C-labeled compounds . Deuterated fatty acids with even numbers of C atoms (C-even) and 13C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-13C2]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa . When Hxd3 was grown on unlabeled hexadecane in the presence of [13C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a 13C label to form its carboxyl group . The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates . These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane . The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-13C2]hexadecane-derived fatty acids contained either two 13C labels located exclusively at their acyl chain termini or none at all . Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end . These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms . We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms .
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