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Genetic Environments of the rmtA Gene in Pseudomonas aeruginosa Clinical Isolates. Kunikazu Yamane, 2004.Nine Pseudomonas aeruginosa strains showing very high levels of resistance to various aminoglycosides have been isolated from clinical specimens in seven separate Japanese hospitals in five prefectures since 1997 . These strains harbor the newly identified 16S rRNA methylase gene (rmtA) . When an rmtA gene probe was hybridized with genomic DNAs of the nine strains digested with EcoRI, two distinct patterns were observed . The 11.1- and 15.8-kb regions containing the rmtA genes of strains AR-2 and AR-11, respectively, were sequenced and compared . In strain AR-2, a transposase gene-like sequence (sequence 1) and a probable tRNA ribosyltransferase gene (orfA) were located upstream of rmtA, and a Na+/H+ antiporter gene-like sequence (sequence 2) was identified downstream of rmtA . This 6.2-kbp insert (the rmtA locus) was flanked by 262-bp   elements . Part of the orfQ gene adjacent to an inverted repeat was found outside of the rmtA locus . In strain AR-11, the rmtA gene and sequence 2 were found, but the 5' end of the orfA gene was truncated and replaced with IS6100 . An orfQ-orfI region was present on each side of the rmtA gene in strain AR-11 . The G+C content of the rmtA gene was about 55%, and since the newly identified rmtA gene may well be mediated by some mobile genetic elements such as Tn5041, further dissemination of the rmtA gene could become an actual clinical problem in the near future .
Cloning, Overexpression, and Characterization of a Novel Thermostable Penicillin G Acylase from Achromobacter xylosoxidans: Probing the Molecular Basis for Its High Thermostability. Gang Cai, 2004.The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli . The pgaW gene contains an open reading frame of 2,586 nucleotides . The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens, and Escherichia coli . Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60°C and its half-life of inactivation at 55°C is four times longer than that of another previously reported thermostable PGA from Alcaligenes faecalis (R . M . D . Verhaert, A . M . Riemens, J . V . R . Laan, J . V . Duin, and W . J . Quax, Appl . Environ . Microbiol . 63:3412-3418, 1997) . To our knowledge, this is the most thermostable PGA ever characterized . To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed . The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability . The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry . VGJ  , a Novel Filamentous Phage of Vibrio cholerae, Integrates into the Same Chromosomal Site as CTX.Javier Campos, 2003.We describe a novel filamentous phage, designated VGJ , isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested . The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX of V . cholerae and Ff phages of Escherichia coli . The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide . VGJ, like other filamentous phages, uses a type IV pilus to infect V . cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin . VGJ-infected V . cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages . Once inside a cell, VGJ is able to integrate its genome into the same chromosomal attB site as CTX, entering into a lysogenic state . Additionally, we found an attP structure in VGJ, which is also conserved in several lysogenic filamentous phages from different bacterial hosts . Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V . cholerae El Tor .
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