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DNA Sequence Analysis of Regions Surrounding blaCMY-2 from Multiple Salmonella Plasmid Backbones. W. P. Giles, 2004.The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry the blaCMY-2 gene . In the present study, the distribution of these four known blaCMY-2-carrying plasmids among 35 ceftriaxone-resistant Salmonella isolates obtained from 1998 to 2001 was examined . Twenty-three of these isolates were Salmonella enterica serotype Newport, 10 were Salmonella enterica serotype Typhimurium, 1 was Salmonella enterica serotype Agona, and 1 was Salmonella enterica serotype Reading . All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively . Both the serotype Agona and serotype Reading isolates carried type A plasmids . None of the isolates carried a type D plasmid . Hybridization data suggested that plasmid types A and C were highly related replicons . DNA sequencing revealed that the region surrounding blaCMY-2 was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surrounding blaCMY-5 from the Klebsiella oxytoca plasmid pTKH11 . These findings are consistent with a model in which blaCMY-2 has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multiple Salmonella chromosomal backbones . Protein Expression by Streptococcus mutans during Initial Stage of Biofilm Formation. J. Welin, 2004.Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents . In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface . In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface . Log-phase planktonic cells of S . mutans were allowed to adhere to a glass slide for 2 h in the presence of a 14C-amino acid mixture . Nonadhered cells were washed away, and the adhered cells were removed by sonication . The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis . Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished  1.3-fold in the biofilm cells . Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis . Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting . The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface .
Subunit Topology of Two 20S Proteasomes from Haloferax volcanii. Steven J. Kaczowka, 2003.Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (  1,
2, and ß) which are classified in the 20S proteasome superfamily . The
1 and ß proteins alone form active 20S proteasomes; the role of
2, however, is not clear . To address this,
2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H . volcanii . The
2 protein copurified with
1 and ß in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described
1-ß proteasome . Supplementing buffers with 10 mM CaCl2 stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis . This facilitated the discovery that wild-type H . volcanii synthesizes more than one type of 20S proteasome . Two 20S proteasomes, the
1-ß and
1-2-ß proteasomes, were identified during stationary phase . Cross-linking of these enzymes, coupled with available structural information, suggested that the
1-ß proteasome was a symmetrical cylinder with
1 rings on each end . In contrast, the
1-2-ß proteasome appeared to be asymmetrical with homo-oligomeric
1 and
2 rings positioned on separate ends . Inter--subunit contacts were only detected when the ratio of
1 to
2 was perturbed in the cell using recombinant technology . These results support a model that the ratio of
proteins may modulate the composition and subunit topology of 20S proteasomes in the cell .
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