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Involvement of Streptococcus gordonii Beta-Glucoside Metabolism Systems in Adhesion, Biofilm Formation, and In Vivo Gene Expression. Ali O. Kiliç, 2004.Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA) . To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed . To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions . Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides . Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants . Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci . Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium . Therefore, genes associated with beta-glucoside metabolism may regulate S . gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization . Complete Nucleotide Sequence of a 92-Kilobase Plasmid Harboring the CTX-M-15 Extended-Spectrum Beta-Lactamase Involved in an Outbreak in Long-Term-Care Facilities in Toronto, Canada. David A. Boyd, 2004.A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002 . We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain . Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions . The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn10 . The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS26 . All drug resistance genes found in pC15-1a, including the beta-lactamase genes blaCTX-M-15, blaOXA-1, and blaTEM-1, the tetracycline resistance gene tetA, and aminoglycoside resistance genes aac(6')-Ib and aac(3)-II, are located in the MDR . The blaCTX-M-15 gene was found downstream of ISEcp1as part of a transposition unit, as determined from the surrounding sequence . Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada . Comparison of pC15-1a and pCTX15, found in an E . coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including blaCTX-M-15, blaTEM-1, blaOXA-1, tetA, and aac(6')-Ib . Mineralization of Individual Congeners of Linear Alkylbenzenesulfonate by Defined Pairs of Heterotrophic Bacteria. David Schleheck, 2004.Parvibaculum lavamentivorans DS-1T utilized the commercial surfactant linear alkylbenzenesulfonate (LAS) (20 congeners with C10 to C13 side chains) as a carbon and energy source by shortening the side chain, and sulfophenylcarboxylates (SPCs) and similar compounds (e.g.,  ,ß-unsaturated SPCs [SPC-2Hs]) were excreted with quantitative recovery of the sulfophenyl moiety . 2-(4-Sulfophenyl)decane (2-C10-LAS) was converted largely to 3-(4-sulfophenyl)butyrate (3-C4-SPC), as were 2-C12-LAS and 2-C14-LAS; the other products were 5-C6-SPC (SPC+2C) and 3-C4-SPC-2H . 2-C11-LAS was converted largely to 4-C5-SPC with the corresponding SPC+2C and SPC-2H; similarly, 3-C12-LAS yielded 4-C6-SPC with the corresponding SPC+2C and SPC-2H . This pattern of products confirmed that LAS is degraded by
 -oxygenation and chain shortening through ß-oxidation . At least nine major SPCs were formed from commercial LAS . The novel isolates Comamonas testosteroni SPB-2 and KF-1 utilized 3-C4-SPC; Delftia acidovorans SPH-1 utilized 4-C6-SPC enantioselectively . The substrate-dependent oxygen uptake of whole cells of strain SPB-2 indicated that there was inducible oxygenation of 3-C4-SPC and of 4-sulfophenol in whole cells of the strains of C . testosteroni during growth with 3-C4-SPC or 4-sulfophenol . The degradative pathways apparently involved 4-sulfocatechol and 4-sulfocatechol 1,2-dioxygenase . Strain SPB-2 and strain DS-1T grew together in LAS-salts medium, and only seven of the nine major SPCs were recovered . Strain SPB-2 utilized 3-C4-SPC, 3-C5-SPC, and 3-C4-SPC-2H . Strain SPH-1 grew together with strain DS-1T in LAS-salts medium, and a different set of seven major SPCs was recovered . Strain SPH-1 utilized 4-C6-SPC, 4-C5-SPC, 4-C6-SPC-2H, and 4-C5-SPC-2H . A three-member community consisting of strains DS-1T, SPB-2, and SPH-1 utilized four major SPCs . We inferred that this community mineralized the major SPCs derived from 8 of the 20 LAS congeners .
Differential Regulation of Twitching Motility and Elastase Production by Vfr in Pseudomonas aeruginosa. Scott A. Beatson, 2002.Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa . We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr . Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production . We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S . A . Beatson, C . B . Whitchurch, A . B . T . Semmler, and J . S . Mattick, J . Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr . This allele (Vfr  EQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility . Furthermore, structural analysis of Vfr and VfrEQERS in relation to E . coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrEQERS is only capable of responding to cAMP . We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket .
Characterization of the Vibrio vulnificus putAP Operon, Encoding Proline Dehydrogenase and Proline Permease, and Its Differential Expression in Response to Osmotic Stress. Jeong Hyun Lee, 2003.The Vibrio vulnificus putAP genes encoding a proline dehydrogenase and a proline permease are transcribed in the same direction . Proline dehydrogenase activity and the level of putA transcript were determined to reach a maximum in exponential phase and were then repressed when growth slowed down . Northern blotting and primer extension analyses revealed that transcription of putAP genes results in two different transcripts, transcript A (putA transcript) and transcript AP (putAP transcript) . Expression of putAP genes was directed by two promoters, promoter PputA and promoter PputAP . A crp null mutation decreased proline dehydrogenase activity and the level of the put transcripts, indicating that transcription of putAP is under the positive control of cyclic AMP receptor protein . Proline dehydrogenase and the level of both put transcripts were increased by proline but repressed by glutamate . In contrast, the level of transcript A, not transcript AP, increased when proline dehydrogenase was induced by NaCl . Since PputA activity, not PputAP activity, was increased by NaCl, it is apparent that transcript A and transcript AP are transcribed through PputA and PputAP, respectively . Cells challenged with NaCl and various hyperosmotic stresses accumulated higher levels of glutamate than control cells, indicating that glutamate is a compatible solute in V . vulnificus .
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