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Clinical Strains of Pseudomonas aeruginosa Overproducing MexAB-OprM and MexXY Efflux Pumps Simultaneously. Catherine Llanes, 2004.Simultaneous overexpression of the MexAB-OprM and MexXY efflux systems was demonstrated by real-time reverse transcription-PCR and immunoblotting experiments for 12 multiresistant clinical isolates of Pseudomonas aeruginosa . DNA sequencing analysis showed that nine of these strains (named agrZ mutants) harbored mutations in mexZ, the product of which downregulates the expression of the mexXY operon . In addition, 8 of the 12 strains exhibited mutations in genes known to control transcription of the mexAB-oprM operon . Four of them were nalB mutants with alterations in the repressor gene mexR, three of them appeared to be nalC mutants deficient in gene PA3721 and overexpressing gene PA3720, and one strain was a nalB nalC double mutant . For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the expression level of mexA or mexX, and (iii) resistance to effluxed antibiotics . Finally, three isolates, named agrW mutants, overproduced MexXY and had an intact mexZ gene, and four strains overproduced MexAB-OprM and had intact mexR and PA3721 genes (nalD mutants) . These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY . Cloning and Expression of clt Genes Encoding Milk-Clotting Proteases from Myxococcus xanthus 422. M. Poza, 2004.The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames . Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E . coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems . Contribution of pilA to Competitive Colonization of the Squid Euprymna scolopes by Vibrio fischeri. Eric V. Stabb, 2003.Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis . Hatchling squid lack these bacterial symbionts, and V . fischeri strains must compete to occupy this privileged niche . We cloned a V . fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins . Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly . Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene . In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization . In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing . The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V . fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used . ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain . V . fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V . fischeri isolates apparently possess pilA . The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria .
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