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Inactivation of ompX Causes Increased Interactions of Type 1 Fimbriated Escherichia coli with Abiotic Surfaces. Karen Otto, 2004.During the initial steps of biofilm formation, bacteria have to adapt to a major change in their environment . The adhesion-induced phenotypic changes in a type 1 fimbriated Escherichia coli strain included reductions in the levels of several outer membrane proteins, one of which was identified as OmpX . Here, the phenotypes of mutant strains that differ at the ompX locus were studied with regard to adhesion, cell surface properties, and resistance to stress and antimicrobial compounds . The kinetics of adhesion were measured online by an extended quartz crystal microbalance technique for wild-type and mutant strains with a fimbriated or nonfimbriated background . Deletion of ompX led to significantly increased cell-surface contact in fimbriated strains but to decreased cell-surface contact in a nonfimbriated strain . Phenotypic characterization of the ompX mutant demonstrated that ompX interferes with proper regulation of cell surface structures that play a key role in mediating firm contact of the cell with a surface (i.e., type 1 fimbriae, flagellae, and exopolysaccharides) . These phenotypic changes were accompanied by increased tolerance to several antibiotic compounds and sodium dodecyl sulfate . Based on these results, we propose that changes in the composition of outer membrane proteins during fimbria-mediated adhesion may be part of a coordinated adaptive response to the attached mode of growth . Aquaporin-Mediated Improvement of Freeze Tolerance of Saccharomyces cerevisiae Is Restricted to Rapid Freezing Conditions. An Tanghe, 2004.Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications . In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions . We found that the difference in the freezing rate is apparently responsible for the difference in the results . We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect . Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested . Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity . In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions . If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly . Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought . Overexpression of the MtrC-MtrD-MtrE Efflux Pump Due to an mtrR Mutation Is Required for Chromosomally Mediated Penicillin Resistance in Neisseria gonorrhoeae. Wendy L. Veal, 2002.The importance of the mtrCDE-encoded efflux pump in conferring chromosomally mediated penicillin resistance on certain strains of Neisseria gonorrhoeae was determined by using genetic derivatives of penicillin-sensitive strain FA19 bearing defined mutations (mtrR, penA, and penB) donated by a clinical isolate (FA6140) expressing high-level resistance to penicillin and antimicrobial hydrophobic agents (HAs) . When introduced into strain FA19 by transformation, a single base pair deletion in the mtrR promoter sequence from strain FA6140 was sufficient to provide high-level resistance to HAs (e.g., erythromycin and Triton X-100) but only a twofold increase in resistance to penicillin . When subsequent mutations in penA and porIB were introduced from strain FA6140 into strain WV30 (FA19 mtrR) by transformation, resistance to penicillin increased incrementally up to a MIC of 1.0 µg/ml . Insertional inactivation of the gene (mtrD) encoding the membrane transporter component of the Mtr efflux pump in these transformant strains and in strain FA6140 decreased the MIC of penicillin by 16-fold . Genetic analyses revealed that mtrR mutations, such as the single base pair deletion in its promoter, are needed for phenotypic expression of penicillin and tetracycline resistance afforded by the penB mutation . As penB represents amino acid substitutions within the third loop of the outer membrane PorIB protein that modulate entry of penicillin and tetracycline, the results presented herein suggest that PorIB and the MtrC-MtrD-MtrE efflux pump act synergistically to confer resistance to these antibiotics . Global Regulation by gidA in Pseudomonas syringae. Thomas G. Kinscherf, 2002.Analysis of two virulence mutants of Pseudomonas syringae B728a revealed that the Tn 5 sites of insertion were within the gidA open reading frame (ORF) . These mutations were pleiotropic, affecting diverse phenotypic traits, such as lipodepsipeptide (syringomycin and syringopeptin) antibiotic production, swarming, presence of fluorescent pigment, and virulence . Site-specific recombination of a disrupted gidA gene into the chromosome resulted in the same phenotypic pattern as transposon insertion . Mutant phenotypes were restored by the gidA ORF on a plasmid . The salA gene, a copy number suppressor of the syringomycin-deficient phenotype in gacS and gacA mutants, was also found to suppress the antibiotic-negative phenotypes of gidA mutants, suggesting that gidA might play some role in salA regulation . Reporter studies with chromosomal salA-lacZ translational fusions confirmed that salA reporter expression decreased approximately fivefold in a gidA mutant background, with a concurrent decrease in the expression of the syringomycin biosynthetic reporter fusion syrB-lacZ. Wild-type levels of reporter expression were restored by supplying an intact gidA gene on a plasmid . Often described as being involved in cell division, more recent evidence suggests a role for gidA in moderating translational fidelity, suggesting a mechanism by which global regulation might occur . The gidA gene is essentially universal in the domains Bacteria and Eucarya but has no counterparts in Archaea, probably reflecting specific differences in the translational machinery between the former and latter domains . Regulation of Expression of the 2-Deoxy-D-Ribose Utilization Regulon, deoQKPX, from Salmonella enterica Serovar Typhimurium. Mette Christensen, 2003.Salmonella enterica, in contrast to Escherichia coli K12, can use 2-deoxy-D-ribose as the sole carbon source . The genetic determinants for this capacity in S . enterica serovar Typhimurium include four genes, of which three, deoK, deoP, and deoX, constitute an operon . The fourth, deoQ, is transcribed in the opposite direction . The deoK gene encodes deoxyribokinase . In silico analyses indicated that deoP encodes a permease and deoQ encodes a regulatory protein of the deoR family . The deoX gene product showed no match to known proteins in the databases . Deletion analyses showed that both a functional deoP gene and a functional deoX gene were required for optimal utilization of deoxyribose . Using gene fusion technology, we observed that deoQ and the deoKPX operon were transcribed from divergent promoters located in the 324-bp intercistronic region between deoQ and deoK . The deoKPX promoter was 10-fold stronger than the deoQ promoter, and expression was negatively regulated by DeoQ as well as by DeoR, the repressor of the deoxynucleoside catabolism operon . Transcription of deoKPX but not of deoQ was regulated by catabolite repression . Primer extension analysis identified the transcriptional start points of both promoters and showed that induction by deoxyribose occurred at the level of transcription initiation . Gel retardation experiments with purified DeoQ illustrated that it binds independently to tandem operator sites within the deoQ and deoK promoter regions with Kd values of 54 and 2.4 nM, respectively .
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