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Phosphorylation of the Pseudomonas aeruginosa Response Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility. Cynthia B. Whitchurch, 2002.The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa . In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined . The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P . aeruginosa strains PAK and PAO1 . Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation . However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function . algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis . In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA . These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P . aeruginosa . Roles of NhaA, NhaB, and NhaD Na+/H+ Antiporters in Survival of Vibrio cholerae in a Saline Environment. Katia Herz, 2003.Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity . In this work, we investigated the role of three Na+/H+ antiporters on the survival of V . cholerae in a saline environment . We have previously cloned the Vc-nhaA gene encoding the V . cholerae homolog of Escherichia coli . Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E . coli. We showed that both Vc-NhaA and Vc-NhaB confer Na+ resistance and that Vc-NhaA displays an antiport activity in E . coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E . coli NhaA . To determine the roles of the Na+/H+ antiporters in V . cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants) . In contrast to E . coli, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V . cholerae did not alter the bacterial exponential growth in the presence of high Na+ concentrations and had only a slight effect in the stationary phase . In contrast, a pronounced and similar Li+-sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth . By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na+ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na+/H+ antiporter activity becomes essential for the resistance of V . cholerae to Na+ at alkaline pH . Since the ion pump NQR is Na+ specific, we suggest that its activity masks the Na+/H+ but not the Li+/H+ antiporter activities . Our results indicate that the Na+ resistance of the human pathogen V . cholerae requires a complex molecular system involving multiple antiporters and the NQR pump . Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection. Courtney L. Frazier, 2003.Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria . Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome . Integrants were readily identified and maintained in the absence of a selectable marker . Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans . Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene . Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant . Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci . Characterization and In Situ Carbon Metabolism of Phototrophic Consortia. Jens Glaeser, 2003.A dense population of the phototrophic consortium "Pelochromatium roseum" was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany) . Fluorescence in situ hybridization revealed that the brown epibionts of "P . roseum" constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline . Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low . The 465-bp 16S rRNA gene sequence of the epibiont of "P . roseum" was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR . The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria . Maximum light-dependent H14CO3- fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria . The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (  13C) of their specific biomarkers . Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl) e and smaller amounts of BChl a and d and chlorophyll a in the chemocline . Unexpectedly, isorenieratene and ß-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent . Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their
13C values were determined . Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in 13C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria . The difference between the
13C values of farnesol, the major esterifying alcohol of BChl e, and CO2 was -7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of "P . roseum" in situ is photoautotrophic .
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