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Vibrio fischeri LuxS and AinS: Comparative Study of Two Signal Synthases. Claudia Lupp, 2004.Vibrio fischeri possesses two acyl-homoserine lactone quorum-sensing systems, ain and lux, both of which are involved in the regulation of luminescence gene expression and are required for persistent colonization of the squid host, Euprymna scolopes . We have previously demonstrated that the ain system induces luminescence at cell densities that precede lux system activation . Our data suggested that the ain system both relieves repression and initially induces the lux system, thereby achieving sequential induction of gene expression by these two systems . Analysis of the V . fischeri genome revealed the presence of a putative third system based on the enzyme LuxS, which catalyzes the synthesis of the Vibrio harveyi autoinducer 2 (AI-2) . In this study, we investigated the impact of V . fischeri LuxS on luminescence and colonization competence in comparison to that of the ain system . Similar to the ain system, inactivation of the AI-2 system decreased light production in culture, but not in the squid host . However, while an ainS mutant produces no detectable light in culture, a luxS mutant expressed approximately 70% of wild-type luminescence levels . A mutation in luxS alone did not compromise symbiotic competence of V . fischeri; however, levels of colonization of an ainS luxS double mutant were reduced to 50% of the already diminished level of ainS mutant colonization, suggesting that these two systems regulate colonization gene expression synergistically through a common pathway . Introduction of a luxO mutation into the luxS and ainS luxS background could relieve both luminescence and colonization defects, consistent with a model in which LuxS, like AinS, regulates gene expression through LuxO . Furthermore, while luxS transcription appeared to be constitutive and the AI-2 signal concentration did not change dramatically, our data suggest that ainS transcription is autoregulated, resulting in an over 2,000-fold increase in signal concentration as culture density increased . Taken together, these data indicate that V . fischeri LuxS affects both luminescence regulation and colonization competence; however, its quantitative contribution is small when compared to that of the AinS signal . Identification of Lactococcus lactis Genes Required for Bacteriophage Adsorption. Kitt Dupont, 2004.The aim of this work was to identify genes in Lactococcus lactis subsp . lactis IL1403 and Lactococcus lactis subsp . cremoris Wg2 important for adsorption of the 936-species phages bIL170 and  645, respectively . Random insertional mutagenesis of the two L . lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected . In L . lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L . lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L . lactis IL1403 . rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions . Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L . lactis IL1403 . This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS) . Binding and infection studies showed that
645 binds to and infects L . lactis Wg2, L . lactis IL1403, and L . lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L . lactis IL1403 and cannot infect Wg2 . These results indicate that
645 binds to a WPS structure present in both L . lactis IL1403 and L . lactis Wg2, whereas bIL170 binds to another WPS structure not present in L . lactis Wg2 . Binding of bIL170 and
645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and
645 that showed no homology in the C-terminal part .
Uptake of the ß-Lactam Precursor  -Aminoadipic Acid in Penicillium chrysogenum Is Mediated by the Acidic and the General Amino Acid Permease.Hein Trip, 2004.External addition of the ß-lactam precursor -aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular
-aminoadipic acid concentration and an increase in penicillin production . The exact route for
-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process . Their corresponding genes, PcGAP1 and PcDIP5, of P . chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted . Transport assays show that both PcGap1 and PcDip5 mediated the uptake of
-aminoadipic acid, although PcGap1 showed a higher affinity for
-aminoadipic acid than PcDip5 (Km values, 230 and 800 µM, respectively) . Leucine strongly inhibits
-aminoadipic acid transport via PcGap1 but not via PcDip5 . This difference was exploited to estimate the relative contribution of each transport system to the
-aminoadipic acid flux in ß-lactam-producing P . chrysogenum . The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the
-aminoadipic acid flux .
The MexR Repressor of the mexAB-oprM Multidrug Efflux Operon in Pseudomonas aeruginosa: Characterization of Mutations Compromising Activity. Lateef Adewoye, 2002.Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon . MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize . Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding . 2,5-Dialkylresorcinol Biosynthesis in Pseudomonas aurantiaca: Novel Head-to-Head Condensation of Two Fatty Acid-Derived Precursors. Brian Nowak-Thompson, 2003.2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by Pseudomonas aurantiaca (Pseudomonas fluorescens BL915) . We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a ß-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in Escherichia coli. Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster . Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of ß-ketoacyl thioester intermediates . Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order Cytophagales . Degradation and Transformability of DNA from Transgenic Leaves. MariaTeresa Ceccherini, 2003.The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum . Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene . These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues . Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil . Citrate Synthase Mutants of Sinorhizobium fredii USDA257 Form Ineffective Nodules with Aberrant Ultrastructure. Hari B. Krishnan, 2003.The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen . Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle . We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes . The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici . Southern blot analysis revealed that the fast-growing S . fredii strains and Rhizobium sp . strain NGR234 contained a single copy of the gene located in the bacterial chromosome . S . fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean . Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids . The infected cells contained large vacuoles and prominent starch grains . Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected . The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced . A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above . Our results demonstrate that a functional citrate synthase gene of S . fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation .
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