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Control of Glucose- and NaCl-Induced Biofilm Formation by rbf in Staphylococcus aureus. Yong Lim, 2004.Both Staphylococcus aureus and S . epidermidis are capable of forming biofilm on biomaterials . We used Tn917 mutagenesis to identify a gene, rbf, affecting biofilm formation in S . aureus NCTC8325-4 . Sequencing revealed that Rbf contained a consensus region signature of the AraC/XylS family of regulators, suggesting that Rbf is a transcriptional regulator . Insertional duplication inactivation of the rbf gene confirmed that the gene was involved in biofilm formation on polystyrene and glass . Phenotypic analysis of the wild type and the mutant suggested that the rbf gene mediates the biofilm formation of S . aureus at the multicellular aggregation stage rather than at initial attachment . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the mutation resulted in the loss of an  190-kDa
protein . Biofilm production by the mutant could be restored by
complementation with a 2.5-kb DNA fragment containing the rbf
gene . The rbf-specific mutation affected the induction of
biofilm formation by glucose and a high concentration of NaCl but not
by ethanol . The mutation did not affect the transcription of the
ica genes previously shown to be required for biofilm formation .
Taken together, our results suggest that the rbf gene is
involved in the regulation of the multicellular aggregation step of
S . aureus biofilm formation in response to glucose and salt
and that this regulation may be mediated through the 190-kDa protein .
Host Physiology and Pathogenic Variation of Cochliobolus heterostrophus Strains with Mutations in the G Protein Alpha Subunit, CGA1. Ofir Degani, 2004. Global Genomic Analysis of AlgU (  E)-Dependent Promoters (Sigmulon) in Pseudomonas aeruginosa and Implications for Inflammatory Processes in Cystic Fibrosis.Aaron M. Firoved, 2002.The conversion of Pseudomonas aeruginosa to the mucoid phenotype coincides with the establishment of chronic respiratory infections in cystic fibrosis (CF) . A major pathway of conversion to mucoidy in clinical strains of P . aeruginosa is dependent upon activation of the alternative sigma factor AlgU (P . aeruginosa E) . Here we initiated studies of AlgU-dependent global expression patterns in P . aeruginosa in order to assess whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are turned on during conversion to mucoidy . Using genomic information and the consensus AlgU promoter sequence, we identified 35 potential AlgU (E) promoter sites on the P . aeruginosa chromosome . Each candidate promoter was individually tested by reverse transcription and mRNA 5'-end mapping using RNA isolated from algU+ and algU::Tcr mutant cells . A total of 10 new AlgU-dependent promoters were identified, and the corresponding mRNA start sites were mapped . Two of the 10 newly identified AlgU promoters were upstream of predicted lipoprotein genes . Since bacterial lipoproteins have been implicated as inducers of inflammatory pathways, we tested whether lipopeptides corresponding to the products of the newly identified AlgU-dependent lipoprotein genes, lptA and lptB, had proinflammatory activity . In human peripheral blood monocyte-derived macrophages the peptides caused production of interleukin-8, a proinflammatory chemokine typically present at excessively high levels in the CF lung . Our studies show how genomic information can be used to uncover on a global scale the genes controlled by a given
factor (collectively termed here sigmulon) using conventional molecular tools . In addition, our data suggest the existence of a previously unknown connection between conversion to mucoidy and expression of lipoproteins with potential proinflammatory activity . This link may be of significance for infections and inflammatory processes in CF .
In Vitro and In Vivo Functional Activity of Chlamydia MurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance. Andrea J. McCoy, 2003.Organisms of Chlamydia spp . are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion . The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG . In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis . The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli . After transduction of a lethal  murA mutation into the strain, viability of the E . coli strain became dependent upon expression of the C . trachomatis murA. DNA sequence analysis of murA from C . trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA . In E . coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA . In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin . Growth of C . trachomatis was also resistant to fosfomycin . Moreover, fosfomycin resistance was imparted to the E . coli strain expressing the chlamydial murA . Conversion of C . trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA . mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C . trachomatis infection . Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp . We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division . Elucidating the existence of PG in Chlamydia spp . is of significance for the development of novel antibiotics targeting the chlamydial cell wall .
A Combined Model To Predict the Functionality of the Bacteriocin-Producing Lactobacillus sakei Strain CTC 494. Frédéric Leroy, 2003.The use of bacteriocin-producing lactic acid bacteria for improved food fermentation processes seems promising . However, lack of fundamental knowledge about the functionality of bacteriocin-producing strains under food fermentation conditions hampers their industrial use . Predictive microbiology or a mathematical estimation of microbial behavior in food ecosystems may help to overcome this problem . In this study, a combined model was developed that was able to estimate, from a given initial situation of temperature, pH, and nutrient availability, the growth and self-inhibition dynamics of a bacteriocin-producing Lactobacillus sakei CTC 494 culture in (modified) MRS broth . Moreover, the drop in pH induced by lactic acid production and the bacteriocin activity toward Listeria as an indicator organism were modeled . Self-inhibition was due to the depletion of nutrients as well as to the production of lactic acid . Lactic acid production resulted in a pH drop, an accumulation of toxic undissociated lactic acid molecules, and a shift in the dissociation degree of the growth-inhibiting buffer components . The model was validated experimentally . Population Structure of Alexandrium (Dinophyceae) Cyst Formation-Promoting Bacteria in Hiroshima Bay, Japan. Masao Adachi, 2003.A total of 31 bacterial isolates that have potential Alexandrium cyst formation-promoting activity (Alex-CFPB) were isolated from Hiroshima Bay (Japan), which is characterized by seasonal blooms of the toxic dinoflagellate Alexandrium tamarense . The population structure of Alex-CFPB was analyzed by means of restriction fragment length polymorphism analysis of the 16S rRNA genes (16S rDNA) . Fourteen ribotypes, A to N, were observed among the 31 isolates of Alex-CFPB by using four restriction enzymes, MboI, HhaI, RsaI and BstUI . Among them, seven isolates, which were obtained from the seawater samples taken during the peak and termination periods of the A . tamarense bloom in 1998, belonged to ribotype A . This result suggests that bacterial strains of ribotype A may be dominant in the Alex-CFPB assemblages during these periods . The partial 16S rDNA-based phylogenetic tree of 10 ribotypes studied showed that nine of them fell into the Rhodobacter group of the  subclass of the Proteobacteria . Eight of nine ribotypes of the Rhodobacter group fell into the lineage of the Roseobacter subgroup, and one fell into the Rhodobacter subgroup . The non-Rhodobacter group type fell into the Marinobacterium-Neptunomonas-Pseudomonas group of the
 -Proteobacteria . Isolates of Alex-CFPB ribotypes A and C do not have clear growth-promoting activities but have strong cyst formation-promoting activities (CFPAs) under our laboratory conditions . These results show that the Alex-CFPB assemblage may consist of various bacteria that belong mainly to the Roseobacter group and have strong CFPAs . These results suggest that not only the Alexandrium cyst formation-inhibiting bacteria (Alex-CFIB) reported previously but also Alex-CFPB, especially bacteria of ribotype A, may play significant roles in the process of encystment and bloom dynamics of Alexandrium in the natural environment .
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