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DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis. Giuseppe Amati, 2004.Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth . The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, suchas competence development, the production of exoenzymes, andmotility . The degU32[Hy] mutation extends the half-life of the phosphorylated form of DegU [DegU-P]; this in turn increasesthe production of alkaline protease, levan-sucrase, and otherexoenzymes and inhibits motility and the production of flagella.The expression of the flagellum-specific sigma factor SigD,of the flagellin gene hag, and of the fla-che operon is stronglyreduced in a degU32[Hy] genetic background . To investigate themechanism of action of DegU-P on motility, we isolated mutantsof degU32[Hy] that completely suppressed the motility deficiency.The mutations were genetically mapped and characterized by PCRand sequencing . Most of the mutations were found to delete atranscriptional termination signal upstream of the main flagellaroperon, fla-che, thus allowing transcriptional readthrough fromthe cod operon . Two additional mutations improved the  A-dependent
promoter sequence of the fla-che operon . Using an electrophoretic
mobility shift assay, we have demonstrated that purified DegU
binds specifically to the PA promoter region of the fla-che
operon . The data suggest that DegU represses transcription of
the fla-che operon, and they indicate a central role of the
operon in regulating the synthesis and assembly of flagella.
Lack of Evidence that DNA in Antibiotic Preparations Is a Source of Antibiotic Resistance Genes in Bacteria from Animal or Human Sources. Susanna K. P. Lau, 2004.Although DNA encoding antibiotic resistance has been discovered in antibiotic preparations, its significance for the development of antibiotic resistance in bacteria is unknown . No phylogenetic evidence was obtained for recent horizontal transfer of antibiotic resistance genes from antibiotic-producing organisms to bacteria from human or animal sources . Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134. Tai Man Louie, 2002.Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP) . Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized . In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate . We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts . Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ) . The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases . Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively . The three genes were individually inactivated in JMP134 . The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ . Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ . For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase . TcpC produced in E . coli oxidized 6-CHQ to 2-chloromaleylacetate . Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC . Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis . The function of TcpB remains unknown . A Mutation in the Essential Gene gmk (Encoding Guanlyate Kinase) Generates a Requirement for Adenine at Low Temperature in Salmonella enterica. Brian J. Beck, 2003.In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides . Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature . Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher Km for ATP than the wild-type enzyme . The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference . We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes . Transcriptional Regulation of Vibrio cholerae Hemagglutinin/Protease by the Cyclic AMP Receptor Protein and RpoS. Anisia J. Silva, 2004.Vibrio cholerae secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HA/protease), which is encoded by hapA and displays a broad range of potentially pathogenic activities . Production of HA/protease requires transcriptional activation by the quorum-sensing regulator HapR . In this study we demonstrate that transcription of hapA is growth phase dependent and specifically activated in the deceleration and stationary growth phases . Addition of glucose in these phases repressed hapA transcription by inducing V . cholerae to resume exponential growth, which in turn diminished the expression of a rpoS-lacZ transcriptional fusion . Contrary to a previous observation, we demonstrate that transcription of hapA requires the rpoS-encoded  s factor . The cyclic AMP (cAMP) receptor protein (CRP) strongly enhanced hapA transcription in the deceleration phase . Analysis of rpoS and hapR mRNA in isogenic CRP+ and CRP– strains suggested that CRP enhances the transcription of rpoS and hapR . Analysis of strains containing hapR-lacZ and hapA-lacZ fusions confirmed that hapA is transcribed in response to concurrent quorum-sensing and nutrient limitation stimuli . Mutations inactivating the stringent response regulator RelA and the HapR-controlled AphA regulator did not affect HA/protease expression . Electrophoretic mobility shift experiments showed that pure cAMP-CRP and HapR alone do not bind the hapA promoter . This result suggests that HapR activation of hapA differs from its interaction with the aphA promoter and could involve additional factors .
Bacterial Diversity and Sulfur Cycling in a Mesophilic Sulfide-Rich Spring. Mostafa S. Elshahed, 2003.An artesian sulfide- and sulfur-rich spring in southwestern Oklahoma is shown to sustain an extremely rich and diverse microbial community . Laboratory incubations and autoradiography studies indicated that active sulfur cycling is occurring in the abundant microbial mats at Zodletone spring . Anoxygenic phototrophic bacteria oxidize sulfide to sulfate, which is reduced by sulfate-reducing bacterial populations . The microbial community at Zodletone spring was analyzed by cloning and sequencing 16S rRNA genes . A large fraction (83%) of the microbial mat clones belong to sulfur- and sulfate-reducing lineages within  -Proteobacteria, purple sulfur
 -Proteobacteria,
 -Proteobacteria, Chloroflexi, and filamentous Cyanobacteria of the order Oscillatoria as well as a novel group within
-Proteobacteria . The 16S clone library constructed from hydrocarbon-exposed sediments at the source of the spring had a higher diversity than the mat clone library (Shannon-Weiner index of 3.84 compared to 2.95 for the mat), with a higher percentage of clones belonging to nonphototrophic lineages (e.g., Cytophaga, Spirochaetes, Planctomycetes, Firmicutes, and Verrucomicrobiae) . Many of these clones were closely related to clones retrieved from hydrocarbon-contaminated environments and anaerobic hydrocarbon-degrading enrichments . In addition, 18 of the source clones did not cluster with any of the previously described microbial divisions . These 18 clones, together with previously published or database-deposited related sequences retrieved from a wide variety of environments, could be clustered into at least four novel candidate divisions . The sulfate-reducing community at Zodletone spring was characterized by cloning and sequencing a 1.9-kb fragment of the dissimilatory sulfite reductase (DSR) gene . DSR clones belonged to the Desulfococcus-Desulfosarcina-Desulfonema group, Desulfobacter group, and Desulfovibrio group as well as to a deeply branched group in the DSR tree with no representatives from cultures . Overall, this work expands the division-level diversity of the bacterial domain and highlights the complexity of microbial communities involved in sulfur cycling in mesophilic microbial mats .
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