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Genes Involved in Formation of Structured Multicellular Communities by Bacillus subtilis. Steven S. Branda, 2004.The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization . Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation . To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B . subtilis genome . This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation . This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media . In this way we identified six genes that are involved in the development of multicellular communities . These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function) . Further analysis revealed that these six genes play different roles in B . subtilis community development . Movement of Viruses between Biomes. Emiko Sano, 2004.Viruses are abundant in all known ecosystems . In the present study, we tested the possibility that viruses from one biome can successfully propagate in another . Viral concentrates were prepared from different near-shore marine sites, lake water, marine sediments, and soil . The concentrates were added to microcosms containing dissolved organic matter as a food source (after filtration to allow 100-kDa particles to pass through) and a 3% (vol/vol) microbial inoculum from a marine water sample (after filtration through a 0.45-µm-pore-size filter) . Virus-like particle abundances were then monitored using direct counting . Viral populations from lake water, marine sediments, and soil were able to replicate when they were incubated with the marine microbes, showing that viruses can move between different ecosystems and propagate . These results imply that viruses can laterally transfer DNA between microbes in different biomes . Characterization of Two Bacillus thuringiensis Genes Identified by In Vivo Screening of Virulence Factors. Sinda Fedhila, 2004.Bacillus thuringiensis vegetative cells are known to be highly pathogenic when injected into the hemocoel of susceptible insect larvae . This pathogenicity is due to the capacity of B . thuringiensis to cause septicemia in the host . We screened a B . thuringiensis mini-Tn10 insertion library for loss of virulence against Bombyx mori larvae on injection into the hemocoel . Three clones with attenuated virulence were isolated, corresponding to two different mini-Tn10 insertions mapping to the yqgB/yqfZ locus . Single disruptions of the yqgB and yqfZ genes did not affect virulence against B . mori . In contrast, the inactivation of both genes simultaneously reproduced the effect of the mini-Tn10 insertion and resulted in a significant delay to infection . The double  yqgB
yqfZ mutant was also nonmotile, and its growth was affected at 25°C . We analyzed lacZ transcriptional fusions and detected promoter activity upstream from yqgB at 25 and 37°C . Overall, our findings suggest that the yqgB and yqfZ genes encode adaptive factors that may act in synergy, enabling the bacteria to cope with the physical environment in vivo, facilitating colonization of the host .
Characterization of a Bacillus subtilis Thermosensitive Teichoic Acid-Deficient Mutant: Gene mnaA (yvyH) Encodes the UDP-N-Acetylglucosamine 2-Epimerase. Blazenka Soldo, 2002.The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G  T-A transitions in the mnaA gene . At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content . MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit .
Postdivisional Synthesis of the Sporosarcina ureae DNA Translocase SpoIIIE either in the Mother Cell or in the Prespore Enables Bacillus subtilis To Translocate DNA from the Mother Cell to the Prespore. Vasant K. Chary, 2003.The differentiation of vegetative cells of Bacillus subtilis into spores involves asymmetric cell division, which precedes complete chromosome partitioning . The DNA translocase SpoIIIE is required to translocate the origin distal 70% of the chromosome from the larger mother cell into the smaller prespore, the two cells that result from the division . We have tested the effect of altering the time and location of SpoIIIE synthesis on spore formation . We have expressed the spoIIIE homologue from Sporosarcina ureae in B . subtilis under the control of different promoters . Expression from either a weak mother cell-specific (  E) promoter or a weak prespore-specific (F) promoter partly complemented the sporulation defect of a spoIIIE36 mutant; however, expression from a strong prespore-specific (F) promoter did not . DNA translocation from the mother cell to the prespore was assayed using spoIIQ-lacZ inserted at thrC; transcription of spoIIQ occurs only in the prespore . Translocation of thrC::spoIIQ-lacZ into the prespore occurred efficiently when spoIIIESu was expressed from the weak
E- or
F-controlled promoters but not when it was expressed from the strong
F-controlled promoter . It is speculated that the mechanism directing SpoIIIE insertion into the septum in the correct orientation may accommodate slow postseptational, prespore-specific SpoIIIE synthesis but may be swamped by strong prespore-specific synthesis .
Rapid Identification of Listeria Species by Using Restriction Fragment Length Polymorphism of PCR-Amplified 23S rRNA Gene Fragments. Delphine Paillard, 2003.A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level . Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA . S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI . This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants . All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium . The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua . Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures . Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains . The 158 single identifiable isolates were 92 L . monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L . innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain . The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species . Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology .
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