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Surface-Associated Flagellum Formation and Swarming Differentiation in Bacillus subtilis Are Controlled by the ifm Locus. Sonia Senesi, 2004.Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures.No information is available regarding the regulation of flagellar formation in B . subtilis in response to contact with a solid surface . One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates . This study describes the swarming abilityof a B . subtilis hypermotile mutant harboring a mutation inthe ifm locus that has long been known to affect the degreeof flagellation and motility in liquid media . On solid media,the mutant produces elongated and hyperflagellated cells displayinga 10-fold increase in extracellular flagellin . In contrast tothe mutant, the parental strain, as well as other laboratorystrains carrying a wild-type ifm locus, fails to activate aswarm response . Furthermore, it stops to produce flagella whentransferred from liquid to solid medium . Evidence is providedthat the absence of flagella is due to the lack of flagellingene expression . However, restoration of flagellin synthesisin cells overexpressing  D
or carrying a deletion of flgM doesnot recover the ability to
assemble flagella . Thus, the ifmgene plays a determinantal
role in the ability of B . subtilisto contact with solid
surfaces.
Multiple Paths for Nonphysiological Transport of K+ in Escherichia coli. Ed T. Buurman, 2004.Mutants of Escherichia coli lacking all of the known saturable K+ transport systems, "triple mutants," require elevated K+ concentrations for growth . K+ transport activity in such mutants, called TrkF activity, has low substrate specificity and a low rate that increases with increasing external pH . Attempts to isolate mutants requiring even higher concentrations of K+ failed, implying that either TrkF is essential or is composed of multiple minor K+ transport activities . Instead, we sought mutations that allowed triple mutants to grow at lower K+ concentrations . Mutations so identified include ones altering MscL, the large mechanosensitive channel, or Opp, the oligopeptide permease . However, a possible contribution of wild-type Opp and MscL to TrkF activity was not proven . In contrast, expression of wild-type ProP, TrkG, and TrkH proteins increased uptake when encoded on multicopy plasmids . In all of these situations, the driving force for K+ appeared to be the transmembrane electric potential, and in most cases substrate specificity was low; these are characteristics of TrkF activity . These results support the view that TrkF is composed of multiple, "aberrant" K+ transport activities, i.e., paths that, regardless of their physiological function, allow K+ to cross the cell membrane by a uniport process . Tetracycline Resistance in Chlamydia suis Mediated by Genomic Islands Inserted into the Chlamydial inv-Like Gene. Jae Dugan, 2004.Many strains of Chlamydia suis, a pathogen of pigs, express a stable tetracycline resistance phenotype . We demonstrate that this resistance pattern is associated with a resistance gene, tet(C), in the chlamydial chromosome . Four related genomic islands were identified in seven tetracycline-resistant C . suis strains . All resistant isolates carry the structural gene tet(C) and the tetracycline repressor gene tetR(C) . The islands share significant nucleotide sequence identity with resistance plasmids carried by a variety of different bacterial species . Three of the four tet(C) islands also carry a novel insertion sequence that is homologous to the IS605 family of insertion sequences . In each strain, the resistance gene and associated sequences are recombined into an identical position in a gene homologous to the inv gene of the yersiniae . These genomic islands represent the first examples of horizontally acquired DNA integrated into a natural isolate of chlamydiae or within any other obligate intracellular bacterium . Liquid Serial Dilution Is Inferior to Solid Media for Isolation of Cultures Representative of the Phylum-Level Diversity of Soil Bacteria. Liesbeth Schoenborn, 2004. FtsA Mutants of Bacillus subtilis Impaired in Sporulation. Jennifer T. Kemp, 2002.Spore formation in Bacillus subtilis involves a switch in the site of cell division from the midcell to a polar position . Both medial division and polar division are mediated in part by the actin-like, cytokinetic protein FtsA . We report the isolation of an FtsA mutant (FtsAD265G) that is defective in sporulation but is apparently unimpaired in vegetative growth . Sporulating cells of the mutant reach the stage of asymmetric division but are partially blocked in the subsequent morphological process of engulfment . As judged by fluorescence microscopy and electron microscopy, the FtsAD265G mutant produces normal-looking medial septa but immature (abnormally thin) polar septa . The mutant was unimpaired in transcription under the control of Spo0A, the master regulator for entry into sporulation, but was defective in transcription under the control of  F, a regulatory protein whose activation is known to depend on polar division . An amino acid substitution at a residue (Y264) adjacent to D265 also caused a defect in sporulation . D265 and Y264 are conserved among endospore-forming bacteria, raising the possibility that these residues are involved in a sporulation-specific protein interaction that facilitates maturation of the sporulation septum and the activation of
F .
A Sinorhizobium meliloti Lipopolysaccharide Mutant Altered in Cell Surface Sulfation. David H. Keating, 2002.The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates . Nodule development requires a bacterial signal referred to as Nod factor, which in Sinorhizobium meliloti is a ß-(1,4)-linked tetramer of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the nonreducing end and a critical 6-O-sulfate at the reducing end . This sulfate modification requires the action of three gene products: nodH, which catalyzes the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate, 3'-phosphoadenosine-5'-phosphosulfate . It was previously reported that S . meliloti cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ . We have further characterized this unique form of bacterial carbohydrate modification . Our studies have determined that one of the nodPQ mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation . We cloned the gene affected by this mutation (referred to as lps-212) and found it to be an allele of lpsL, a gene previously predicted to encode a UDP-glucuronic acid epimerase . We demonstrated that lpsL encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212 mutant . The lps-212 mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo . Finally, we determined that the lps-212 mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely . Increased Production of Folate by Metabolic Engineering of Lactococcus lactis. Wilbert Sybesma, 2003.The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form . Only small amounts of the produced folate are released in the extracellular medium . Five genes involved in folate biosynthesis were identified in a folate gene cluster in L . lactis MG1363: folA, folB, folKE, folP, and folC . The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I . The overexpression of folKE in L . lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold . The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell . The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis . Effects of Iron Limitation on Adherence and Cell Surface Carbohydrates of Corynebacterium diphtheriae Strains. Lílian de Oliveira Moreira, 2003.Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment . The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp . mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells . A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl3 . The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively . Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells . Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions . Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface . Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in D-mannose and D-galactose residues on the surfaces of both strains . Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C . diphtheriae surface carbohydrate moieties . The significant changes in the degree of lectin binding specific for D-mannose, D-galactose and sialic acid residues may have an effect on binding of host cells . The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems . For C . diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes .
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