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Denitrification Genes Regulate Brucella Virulence in Mice. Seung-Hun Baek, 2004.Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world . Genome sequencing of B . suis and B . melitensis revealed that both are complete denitrifiers . To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrificationin the closely related B . neotomae was undertaken . In contrastto B . suis and B . melitensis, it was found that B . neotomae is a partial denitrifier that can reduce nitrate to nitritebut no further . Examination of the B . neotomae genome showedthat a deletion in the denitrification gene cluster resultedin complete loss of nirV and the partial deletion of nirK and nnrA . Even though the nor operon is intact, a norC-lacZ promoterfusion was not expressed in B . neotomae . However, the norC-lacZfusion was expressed in the related denitrifier Agrobacteriumtumefaciens, suggesting that the lack of expression in B . neotomaeis due to inactivation of NnrA . A narK-lacZ promoter fusionwas found to exhibit nitrate-dependent expression consistentwith the partial denitrifier phenotype . Complementation of thedeleted region in B . neotomae by using nirK, nirV, and nnrAfrom B . melitensis restored the ability of B . neotomae to reducenitrite . There was a significant difference in the death ofIRF-1–/– mice when infected with B . neotomae containingnirK, nirV, and nnrA and those infected with wild-type B . neotomae.The wild-type strain killed all the infected mice, whereas mostof the mice infected with B . neotomae containing nirK, nirV, and nnrA survived. Identification and Characterization of Transposable Elements of Paracoccus pantotrophus. Dariusz Bartosik, 2003.We studied diversity and distribution of transposable elements residing in different strains (DSM 11072, DSM 11073, DSM 65, and LMD 82.5) of a soil bacterium Paracoccus pantotrophus (  -Proteobacteria) . With application of a shuttle entrapment vector pMEC1, several novel insertion sequences (ISs) and transposons (Tns) have been identified . They were sequenced and subjected to detailed comparative analysis, which allowed their characterization (i.e., identification of transposase genes, terminal inverted repeats, as well as target sequences) and classification into the appropriate IS or Tn families . The frequency of transposition of these elements varied and ranged from 10-6 to 10-3 depending on the strain . The copy number, localization (plasmid or chromosome), and distribution of these elements in the Paracoccus species P . pantotrophus, P . denitrificans, P . methylutens, P . solventivorans, and P . versutus were analyzed . This allowed us to distinguish elements that are common in paracocci (ISPpa2, ISPpa3—both of the IS5 family—and ISPpa5 of IS66 family) as well as strain-specific ones (ISPpa1 of the IS256 family, ISPpa4 of the IS5 family, and Tn3434 and Tn5393 of the Tn3 family), acquired by lateral transfer events . These elements will be of a great value in the design of new genetic tools for paracocci, since only one element (IS1248 of P . denitrificans) has been described so far in this genus .
Characterization of Three New Competence-Regulated Operons in Haemophilus influenzae. Timothy M. VanWagoner, 2004.Haemophilus influenzae is one of a growing number of bacteria in which the natural ability to uptake exogenous DNA for potential genomic transformation has been recognized . To date, several operons involved in transformation in this organism have been described . These operons are characterized by a conserved 22-bp regulatory element upstream of the first gene and are induced coincident with transfer from rich to nutrient-depleted media . The previously identified operons comprised genes encoding proteins that include members of the type II secretion system and type IV pili, shown to be essential for transformation in other bacteria, and other proteins previously identified as required for transformation in H . influenzae . In the present study, three novel competence operons were identified by comparative genomics and transcriptional analysis . These operons have been further characterized by construction of null mutants and examination of the resulting transformation phenotypes . The putative protein encoded by the HI0366 gene was shown to be essential for DNA uptake, but not binding, and is homologous to a protein shown to be required for pilus biogenesis and twitching motility in Pseudomonas aeruginosa . An insertion in HI0939 abolished both DNA binding and uptake . The predicted product of this gene shares characteristics with PulJ, a pseudopilin involved in pullulanase export in Klebsiella oxytoca . Bacterial Conversion of Hydroxylamino Aromatic Compounds by both Lyase and Mutase Enzymes Involves Intramolecular Transfer of Hydroxyl Groups. Lloyd J. Nadeau, 2003.Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds . The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism) . The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer . The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp . strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism . GC-MS analysis of the reaction products formed in H218O did not indicate any 18O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R . eutropha JMP134 . During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp . strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was 18O labeled . The other hydroxyl group in the product must have come from the substrate . The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol . The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups . Biofilm Formation and the Presence of the Intercellular Adhesion Locus ica among Staphylococci from Food and Food Processing Environments. Trond Møretrø, 2003.In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence . Biofilm formation may also be of importance for survival and virulence in food-related staphylococci . In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene . A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively . Glucose and sodium chloride stimulated biofilm formation . The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested . The presence of icaA was positively correlated with strong biofilm formation . The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100% . Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms . Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants .
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