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Intraspecific Phylogeny and Lineage Group Identification Based on the prfA Virulence Gene Cluster of Listeria monocytogenes  .Todd J. Ward, 2004.Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years . In order to test hypotheses regarding L . monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L . monocytogenes isolates . The results of the multigene phylogenetic analyses confirm that L . monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3 . In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested . However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment . Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L . monocytogenes lineages may represent distinct species within the framework of the cohesion species concept . Metabolism of Zearalenone by Genetically Modified Organisms Expressing the Detoxification Gene from Clonostachys rosea. Naoko Takahashi-Ando, 2004.Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101 . An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli . Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized . Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45°C and pH 10.5 (kcat at 30°C, 0.51 s–1) . The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors . ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the kcat was highest with zearalanone (1.5 s–1) and lowest with ß-zearalenol (0.075 s–1) . EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity . Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions . We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN . While recombinant E . coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, ß-zearalenol, by an as-yet-unidentified reductase and remained in the medium . Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H . El-Nezami, N . Polychronaki, S . Salminen, and H . Mykkuäne, Appl . Environ . Microbiol . 68:3545-3549, 2002) . An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed . A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm Formation. Yung-Hua Li, 2002.In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J . Bacteriol . 183:897-908, 2001) . Here, we demonstrate that this system also plays a role in the ability of S . mutans to initiate biofilm formation . To test this hypothesis, S . mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation . The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy . The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm . The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass . Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms . The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP . Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea . We conclude that the quorum-sensing signaling system essential for genetic competence in S . mutans is important for the formation of biofilms by this gram-positive organism . Population Genetic Structure of Legionella pneumophila Inferred from RNA Polymerase Gene (rpoB) and DotA Gene (dotA) Sequences. Kwan Soo Ko, 2002. Identification of MpaA, an Amidase in Escherichia coli That Hydrolyzes the  -D-Glutamyl-meso-Diaminopimelate Bond in Murein Peptides.Tsuyoshi Uehara, 2003.MpaA amidase was identified in Escherichia coli by its amino acid sequence homology with the ENP1 endopeptidase from Bacillus sphaericus . The enzymatic activity of MpaA, i.e., hydrolysis of the -D-glutamyl-diaminopimelic acid bond in the murein tripeptide L-alanyl--D-glutamyl-meso-diaminopimelic acid, was demonstrated in the cell extract of a strain expressing mpaA from a multicopy plasmid . An mpaA mpl (murein peptide ligase) double mutant accumulated large amounts of murein tripeptide in its cytoplasm, consistent with the premise that MpaA degrades the tripeptide if its recycling via the peptidoglycan biosynthetic pathway is blocked .
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