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Comparative Whole-Genome Hybridization Reveals Genomic Islands in Brucella Species. Gireesh Rajashekara, 2004.Brucella species are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans . Based on host preference, the genus is divided into six species . Brucella abortus, B . melitensis, and B . suis are pathogenic to humans, whereas B . ovis and B . neotomae are nonpathogenic to humans and B . canis human infections are rare . Limited genome diversity exists among Brucella species . Comparison of Brucella species whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction . To facilitate such studies, we used the complete genome sequence of B . melitensis 16M, the species highly pathogenic to humans, to construct a genomic microarray . Hybridization of labeled genomic DNA from Brucella species to this microarray revealed a total of 217 open reading frames (ORFs) altered in five Brucella species analyzed . These ORFs are often found in clusters (islands) in the 16M genome . Examination of the genomic context of these islands suggests that many are horizontally acquired . Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species . These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certain Brucella species for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences . Nosocomial Outbreak of Extended-Spectrum ß-Lactamase SHV-5-Producing Isolates of Pseudomonas aeruginosa in Athens, Greece. Laurent Poirel, 2004.Seven nonrepetitive Pseudomonas aeruginosa isolates producing the clavulanic acid-inhibited extended-spectrum ß-lactamase SHV-5 were isolated in the same hospital in Athens, Greece, from 1998 to 2002 . All isolates except one were clonally related, and the blaSHV-5 gene was chromosomally located . This study underlined that this gene, which is widespread in Enterobacteriaceae in Greece, may disseminate also in P . aeruginosa . Phylogenetic Analysis Reveals Multiple Lateral Transfers of Adenosine-5'-Phosphosulfate Reductase Genes among Sulfate-Reducing Microorganisms. Michael W. Friedrich, 2002.Lateral gene transfer affects the evolutionary path of key genes involved in ancient metabolic traits, such as sulfate respiration, even more than previously expected . In this study, the phylogeny of the adenosine-5'-phosphosulfate (APS) reductase was analyzed . APS reductase is a key enzyme in sulfate respiration present in all sulfate-respiring prokaryotes . A newly developed PCR assay was used to amplify and sequence a fragment (  900 bp) of the APS reductase gene, apsA, from a taxonomically wide range of sulfate-reducing prokaryotes (n = 60) . Comparative phylogenetic analysis of all obtained and available ApsA sequences indicated a high degree of sequence conservation in the region analyzed . However, a comparison of ApsA- and 16S rRNA-based phylogenetic trees revealed topological incongruences affecting seven members of the Syntrophobacteraceae and three members of the Nitrospinaceae, which were clearly monophyletic with gram-positive sulfate-reducing bacteria (SRB) . In addition, Thermodesulfovibrio islandicus and Thermodesulfobacterium thermophilum, Thermodesulfobacterium commune, and Thermodesulfobacterium hveragerdense clearly branched off between the radiation of the
 -proteobacterial gram-negative SRB and the gram-positive SRB and not close to the root of the tree as expected from 16S rRNA phylogeny . The most parsimonious explanation for these discrepancies in tree topologies is lateral transfer of apsA genes across bacterial divisions . Similar patterns of insertions and deletions in ApsA sequences of donor and recipient lineages provide additional evidence for lateral gene transfer . From a subset of reference strains (n = 25), a fragment of the dissimilatory sulfite reductase genes (dsrAB), which have recently been proposed to have undergone multiple lateral gene transfers (M . Klein et al., J . Bacteriol . 183:6028–6035, 2001), was also amplified and sequenced . Phylogenetic comparison of DsrAB- and ApsA-based trees suggests a frequent involvement of gram-positive and thermophilic SRB in lateral gene transfer events among SRB .
Genetic Analysis of the AdnA Regulon in Pseudomonas fluorescens: Nonessential Role of Flagella in Adhesion to Sand and Biofilm Formation. Eduardo A. Robleto, 2003.AdnA is a transcription factor in Pseudomonas fluorescens that affects flagellar synthesis, biofilm formation, and sand adhesion . To identify the AdnA regulon, we used a promoterless Tn5-lacZ element to study the phenotypes of insertion mutants in the presence and absence of AdnA . Of 12,000 insertions, we identified seven different putative open reading frames (ORFs) activated by AdnA (named aba for activated by AdnA) . aba120 and aba177 showed homology to flgC and flgI, components of the basal body of the flagella in Pseudomonas aeruginosa . Two other insertions, aba18 and aba51, disrupted genes affecting chemotaxis . The mutant loci aba160 (possibly affecting lipopolysaccharide synthesis) and aba175 (unknown function) led to loss of flagella . The mutant bearing aba203 became motile when complemented with adnA, but the mutated gene showed no similarity to known genes . Curiously, aba18, aba51, aba160, and aba203 mutants formed biofilms even in the absence of AdnA, suppressing the phenotype of the adnA deletion mutant . The combined findings suggest that flagella are nonessential for sand attachment or biofilm formation . Sequence and promoter analyses indicate that AdnA affects at least 23 ORFs either directly or by polar effects . These results support the concept that AdnA regulates cell processes other than those directly related to flagellar synthesis and define a broader cadre of genes in P . fluorescens than that described so far for its homolog, FleQ, in P . aeruginosa . Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of Vibrio cholerae. Manrong Li, 2003.Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene . The ctx genes reside in the genome of a filamentous phage (CTX  ), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPI . In order to determine the prevalence of horizontal transfer of VPI and CTX among nonepidemic (non-O1 and non-O139 serogroups) V . cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB . In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster . Seven of the 11 VPI+ strains have also acquired the CTX . Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTX prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains . The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements . In addition, several CTX prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains . These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTX independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain .
Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States. Guiqing Wang, 2003.The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy . In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B . burgdorferi, in field-collected Ixodes scapularis ticks . The sensitivity of qPCR for detection of B . burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer . Of the 498 I . scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay . The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick . No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B . burgdorferi (P = 0.39) . A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed . From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice . These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B . burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes .
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