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Extensive Genomic Diversity in Pathogenic Escherichia coli and Shigella Strains Revealed by Comparative Genomic Hybridization Microarray . Satoru Fukiya, 2004.Escherichia coli, including the closely related genus Shigella, is a highly diverse species in terms of genome structure . Comparative genomic hybridization (CGH) microarray analysis was used to compare the gene content of E . coli K-12 with the gene contents of pathogenic strains . Missing genes in a pathogen were detected on a microarray slide spotted with 4,071 open reading frames (ORFs) of W3110, a commonly used wild-type K-12 strain . For 22 strains subjected to the CGH microarray analyses 1,424 ORFs were found to be absent in at least one strain . The common backbone of the E . coli genome was estimated to contain about 2,800 ORFs . The mosaic distribution of absent regions indicated that the genomes of pathogenic strains were highly diversified becasue of insertions and deletions . Prophages, cell envelope genes, transporter genes, and regulator genes in the K-12 genome often were not present in pathogens . The gene contents of the strains tested were recognized as a matrix for a neighbor-joining analysis . The phylogenic tree obtained was consistent with the results of previous studies . However, unique relationships between enteroinvasive strains and Shigella, uropathogenic, and some enteropathogenic strains were suggested by the results of this study . The data demonstrated that the CGH microarray technique is useful not only for genomic comparisons but also for phylogenic analysis of E . coli at the strain level . Mutational Analysis of Mesentericin Y105, an Anti-Listeria Bacteriocin, for Determination of Impact on Bactericidal Activity, In Vitro Secondary Structure, and Membrane Interaction. Dany Morisset, 2004.Mesentericin Y105 is a 37-residue bacteriocin produced by Leuconostoc mesenteroides Y105 that displays antagonistic activity against gram-positive bacteria such as Enterococcus faecalis and Listeria monocytogenes . It is closely related to leucocin A, an antimicrobial peptide containing ß-sheet and  -helical structures . To analyze structure-function relationships and the mode of action of this bacteriocin, we generated a collection of mesentericin derivatives . Mutations were obtained mostly by PCR random mutagenesis, and the peptides were produced by an original system of heterologous expression recently described (D . Morisset and J . Frère, Biochimie 84:569-576, 2002) . Ten derivatives were obtained displaying modifications at eight different positions in the mesentericin Y105 sequence . Purified peptides were incorporated into lysophosphatidylcholine micelles and analyzed by circular dichroism . The
-helical contents of these peptides were compared and related to their respective bactericidal activities . Moreover, studies of the intrinsic fluorescence of tryptophan residues naturally occurring at positions 18 and 37 revealed information about insertion of the peptides in micelles . A model for the mode of action of mesentericin Y105 and related bacteriocins is proposed .
The Unique tuf2 Gene from the Kirromycin Producer Streptomyces ramocissimus Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu. Lian N. Olsthoorn-Tieleman, 2002.Streptomyces ramocissimus, the producer of elongation factor Tu (EF-Tu)-targeted antibiotic kirromycin, contains three divergent tuf-like genes, with tuf1 encoding regular kirromycin-sensitive EF-Tu1; the functions of tuf2 and tuf3 are unknown . Analysis of the tuf gene organization in nine producers of kirromycin-type antibiotics revealed that they all contain homologues of tuf1 and sometimes of tuf3 but that tuf2 was found in S . ramocissimus only . The tuf2-flanking regions were sequenced, and the two tuf2-surrounding open reading frames were shown to be oriented in opposite directions . In vivo transcription analysis of the tuf2 gene displayed an upstream region with bidirectional promoter activity . The transcription start site of tuf2 was located approximately 290 nucleotides upstream of the coding sequence . Very small amounts of tuf2 transcripts were detected in both liquid- and surface-grown cultures of S . ramocissimus, consistent with the apparent absence of EF-Tu2 in total protein extracts . The tuf2 transcript level was not influenced by the addition of kirromycin to exponentially growing cultures . To assess the function of S . ramocissimus EF-Tu2, the protein was overexpressed in Streptomyces coelicolor LT2 . This strain is a J1501 derivative containing His6-tagged EF-Tu1 as the sole EF-Tu species, which facilitated the separation of EF-Tu2 from the interfering EF-Tu1 . S . ramocissimus EF-Tu1 and EF-Tu2 were indistinguishable in their ability to stimulate protein synthesis in vitro and exhibited the same kirromycin sensitivity, which excludes the possibility that EF-Tu2 is directly involved in the kirromycin resistance mechanism of S . ramocissimus . Unusual Integrase Gene Expression on the clc Genomic Island in Pseudomonas sp . Strain B13. V. Sentchilo, 2003.An unusual type of gene expression from an integrase promoter was found in cultures of the bacterium Pseudomonas sp . strain B13 . The promoter controls expression of the intB13 integrase gene, which is present near the right end of a 105-kb conjugative genomic island (the clc element) encoding catabolism of aromatic compounds . The enzymatic activity of integrase IntB13 is essential for site-specific integration of the clc element into the bacterial host's chromosome . By creating transcription fusions between the intB13 promoter and the gfp gene, we showed that integrase expression in strain B13 was inducible under stationary-phase conditions but, strangely, occurred in only a small proportion of individual bacterial cells rather than equally in the whole population . Integrase expression was significantly stimulated by growing cultures on 3-chlorobenzoate . High cell density, heat shock, osmotic shock, UV irradiation, and treatment with alcohol did not result in measurable integrase expression . The occurrence of the excised form of the clc element and an increase in the rates of clc element transfer in conjugation experiments correlated with the observed induction of the intB13'-gfp fusion in stationary phase and in the presence of 3-chlorobenzoate . This suggested that activation of the intB13 promoter is the first step in stimulation of clc transfer . To our knowledge, this is the first report of a chlorinated compound's stimulating horizontal transfer of the genes encoding its very metabolism . ISCce1 and ISCce2, Two Novel Insertion Sequences in Clostridium cellulolyticum. Hédia Maamar, 2003.Two new insertion sequences, ISCce1 and ISCce2, were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum . In these insertional mutants, the cipC gene was disrupted either by ISCce1 alone or by both ISCce1 and ISCce2 . ISCce1 is 1,292 bp long and has one open reading frame . The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS481 and IS3 families . Imperfect 23-bp inverted repeats were found near the extremities of ISCce1 . ISCce2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini . The open reading frame encodes a putative 398-amino-acid protein . This protein shows significant levels of identity with transposases belonging to the IS256 family . Upon transposition, both ISCce1 and ISCce2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site . ISCce1 is copied at least 20 times in the genome, as assessed by Southern blot analysis . ISCce2 was found to be mostly inserted into ISCce1 . In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C . cellulolyticum strain used . Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces. G. Douglas Inglis, 2003.This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment . Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms . The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction . With nested or seminested multiplex PCR, Campylobacter coli, C . fetus, C . hyointestinalis, and C . jejuni were detected in all fecal samples inoculated at  104 CFU g-1, and 50 to 83% of the samples inoculated at
103 CFU g-1 were positive . At
102 CFU g-1, C . fetus, C . hyointestinalis, and C . jejuni (17 to 50% of the samples) but not C . coli were detected by PCR . From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures . The most frequently isolated taxa were C . jejuni (152 isolates) and C . lanienae (42 isolates), but isolates of C . fetus subsp . fetus, Arcobacter butzleri, and A . skirrowii also were recovered ( 2 isolates per taxon) . Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested . With genus-specific primers, Campylobacter DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested . With nested primers, C . jejuni and C . lanienae were detected in 25 and 67% of the samples, respectively . In no instance was DNA from either C . coli, C . fetus, or C . hyointestinalis detected in uninoculated bovine feces . PCR was more sensitive than isolation on microbiological media for detecting C . lanienae (17%) but not C . jejuni . Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of Campylobacter species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding .
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