Project description:DNA microarray analysis was used to compare the differential gene-expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22 kb genomic island covering a cluster of 34 genes (LA0186 to LA0219) was actively expressed in both strains but concomitantly up-expressed in strain 56601 in contrast to that of IPAV. RT-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 out of 34 CDSs encoding prophage-like proteins, of which, LA0195 is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II and III, all proximal to LA0195, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter CAT enzyme activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to LA0195 in vitro. These results suggested that LA0195 is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems resided outside of this region within the genome maybe responsible for the differential expression profiling in these two strains. Keywords: Differential expression of L.interrogans 56601 and IPAV in 37Â°C The microarray chip was prepared as previously described (42). In brief, the 3528 protein coding sequences (CDSs) successfully amplified by PCR were printed in triplicate on the slides as probes. Three expression profiling experiments were performed via reverse transcription using Superscript (Invitrogen) and with one experiment having the cDNA of IPAV labeled by Cy3 and of 56601 labeled by Cy5, while the other two having the cDNA of 56601 labeled by Cy3 and of IPAV labeled by Cy5. The unincorporated dye was removed using a QIAquick Nucleotide Removal Kit (Qiagen) as specified by the manufacturer's protocol. Samples were hybridized at 42Â°C for 16h, and then washed as described in the manual. Hybridization experiments were performed in triplicate using cDNA derived from three different cultures of virulent strain 56601 and avirulent strain IPAV grown at 37Â°C. The hybridized slides were scanned and analyzed by Tiffsplit (Agilent) to calculate the signal intensities and to determine the presence or absence of each CDS. The data were then normalized, and their backgrounds were defined using GeneSpring 4.0 (Silicon Genetics). The GeneSpring software was used to further analyze the transcription patterns. To identify genes with significantly altered expression levels, cutoff values for expression level ratios 1.5 were used to filter genes with changes (n-fold) greater than Â±1.5 in three independent biological samples (27, 48, 50). Studentâs t test analysis of variance was used to compare the mean expression levels of the test and reference samples. Genes with significant differential expression levels (P < 0.05) were selected.

Project description:The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 Kb (51% GC) and 131 Kb (56% GC). The genome was used to construct a 385,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10 - 17% absence of genes. CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units. Comparative genomic hybridization highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

Project description:The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 Kb (51% GC) and 131 Kb (56% GC). The genome was used to construct a 385,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10 - 17% absence of genes. CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units. Comparative genomic hybridization highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units. Ten Cronobacter samples were analyzed, including total genomic DNA of six C. sakazakii strains, one C. malonaticus strain, one C. muytjensii strain, one C. dublinensis strain and one C. turicensis strain.

Project description:Shimoni2009 - Escherichia Coli SOS Simple model, involving only the basic components of the circuit, sufficient to explain the peaks in the promoter activities of recA and lexA. This model is described in the article: Stochastic analysis of the SOS response in Escherichia coli. Shimoni Y, Altuvia S, Margalit H, Biham O PloS one. 2009; 4(5):e5363 Abstract: BACKGROUND: DNA damage in Escherichia coli evokes a response mechanism called the SOS response. The genetic circuit of this mechanism includes the genes recA and lexA, which regulate each other via a mixed feedback loop involving transcriptional regulation and protein-protein interaction. Under normal conditions, recA is transcriptionally repressed by LexA, which also functions as an auto-repressor. In presence of DNA damage, RecA proteins recognize stalled replication forks and participate in the DNA repair process. Under these conditions, RecA marks LexA for fast degradation. Generally, such mixed feedback loops are known to exhibit either bi-stability or a single steady state. However, when the dynamics of the SOS system following DNA damage was recently studied in single cells, ordered peaks were observed in the promoter activity of both genes (Friedman et al., 2005, PLoS Biol. 3(7):e238). This surprising phenomenon was masked in previous studies of cell populations. Previous attempts to explain these results harnessed additional genes to the system and deployed complex deterministic mathematical models that were only partially successful in explaining the results. PRINCIPAL FINDINGS: Here we apply stochastic methods, which are better suited for dynamic simulations of single cells. We show that a simple model, involving only the basic components of the circuit, is sufficient to explain the peaks in the promoter activities of recA and lexA. Notably, deterministic simulations of the same model do not produce peaks in the promoter activities. SIGNIFICANCE: We conclude that the double negative mixed feedback loop with auto-repression accounts for the experimentally observed peaks in the promoter activities. In addition to explaining the experimental results, this result shows that including additional regulations in a mixed feedback loop may dramatically change the dynamic functionality of this regulatory module. Furthermore, our results suggests that stochastic fluctuations strongly affect the qualitative behavior of important regulatory modules even under biologically relevant conditions, thus emphasizing the importance of stochastic analysis of regulatory circuits. This model is hosted on BioModels Database and identified by: MODEL2937159804. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Comparison of L. interrogans strains 56601 and IPAV at 37°C

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