Showing papers by "Michael McClelland published in 2005"

Co‐regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ

Abstract: Summary Analysis of the transcriptome of slyA mutant Salmo- nella enterica serovar Typhimurium revealed that many SlyA-dependent genes, including pagC , pagD , ugtL , mig-14 , virK , phoN , pgtE , pipB2 , sopD2 , pagJ and pagK , are also controlled by the PhoP/PhoQ regulatory system. Many SlyA- and PhoP/PhoQ-co- regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicro- bial peptides. Purified His-tagged SlyA binds to the pagC and mig-14 promoters in regions homologous to a previously proposed 'SlyA-box'. The pagC pro- moter lacks a consensus PhoP binding site and does not bind PhoP in vitro , suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low-magnesium PhoP-inducing conditions in which pagC expression is profoundly elevated, how- ever, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti-repressor to allow PhoP acti- vation. The absence of almost all SlyA-activated genes from the Escherichia coli K12 genome sug- gests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmo- nella evolved its distinctive pathogenic lifestyle.

Comparison of the PhoPQ Regulon in Escherichia coli and Salmonella typhimurium

Abstract: The PhoPQ two-component system acts as a transcriptional regulator that responds to Mg2+ starvation both in Escherichia coli and Salmonella typhimurium (Garcia et al. 1996; Kato et al. 1999). By monitoring the availability of extracellular Mg2+, this two-component system allows S. typhimurium to sense the transition from an extracellular environment to a subcellular location. Concomitantly with this transition, a set of virulence factors essential for survival in the intracellular environment is activated by the PhoPQ system (Groisman et al. 1989; Miller et al. 1989). Compared to nonpathogenic strains, such as E. coli K12, the PhoPQ regulon in pathogens must contain target genes specifically contributing to the virulence phenotype. To verify this hypothesis, we compared the composition of the PhoPQ regulon between E. coli and S. typhimurium using a combination of expression experiments and motif data. PhoPQ-dependent genes in both organisms were identified from PhoPQ-related microarray experiments. To distinguish between direct and indirect targets, we searched for the presence of the regulatory motif in the promoter region of the identified PhoPQ-dependent genes. This allowed us to reconstruct the direct PhoPQ-dependent regulons in E. coli K12 and S. typhimurium LT2. Comparison of both regulons revealed a very limited overlap of PhoPQ-dependent genes between both organisms. These results suggest that the PhoPQ system has acquired a specialized function during evolution in each of these closely related species that allows adaptation to the specificities of their lifestyles (e.g., pathogenesis in S. typhimurium).

Differences in Gene Content between Salmonella enterica Serovar Enteritidis Isolates and Comparison to Closely Related Serovars Gallinarum and Dublin

Abstract: Salmonella enterica serovar Enteritidis is often transmitted into the human food supply through eggs of hens that appear healthy. This pathogen became far more prevalent in poultry following eradication of the fowl pathogen S. enterica serovar Gallinarum in the mid-20th century. To investigate whether changes in serovar Enteritidis gene content contributed to this increased prevalence, and to evaluate genetic heterogeneity within the serovar, comparative genomic hybridization was performed on eight 60-year-old and nineteen 10- to 20-year-old serovar Enteritidis strains from various hosts, using a Salmonella-specific microarray. Overall, almost all the serovar Enteritidis genomes were very similar to each other. Excluding two rare strains classified as serovar Enteritidis in the Salmonella reference collection B, only eleven regions of the serovar Enteritidis phage type 4 (PT4) chromosome (sequenced at the Sanger Center) were absent or divergent in any of the other serovar Enteritidis strains tested. The more recent isolates did not have consistent differences from 60-year-old field isolates, suggesting that no large genomic additions on a whole-gene scale were needed for serovar Enteritidis to become more prevalent in domestic fowl. Cross-hybridization of phage genes on the array with related genes in the examined genomes grouped the serovar Enteritidis isolates into two major lineages. Microarray comparisons of the sequenced serovar Enteritidis PT4 to isolates of the closely related serovars Dublin and Gallinarum (biovars Gallinarum and Pullorum) revealed several genomic areas that distinguished them from serovar Enteritidis and from each other. These differences in gene content could be useful in DNA-based typing and in understanding the different phenotypes of these related serovars.

Alternative sigma factor interactions in Salmonella: sigma and sigma promote antioxidant defences by enhancing sigma levels.

Abstract: Hierarchical interactions between alternative sigma factors control sequential gene expression in Gram-positive bacteria, whereas alternative sigma factors in Gram-negative bacteria are generally regarded to direct expression of discrete gene subsets. In Salmonella enterica serovar Typhimurium (S. Typhimurium), sigma(E) responds to extracytoplasmic stress, whereas sigma(H) responds to heat shock and sigma(S) is induced during nutrient limitation. Deficiency of sigma(E), sigma(H) or sigma(S) increases S. Typhimurium susceptibility to oxidative stress, but an analysis of double and triple mutants suggested that antioxidant actions of sigma(E) and sigma(H) might be dependent on sigma(S). Transcriptional profiling of mutant Salmonella lacking sigma(E) revealed reduced expression of genes dependent on sigma(H) and sigma(S) in addition to sigma(E). Further investigation demonstrated that sigma(E) augments sigma(S) levels during stationary phase via enhanced expression of sigma(H) and the RNA-binding protein Hfq, leading to increased expression of sigma(S)-dependent genes and enhanced resistance to oxidative stress. Maximal expression of the sigma(S)-regulated gene katE required sigma(E) in Salmonella-infected macrophages as well as stationary-phase cultures. Interactions between alternative sigma factors permit the integration of diverse stress signals to produce coordinated genetic responses.

WebArray: an online platform for microarray data analysis.

Abstract: Background Many cutting-edge microarray analysis tools and algorithms, including commonly used limma and affy packages in Bioconductor, need sophisticated knowledge of mathematics, statistics and computer skills for implementation. Commercially available software can provide a user-friendly interface at considerable cost. To facilitate the use of these tools for microarray data analysis on an open platform we developed an online microarray data analysis platform, WebArray, for bench biologists to utilize these tools to explore data from single/dual color microarray experiments.

Host Gene Expression Changes and DNA Amplification during Temperate Phage Induction

Abstract: Salmonella enterica serovar Typhimurium LT2 harbors four temperate prophages. The lytic cycle of these phages was induced with hydrogen peroxide or mitomycin C. Microarray analysis was used to monitor the increase in phage genome copy number and the changes in RNA expression. Phage gene transcription was classified temporally, and host genes that responded to hydrogen peroxide, mitomycin C, or phage induction were also identified. A region of the serovar Typhimurium LT2 host genome encompassing hundreds of genes, flanking the Fels-1 lambdoid prophage, was amplified manyfold during lytic induction, presumably due to Fels-1 runoff replication prior to excision, a phenomenon termed escape replication. An excisionase (xis) mutant of Fels-1 also induced escape replication but did not get packaged. Gifsy-1, a lambdoid prophage that does not normally produce escape replication, did so after deletion of either its integrase or excisionase genes. Escape replication is probably widespread; large regions of host genome amplification were also observed after phage induction in serovar Typhimurium strains SL1344 and 14028s at the suspected integration site of prophage genomes.

Genomic Comparisons of Salmonella enterica Serovar Dublin, Agona, and Typhimurium Strains Recently Isolated from Milk Filters and Bovine Samples from Ireland, Using a Salmonella Microarray

Abstract: Salmonella-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical Salmonella enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the Salmonella Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the spv genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.

Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis.

Abstract: The genotype of Salmonella enterica serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whereas the PT4 genome to which they were compared, as well as a PT4 field isolate, had a DNA sequence with some similarity to the bacteriophage ST64b sequence. Phenotype MicroArray analysis indicated that the two PT13A strains and the PT4 field isolate had similar respiratory activity profiles at 37°C. However, the wild-type S. enterica serovar Enteritidis PT13A strain grew significantly better in 20% more of the 1,920 conditions tested when it was assayed at 25°C than the biofilm-forming PT13A strain grew. Statistical analysis of the respiratory activity suggested that S. enterica serovar Enteritidis PT4 had a temperature-influenced dimorphic metabolism which at 25°C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37°C the metabolism was nearly identical to that of the wild-type PT13A strain. Although it is possible that lysogenic bacteriophage alter the balance of phage types on a farm either by lytic competition or by altering the metabolic processes of the host cell in subtle ways, the different physiologies of the S. enterica serovar Enteritidis strains correlated most closely with minor, rather than major, genomic changes. These results strongly suggest that the pandemic of egg-associated human salmonellosis that came into prominence in the 1980s is primarily an example of bacterial adaptive radiation that affects the safety of the food supply.

Cell-type-specific patterns of gene expression

Abstract: Among the methods, compositions, combinations and kits provided herein are those for determining gene expression levels in one or more cell types in heterogeneous cell samples, for identifying genes differentially expressed in different cell types, and for detecting a cell type in a sample from a subject. Also provided herein are methods, compositions, combinations and kits for determining gene expression levels in cells corresponding to phenotypes, and for identifying a phenotype of a subject by detecting differentially expressed genes.

"Promoter array" studies identify cohorts of genes directly regulated by methylation, copy number change, or transcription factor binding in human cancer cells.

Abstract: DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours. Using three statistical criteria, approximately 269 most commonly phosphorylated c-Jun/ATF2-DNA complexes were identified and representative cases were verified by qPCR measurement of ChIP-captured DNA. Expression was correlated at the mRNA and protein levels. The largest functional cohort was 24 genes of known DNA repair function, most of which exhibited increased protein expression indicated coordinate gene regulation. In addition, cell lines of prostate cancer exhibit stable methylation or copy number changes that reflect the alterations of the corresponding primary tumors. 504 (18.5%) promoters showed differential hybridization between immortalized control prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, eight had previously been observed in prostate cancer, and 13 were previously determined methylation targets in other cancers. The vast majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes to study the role of DNA methylation in prostate tumors. Earlier studies using prototype promoter arrays examine approximately 7% of the proximal regulatory sequences while the current gene regulatory events surveyed here occur on a large scale and may rapidly effect the coordinated expression of a large number of genes.

Enhanced microarray performance using low complexity representations of the transcriptome

Abstract: Low abundance mRNAs are more difficult to examine using microarrays than high abundance mRNAs due to the effect of concentration on hybridization kinetics and signal-to-noise ratios. This report describes the use of low complexity representations (LCRs) of mRNA as the targets for cDNA microarrays. Individual sequences in LCRs are more highly represented than in the mRNA populations from which they are derived, leading to favorable hybridization kinetics. LCR targets permit the measurement of abundance changes that are difficult to measure using oligo(dT) priming for target synthesis. An oligo(dT)-primed target and three LCRs detect twice as many differentially regulated genes as could be detected by the oligo(dT)-primed target alone, in an experiment in which serum-starved fibroblasts responded to the reintroduction of serum. Thus, this target preparation strategy considerably increases the sensitivity of cDNA microarrays.

Cell-type-specific pattern of gene expression

Abstract: PROBLEM TO BE SOLVED: To provide methods for determining gene expression levels in one or more cell types in heterogeneous cell samples.SOLUTION: The method can include: (a) a step of determining the relative content of each cell type in two or more related heterogeneous cell samples, wherein at least two of the samples do not contain the same relative content of each cell type; (b) a step of measuring overall levels of one or more gene expression analytes in each sample; (c) a step of determining the regression correlation between the relative content of each cell type and the measured overall levels; and (d) a step of calculating the level of each of the one or more analytes in each cell type according to the regression correlation determined in the step (c), wherein gene expression levels correspond to the calculated levels of analytes.