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Michael McClelland

Publications and source records attributed to Michael McClelland.

At least 37 records · Page 2Linked to original sources
PubMed2005

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

Analysis of the transcriptome of slyA mutant Salmonella 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 antimicrobial peptides. Purified His-tagged SlyA binds to the pagC and mig-14 promoters in regions homologous to a previously proposed 'SlyA-box'. The pagC promoter 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, however, 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 activation. The absence of almost all SlyA-activated genes from the Escherichia coli K12 genome suggests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmonella evolved its distinctive pathogenic lifestyle.

Antimicrobial Cationic Peptides↗
PubMed2005

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

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.

Animals↗
PubMed2005

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

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 degrees 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 degrees 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 degrees C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37 degrees 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.

Animals↗
PubMed2005

Host gene expression changes and DNA amplification during temperate phage induction.

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.

DNA Nucleotidyltransferases↗
PubMed2005

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

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.

Cell Line, Tumor↗
PubMed2005

Survey of differentially methylated promoters in prostate cancer cell lines.

DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5%) showed differential hybridization between immortalized prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, and TSPY) previously observed in prostate cancer and 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, and WIT-1). The 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, and GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

Cell Line, Tumor↗
PubMed2004

Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress.

The NH2-terminal Jun kinases (JNKs) function in diverse roles through phosphorylation and activation of AP-1 components including ATF2 and c-Jun. However, the genes that mediate these processes are poorly understood. A model phenotype characterized by rapid activation of Jun kinase and enhanced DNA repair following cisplatin treatment was examined using chromatin immunoprecipitation with antibodies against ATF2 and c-Jun or their phosphorylated forms and hybridization to promoter arrays. Following genotoxic stress, we identified 269 genes whose promoters are bound upon phosphorylation of ATF2 and c-Jun. Binding did not occur following treatment with transplatin or the JNK inhibitor SP600125 or JNK-specific siRNA. Of 89 known DNA repair genes represented on the array, 23 are specifically activated by cisplatin treatment within 3-6 hr. Thus, the genotoxic stress response occurs at least partly via activation of ATF2 and c-Jun, leading to large-scale coordinate gene expression dominated by genes of DNA repair.

Activating Transcription Factor 2↗
PubMed2004

Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid.

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their approximately 4,400 protein coding sequences: 173 in Paratyphi A and approximately 210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).

Base Sequence↗
PubMed2004

In silico dissection of cell-type-associated patterns of gene expression in prostate cancer.

Prostate tumors are complex entities composed of malignant cells mixed and interacting with nonmalignant cells. However, molecular analyses by standard gene expression profiling are limited because spatial information and nontumor cell types are lost in sample preparation. We scored 88 prostate specimens for relative content of tumor, benign hyperplastic epithelium, stroma, and dilated cystic glands. The proportions of these cell types were then linked in silico to gene expression levels determined by microarray analysis, revealing unique cell-specific profiles. Gene expression differences for malignant and nonmalignant epithelial cells (tumor versus benign hyperplastic epithelium) could be identified without being confounded by contributions from stroma that dominate many samples or sacrificing possible paracrine influences. Cell-specific expression of selected genes was validated by immunohistochemistry and quantitative PCR. The results provide patterns of gene expression for these three lineages with relevance to pathogenetic, diagnostic, and therapeutic considerations.

Gene Expression Profiling↗
PubMed2004

Erwinia toletana sp. nov., associated with Pseudomonas savastanoi-induced tree knots.

Gram-negative bacteria were isolated from knots induced by Pseudomonas savastanoi in olive trees (Olea europaea L.). A total of nine endophytic bacterial strains were isolated, each from inside a different tree knot. Biochemical characterization indicated that all the strains belong to the family Enterobacteriaceae. Phylogenetic analyses of the 16S rRNA genes of these novel isolates revealed that they formed a homogeneous cluster within Erwinia species. DNA signatures of these isolates were identical to those described for the genus Erwinia. The strains formed a homogeneous group as shown by DNA-DNA hybridization analysis and numerical analysis of phenotypic data, clearly differentiated from all species of Erwinia with validly published names. The data provide strong evidence of the differentiation of these strains from the most closely related species. Therefore, these isolates represent a novel species, for which the name Erwinia toletana sp. nov. is proposed. The isolates are available at CFBP, CECT and ATCC. The G+C content is 52+/-0.5 mol%. The type strain is CFBP 6631(T) (=A37(T)=ATCC 700880(T)=CECT 5263(T)).

Bacterial Typing Techniques↗
PubMed2004

Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes.

Swarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide 'slime'. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one-third of the functional genome. The large number of genes showing surface-specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming-specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface-dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.

Bacterial Proteins↗
PubMed2004

Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses.

Vaccination is the most efficient prophylaxis against a variety of infectious diseases. New vaccination strategies rely on the incorporation of effective adjuvants, which stimulate the innate immune response and, in turn, activate the adaptive immune response. It is well established that flagellin induces inflammatory responses through the activation of antigen-presenting cells (APCs). In order to evaluate whether flagellin can serve as a carrier for the development of adjuvants or vaccines, we prepared a flagellin-enhanced green fluorescent protein (EGFP) fusion protein. Our results demonstrate that a flagellin-EGFP fusion protein is capable of stimulating APCs, resulting in the maturation of these cells and secretion of proinflammatory cytokines. Furthermore, APCs pulsed with the flagellin-EGFP fusion protein effectively process and present EGFP antigens. More importantly, animals immunized with the flagellin-EGFP fusion protein developed specific anti-EGFP T-cell responses. In contrast, recombinant EGFP was not able to stimulate APCs, nor did it induce a T-cell response. Thus, recombinant-flagellin fusion proteins may be suitable carriers as adjuvants or vaccines for the development of new vaccination strategies to induce and boost immune responses against infectious diseases and cancer.

Adjuvants, Immunologic↗
PubMed2004

Salmonella enterica serovar Typhi strains from which SPI7, a 134-kilobase island with genes for Vi exopolysaccharide and other functions, has been deleted.

Salmonella enterica serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between pheU and 'pheU (truncated), two genes for tRNA(Phe). SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage. Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion. In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative. We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons. Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single pheU gene; microarrays show that all SPI7 genes are deleted. Two of the strains produce amplicons which have A derived from pheU at bp 27, while five have C derived from 'pheU at this position; thus, the position of the crossover which results in the deletion can be inferred. The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the viaB operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained. We propose that these deletions occurred during storage following isolation.

Antigens, Bacterial↗
PubMed2004

DNA amplification and rearrangements in archival Salmonella enterica serovar Typhimurium LT2 cultures.

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.

Biological Specimen Banks↗
PubMed2004

Host restriction of Salmonella enterica serotype Typhimurium pigeon isolates does not correlate with loss of discrete genes.

The definitive phage types (DT) 2 and 99 of Salmonella enterica serotype Typhimurium are epidemiologically correlated with a host range restricted to pigeons, in contrast to phage types with broader host ranges such as epidemic cattle isolates (DT104 and DT204). To determine whether phage types with broad host range possess genetic islands absent from host-restricted phage types, we compared the genomes of four pigeon isolates to serotype Typhimurium strain LT2 using a DNA microarray. Three of the four isolates tested caused fluid accumulation in bovine ligated ileal loops, but they had reduced colonization of liver and spleen in susceptible BALB/c mice and were defective for intestinal persistence in Salmonella-resistant CBA mice. The genomes of the DT99 and DT2 isolates were extremely similar to the LT2 genome, with few notable differences on the level of complete individual genes. Two large groups of genes representing the Fels-1 and Fels-2 prophages were missing from the DT2 and DT99 phage types we analyzed. One of the DT99 isolates examined was lacking a third cluster of five chromosomal genes (STM1555 to -1559). Results of the microarray analysis were extended using Southern analysis to a collection of 75 serotype Typhimurium clinical isolates of 24 different phage types. This analysis revealed no correlation between the presence of Fels-1, Fels-2, or STM1555 to -1559 and the association of phage types with different host reservoirs. We conclude that serotype Typhimurium phage types with broad host range do not possess genetic islands influencing host restriction, which are absent from the host-restricted pigeon isolates.

Animals↗
PubMed2003

EnteriX 2003: Visualization tools for genome alignments of Enterobacteriaceae.

We describe EnteriX, a suite of three web-based visualization tools for graphically portraying alignment information from comparisons among several fixed and user-supplied sequences from related enterobacterial species, anchored on a reference genome (http://bio.cse.psu.edu/). The first visualization, Enteric, displays stacked pairwise alignments between a reference genome and each of the related bacteria, represented schematically as PIPs (Percent Identity Plots). Encoded in the views are large-scale genomic rearrangement events and functional landmarks. The second visualization, Menteric, computes and displays 1 Kb views of nucleotide-level multiple alignments of the sequences, together with annotations of genes, regulatory sites and conserved regions. The third, a Java-based tool named Maj, displays alignment information in two formats, corresponding roughly to the Enteric and Menteric views, and adds zoom-in capabilities. The uses of such tools are diverse, from examining the multiple sequence alignment to infer conserved sites with potential regulatory roles, to scrutinizing the commonalities and differences between the genomes for pathogenicity or phylogenetic studies. The EnteriX suite currently includes >15 enterobacterial genomes, generates views centered on four different anchor genomes and provides support for including user sequences in the alignments.

Computer Graphics↗
PubMed2003

A non-redundant microarray of genes for two related bacteria.

A microarray with sequences from the annotated open reading frames (ORFs) in Salmonella enterica subspecies 1, serovar Typhimurium was supplemented with annotated chromosomal ORFs from serovar Typhi that are divergent from Typhimurium (>10% DNA sequence divergence). This non- redundant array was used to (i) measure changes in gene copy number in DNA from actively growing versus stationary Typhi and (ii) to reveal the transcriptional response of Typhi to peroxide, a stress similar to that experienced when they are phagocytosed by macrophages. In S.enterica subspecies 1, pairs of genomes differ in the presence or absence of approximately 10% of their genes. An array twice the size of that needed to cover all ORFs for one genome could carry close homologs of all the ORFs for 10 genomes. Non-redundant DNA arrays could be constructed for any group of closely related organisms that differ by the presence and absence of a few genes.

Gene Expression Profiling↗