A novel copper-responsive regulon in Mycobacterium tuberculosis.
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ABSTRACT: In this study, we report the identification of a five-locus copper-inducible regulon in Mycobacterium tuberculosis. The identification of a copper responsive regulon unique to pathogenic Mycobacteria suggests copper homeostasis must be maintained during an infection. WT and mutant Mtb cells were grown in Sauton's minimal media to early stationary phase (OD580 = 1.5) and treated with 500 mM copper sulfate (CuSO4) for four hours or the absence
Project description:In this study, we report the identification of a five-locus copper-inducible regulon in Mycobacterium tuberculosis. The identification of a copper responsive regulon unique to pathogenic Mycobacteria suggests copper homeostasis must be maintained during an infection. WT and mutant Mtb cells were grown in Sauton’s minimal media to early stationary phase (OD580 = 1.5) and treated with 500 mM copper sulfate (CuSO4) for four hours or the absence.
Project description:In this study, we report the identification and characterization of several regulators in Mycobacterium tuberculosis. WT and mutant Mtb cells were grown in Sauton minimal media to early stationary phase (OD580 = 2 and 4)
Project description:Understanding novel mechanism bacteria ustilize in the clinics to become resistant to antibiotics is critical. The study aims to identify genes associated with Vancomycin resistance. Clinical isolates from a single patient with increasing resistance to vancomycin were grown in the presence and absence of vancomycin.Staphylococcus aureus strain 2275 is the reference for this series. S. aureus isolates from a single patient were grown in the presence and absence of vancomycin over a 2 hour time course. RNA samples were extracted at 30, 60 90 and 120 minutes post exposure to antibiotic. Samples were hybridized on aminosilane coated slides with 70-mer oligos comparing mock treated and treated cells to the first strain isolated in the lineage. Differnetial gene expression patterns were determined.
Project description:The goal of this study is the discovery of (a) meaningful phylogenomic relationships among members of this B. cereus/B. anthracis group, and (b) reliable gene-phenotype associations, e.g. recognition of links between genomic traits and the ability of certain strains to cause various forms of disease. We also tried to elucidate genome evolution aspects that may lead to the emergence of variants that are capable (or have the potential) of causing anthrax-like disease. This large-scale comparative genomics approach is unprecedented for this taxonomic group. Dr. A. Hoffmaster (CDC) provided the PFGRC with 73 B. cereus and B. anthracis isolates from the CDC culture collection. Of these, 27 were isolated from patients with severe or systemic disease; ten isolates of this group were obtained from patients (welding factory workers) with anthrax-like disease or from the environment near their workplace. Another set of 26 represented isolates from food-born illnesses. Of the 26 gastrointestinal disease isolates (GIDI), 10 were obtained from patients with diarrhea, whereas another set of 10 had been shown to harbor the emetic (vomit) toxin gene by PCR. The rest of the group consisted of 20 isolates with various phenotypes. All strains were screened for their genomic content using the B. cereus/B. anthracis species microarray. Seventy-three query strains were investigated in this study, with each query strain hybridized against the reference strain, Sterne. Dye-swap experiments were performed with all the 73 strains on both chipA and chipB of the microarray, for a total of four or more hybridizations per query strain. Each 70mer oligo spotted on the B. cereus species microarray is spotted once. Positive controls on the array consist of oligos designed from the sequenced reference genome, Sterne, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.
Project description:The goal of this project was to screen soil samples for bacteria that may harbor B. anthracis virulence-associated genes (VAGs). There is currently no information about the prevalence of these types of organisms in the environment. Due to increased environmental monitoring of select agents by programs such as BioWatch and biodetection systems in place at the United States Post Offices and Department of State locations, it has become critical that we not only better understand the natural range of B. anthracis but also how widespread B. anthracis virulence genes are in environmental communities. Naturally occurring isolates containing the B. anthracis virulence genes could generate false-positive results in tests that detect the anthrax toxins, capsule or their associated genes. Understanding the true diversity and pathogenic potential of Bacillus spp. and particularly the B. cereus group is crucial not only in terms of understanding data from environmental monitoring but also diagnosing patients with clinical presentations similar to anthrax in the future. Severe and fatal disease caused by strains similar to B. anthracis could unnecessarily initiate emergency responses if anthrax was incorrectly suspected. Conversely, these strains may be used as bioterror agents requiring science-based responses; presently our limited understanding of these organisms does not permit data-driven decision making. We have investigated 700 aerobic sporoform soil isolates obtained from two areas in the Southwest of the US. Soil samples from the first site had been taken from public access land approximately 50 meters across from the work site of a fatal pneumonia case in a welding factory. This took place in year 2003 when B. cereus was isolated from a metal worker. The second site was targeted because of a recent case involving a deceased mule suspected to have died of a B. anthracis infection. Soil samples were initially analyzed at the CDC. Isolates were obtained by heating the soil at 65 degrees Celcius for 30 minutes followed by plating on agar media. All isolates were screened by PCR for the presence of B. anthracis genomic traits such as toxin genes (cya, lef and pag) as well as chromosomal markers. All isolates were also tested for their hemolytic activity as well as phage sensitivity. Eighty-four query strains were investigated in this study, with each query strain hybridized against the reference strain, Sterne. Two dye-swap experiments were performed with seventeen strains, for a total of four hybridizations per query strain. The other strains have a single dye experiment, for a total of two hybridizations per query strain. Each 70mer oligo spotted on the B. cereus species microarray is spotted once. Positive controls on the array consist of oligos designed from the sequenced reference genome, Sterne, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.
Project description:This study was aimed to perform comparative expression analysis of both virulent and avirulent strains of F. tularensis with a goal to possibly identify genes unique to low and high virulent strains to understand their role, if any in virulence features of an isolate. Initially, thirty strains were selected representing both type A and type B clades of F. tularensis. Five strains failed to grow on Chamberlain's medium (Chamberlain 1965) and were excluded from the study. All the isolates were grown to mid-log phase in Chamberlain’s medium and the total RNA was collected by standard protocols for expression analysis. The samples were analyzed in duplicates. F. tularensis Schu S4, a known virulent strains was used as a reference strain for this study. Chamberlain RE (1965) "Evaluation of Live Tularemia Vaccine Prepared in a Chemically Defined Medium," Applied Microbiology, 13(2): 232-5 Twenty four query strains and one reference strain (FRAN016 Schu S4) were used in the hybridizations. Most of the hybridizations have more than one biological replicates, resulting in a total of fifty five samples analyzed.
Project description:Comparitive gene expression analysis of wild type and PA 3880 mutant when grown anaerobically and aerobically. Samples were hybridized on aminosilane coated slides with 70-mer oligos.
Project description:Haemophilus influenzae(Hi) is a gram-negative rod shaped bacterium that lives symbiotically in the upper respiratory tract of humans. Capsulated Hi, as well as non-capsulated strains are known to cause a number of significant infections. Recognizing and understanding the links between the phenotypic traits and the genetic background has paramount epidemiological and clinical importance. To date, a panoply of microbiological and molecular biology tools have been developed and utilized by researchers aimed at identifying the evolutionary links among strains and isolates. Comparative genomic hybridization (CGH) has been shown to be a useful tool for screening strains for their genetic content. However, there is a major limitation when CGH is conducted on a microarray based on a single reference genome. CGH results report which genes are present or absent relative to the genome. Hence the information about novel genetic content that the query strain possesses remains obscure. We report here the construction of the first Hi pan-genome microarray representing ca. 4600 features by 70-mers. In addition to those from the Rd strain, new features originate from the unfinished genome sequences present in NCBI database and from our novel gene discovery project efforts using strain HK1212. Genomes of 20strains belonging to different phylogenetic lineages were screened for their gene loss and gain utilizing the species microarray. The results obtained by employing the multistrain species microarray provide comprehensive information about the genomic content of uncharacterized strains. The trees generated by CGH, in general, do not reproduce the phylogeny of a species in terms of vertical evolution, but instead represents the overall relatedness of genomes to one another and provide an assessment about the species genome evolution. Keywords: Comparative Genomic Hybridization Twenty query strains (denoted HKxxxx) were investigated in this study, with each query strain hybridized against the reference strain, KW20. Two dye-swap experiments were performed, for a total of four hybridizations per query strain. Each 70mer oligo spotted on the H. influenzae species microarray is replicated six times. Positive controls on the array consist of oligos designed from the sequenced reference genome, KW20, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.
Project description:In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulator is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a ternary complex with the sensor kinase SaeS and activate the sensor kinase’s phosphatase activity. Efficient activation of the phosphatase activity of SaeS required the presence of both SaeP and SaeQ. When SaeP and SaeQ were expressed, the expression of coagulase, a sae target with low affinity to phosphorylated SaeR, was greatly reduced, while the expression of alpha-hemolysin, a sae target with high affinity to phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by SaeRS TCS, these results suggest that the SaeRS TCS returns to pre-activation state by a negative feedback mechanism. To examine the global effect of SaePQ on sae target gene expression, we treated the wild type strain of USA300-P23 and its P1 promoter mutant with HNP-1 and analyzed the transcription of sae target genes by microarray assays. WT and P1 cells were compared against a each other at the same time point and against a T=0 reference.
Project description:The study aims to compare gene expression patterns of Staphyloccoccus aureus USA300 vs USA300 sae knockout. Samples were hybridized on aminosilane coated slides with 70-mer oligos. 12 arrays total.