Corynebacterium glutamicum ATCC13032 Cells: Control (Wild-Type) vs cg0196 Deletion Mutant
ABSTRACT: Transcriptional profiling of Corynebacterium glutamicum cells comparing wild-type cells with cg0196 deletion mutant cells by site-specific gene deletion using the non-replicable integration vector. cg0196 is gene conding transcriptional regulator related carbon metabolism. Two-condition experiment, Wild vs. Δcg0196 cells. Independently grown and harvested. One replicate per array.
Project description:Otitis media, pneumonia, sinusitis and as well as severe diseases such as meningitis and bacteraemia are related to biofilm-like diseases, in which Streptococcus pneumoniae demonstrated differential and tissue specific gene expressions. In this study, we reported the differential gene expression profile of early in vitro biofilm and planktonic cell in c-DNA microarray analysis. The microarray analysis was performed on total RNA extracted from biofilms grown in 24-well microtiter plate and mid-log grown planktonic cells. To validate the results of microarray, real-time RT-PCR was performed on 13 differentially expressed genes and one constitutively expressed gene from six different functional groups. cDNA-microarray analyses indicated 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Among total differentially expressed gene, almost 50% were hypothetical genes. Of the 46 protein coding genes, 34 showed up-regulation and 16 showed down-regulation in biofilm. The functional annotation showed that many functional categories were differentially regulated in biofilm and planktonic cells, such as genes involve in purine, pyrimidine nucleotide metabolism, RNA/DNA metabolism, amino acid transport and metabolism, translation, transporter protein, carbohydrate transport and metabolism, cell wall biosynthesis, isoprenoid biosynthesis, transcription regulator and cellular process. Streptococcus pneumoniae R6 strain used in this is an unencapsulated and avirulent strain derived from encapsulated serotype 2 pathogenic strain D39. In vitro biofilm formation was carried out in 24-well, flat-bottom, polystyrene microtiter plate (BD falcon, MD, USA) in static model. S. pneumoniae grown up to mid-logarithmic phase in TSB medium was diluted 1:100 with fresh sterile TSB medium supplied with 1% glucose, inoculated 1.5 mL in 24-well microtiter plate and, incubated for 15 hours at 37°C in 5% CO2. After incubation medium was discarded, and the plates were gently washed three times with 1.5 mL sterile, cold phosphate buffer saline (PBS). Adherent cell were scraped and immediately processed for RNA extraction. For planktonic cells RNA extraction, five ml of mid-logarithmic phase cell suspension was pelleted by centrifugation and wash three times with sterile PBS and immediately processed for RNA extraction. All experiments were performed in triplicate (3 independent biological replicates)
Project description:Transcriptional profiling of E.coli O157:H7 cells comparing control untreated cells with PEG8000treated cells Two-condition experiment, Control vs. PEG8000. Biological replicates: 1 control, 1 treated.
Project description:Comparison between Lp Paris wt and rpoN mutant in exponential phase (OD 3.3). We investigated the role of FleQ, FleR, RpoN, and FliA on the regulation of the expression of transmissive traits in L. pneumophila strain Paris at different time points (replicative phase, early transmissive phase, late transmissive phase).
Project description:Investigation of whole genome gene expression to identify overlooked sRNAs and sORFs. Background The completion of numerous genome sequences has introduced an era of whole-genome study. However, many real genes, including small RNAs (sRNAs) and small ORFs (sORFs), are missed in genome annotation. In order to improve genome annotation, we sought to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery or shigellosis. Results Firstly, we identified 64 sRNAs in Shigella which is experimentally validated in other bacteria based on sequence conservation. Secondly, among possible approaches to search for sRNAs, we employed computer-based and tiling array based methods, followed by RT-PCR and northern blots. This allowed us to identify 12 sRNAs in Shigella flexneri strain 301. We also find 29 candidate sORFs. Conclusions This investigation provides an updated and comprehensive annotation of the Shigella genome, increases the expected numbers of sORFs and sRNAs with the corresponding impact on future functional genomics and proteomics studies. Our method can be used for the large scale reannotation of sRNAs and sORFs in any microbe whose genome sequence is available. Study using total RNA recovered from five conditions.
Project description:We performed a gene expression analysis of C. albicans SC5314 planktonic cells exposed to the antifungal peptide ApoEdpL-W. Exponentially-growing C. albicans SC5314 cells in SD at 30°C medium were exposed to 2.5 µM ApoEdpL-W and samples were collected after 10 and 30 min. for transcript profiling
Project description:Transcription profiling of third instar male larvae of two different heteroallelic combinations of ssdp hypomorphic alleles, ssdp[neo48]/ssdp]BG1663] and ssdp/ssdp[BG1663] were compared to each of the corresponding single heterozygotes 22 hybridizations were conducted, these include biological repeats and dye swaps as technical controls.
Project description:Intracellular small-molecule signaling has drawn increasing attention due to its importance in bacterial physiology and host-pathogen interactions. Here, we show that a new quinone-sensing transcriptional regulator, QsbR (quinone-stress sensing and bleomycin resistance regulator), is important for bacterial stress response and virulence in S. aureus. The mutant strain of qsbR exhibits dramatically reduced staphylococcal virulence in a murine abscess model of infection. Moreover, the qsbR mutant shows an accumulation of riboflavin and an increased resistance to quinone stress and bleomycin compared to the wild-type strain Newman. Microarray analysis confirmed that QsbR impacts the expression of genes that are involved in quinone stress response, bleomycin resistance, riboflavin biosynthesis, and pathogenesis. DNase-I footprinting and gel shift assays suggest that QsbR not only directly regulates transcription of genes involved in quinone detoxification and bleomycin resistance, but is also subject to autoregulation by binding to the palindromic consensus sequence GTATAN(5)TATAC in their promoter regions. This binding could be disrupted by quinone treatment in vitro. Mass spectrometric analysis showed that wild-type QsbR, but not QsbRC5S with Cys5 mutated to Ser, could be alkylated at Cys5 by quinone. Further mutagenesis studies revealed that Cys5 plays a key role in quinone sensing as substitution of Cys5 with Ser renders the bacterium more sensitive to quinone stress. Our results demonstrate that QsbR is a quinone-sensing regulator that dramatically impacts staphylococcal virulence and stress response. Sample preparation for transcriptional analysis. For each strain, 2 clones were inoculated in TSB and collected from mid-log or stationary phase. Appropriate volumes from each of the 2 cultures per strain were pooled and added to 2 volumes of RNAprotect reagent (Qiagen). Cultures were centrifuged and cell pellets were stored at 4 ºC until RNA extraction. During the experiment, OD600 and viable cfu per ml were monitored for each of the cultures. This procedure was repeated 2 independent times to generate 2 samples at each time point for each strain.
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:Three different experimental approaches were evaluated for discrimination of genomic variance in and between duplicated sequences using 48 markers in duplicon regions and 17 SNPs in unique sequences previously characterized in another study. We found only the method high-throughput single sperm typing could conclusively resolve the alleles of all markers. Resulting data from single sperm analysis were also used to examine the genetic structure of duplicon markers in the human population. Single sperm typing can be a rapid, efficient and accurate method for initial screening and assessment of genetic variation and for detailed genetic analysis of duplicon markers. Keywords: Genotyping Sixty-five markers including 17 MSVs, 12 PSVs, 19 SIDs and 17 SNPs in unique sequences described in Fredman et al. were selected for study. The samples include 40 genomic DNA samples from four ethnic groups, semen samples from 11 donors, and 10 to 20 sperm from each donor except one, AB012, for whom 65 sperm were analyzed. Both genomic and sperm DNA samples were subject to multiplex amplification followed by microarray analysis. Genotypes were determined by using the Accutyping software. Semen samples were genotyped on both strands. Allele status in these samples were compared and analyzed. The single sperm typing method allowed us to identify markers residing in non-unique sequence, to analyze the detailed genetic structure of the duplicons and to learn whether different alleles are present for the duplicon sequences in the human population.