Unknown,Transcriptomics,Genomics,Proteomics

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Microarray studies of pqqE disruption mutant vs wild type

ABSTRACT: This bacterial genome encodes pyrroloquinoline quinone (PQQ) synthase enzyme required for the synthesis of PQQ in bacteria. The gene pqqE encoding PQQ synthase was annotated in Deinococcus radiodurans R1 genome and synthesis of PQQ was shown in this bacterium. PQQ roles as an antioxidant and as an inducer of protein kinase in bacterial system was shown. The pqqE disuption mutant showed several folds decrease in gamma radiation tolerance of wild type cells and strong impairment of DSB repair. Homology search analysis of PQQ binding domains showed the presence of multiple PQQ binding domains in proteins encoded by five uncharacterized ORFs of this bacterial genome. Many of these proteins are having STK domain of proteins kinases. The effect of pqqE mutation on gene expression under normal and post irradiation conditions was monitored by transcriptome analysis of mutant and compared with wild type cells. All the genes subjected to microarray analysis are as described in the genome sequence of bacterium available at ftp://ftp.ncbi.nih.gov/genomes/bacteria/Deinococcus_radiodurans. Oligonucleotides designed to represent the respective predicted ORFs of Deinococcus radiodurans ((NC_001263, NC_001264, NC_000958, NC_000959) are synthesized and purified by hydrophobic column on HPLC. The oligonucleotides are deposited on to poly-L-Lysine slide in triplicate. Microarrays were scanned using Gene Pix 4000B and hybridization signals were quantified using GenePix Pro 5.1. Prior to channel normalization, microarray outputs were filtered to remove spots of poor signal quality by excluding those data points with a mean intensity by less that 2 standard deviation above background in both channels. Normalization of data and its statistical analysis were carried out in the R computing environment using linear models for microarray data package (Limma). Using this tool, the global LOESS normalization was carried out for each microarray. The 3- replicate spots per gene in each array were used to maximize the robustness of differential expression measurements of each gene via the âlmFitâ function within Limma. Two biological replicates were carried out for transcriptome analysis of pqqE mutant strain against wild type cells.

ORGANISM(S): Deinococcus radiodurans R1

SUBMITTER: Y Hua

PROVIDER: E-GEOD-17724 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Bacterial cells lacking pyrroloquinoline quinone (PQQ) were sensitive to gamma radiation as compared to wild type cells. Deinococcus radiodurans genome encode for five putative PQQ binding STK domain-containing proteins. The comparison of gamma radiation response of all five-deletion mutants with wild type suggested that dr2518 mutant was hypersensitive to gamma radiation as compared to wild type and other mutants. DR2518 is an auto kinase. Hence, the effect of dr2518 mutation on cellular response of Deinococcus radiodurans R1, to DNA damage and subsequently to its genome functions under both normal and post irradiation growth condition was further examined. Transcriptome analysis of dr2518 mutant grown under normal conditions and post-irradiated cells was done and compared with wild type cells. Mutant lacking dr2518 become extremely sensitive to gamma radiation and complete arrest in DSB repair. These cells were therefore checked for gene expression profile in native growth and gamma irradiated growth conditions All the genes subjected to microarray analysis are as described in the genome sequence of bacterium available at ftp://ftp.ncbi.nih.gov/genomes/bacteria/Deinococcus_radiodurans. Oligonucleotides designed to represent the respective predicted ORFs of Deinococcus radiodurans ((NC_001263, NC_001264, NC_000958, NC_000959) are synthesized and purified by hydrophobic column on HPLC. The oligonucleotides are deposited on to poly-L-Lysine slide in triplicate. Microarrays were scanned using Gene Pix 4000B and hybridization signals were quantified using GenePix Pro 5.1. Prior to channel normalization, microarray outputs were filtered to remove spots of poor signal quality by excluding those data points with a mean intensity by less that 2 standard deviation above background in both channels. Normalization of data and its statistical analysis were carried out in the R computing environment using linear models for microarray data package (Limma). Using this tool, the global LOESS normalization was carried out for each microarray. The 3- replicate spots per gene in each array were used to maximize the robustness of differential expression measurements of each gene via the âlmFitâ function within Limma. Two biological replicates were analysed for global gene expression profile in dr2518 mutant against wild type cells.

Project description:Deinococcus radiodurans exposed to higher doses of gamma radiation showed induced levels of DRB0091 polypeptide annotated as a putative response regulator in an operon expressing downstream to DRB0090, histidine kinase. Deletion of drB0090 generated double mutation for both of these genes, which showed decreased tolerance to DNA damage and impairment in DSB repair. Recombinant DR0090 protein showed phosphotransferase activity on its cognate response regulator, DRB0091. The drb0090 deletion mutant was checked for its role in regulation of gene expression in response to DNA damage in Deinococcus radiodurans R1. The gene expression profiling of this mutant was carried out by complete transcriptome analysis of these cells grown under normal as well as upon gamma-irradiated conditions and compared with wild type cells. DRB0090/DRB0091 mutant showed extreme sensitivity to gamma radiation and was used for Gene expression profiling using microarray and compared with wild type data. All the genes subjected to microarray analysis are as described in the genome sequence of bacterium available at ftp://ftp.ncbi.nih.gov/genomes/bacteria/Deinococcus_radiodurans. Oligonucleotides designed to represent the respective predicted ORFs of Deinococcus radiodurans ((NC_001263, NC_001264, NC_000958, NC_000959) are synthesized and purified by hydrophobic column on HPLC. The oligonucleotides are deposited on to poly-L-Lysine slide in triplicate. Microarrays were scanned using Gene Pix 4000B and hybridization signals were quantified using GenePix Pro 5.1. Prior to channel normalization, microarray outputs were filtered to remove spots of poor signal quality by excluding those data points with a mean intensity by less that 2 standard deviation above background in both channels. Normalization of data and its statistical analysis were carried out in the R computing environment using linear models for microarray data package (Limma). Using this tool, the global LOESS normalization was carried out for each microarray. The 3- replicate spots per gene in each array were used to maximize the robustness of differential expression measurements of each gene via the âlmFitâ function within Limma. Two biological replicates were analysed for global gene expression profile in drb0090 mutant against wild type cells.

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Microarray studies of pqqE disruption mutant vs wild type

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