Unknown,Transcriptomics,Genomics,Proteomics

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Expression profiling of P. stutzeri A1501 treated with 20mM ammonia shock for 10 min

ABSTRACT: A whole genome DNA microarray was used to undertake a global transcriptional analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501. The aim of this study was to identify the genes that are up-regulated under nitrogen fixation conditions and rapidly down-regulated as soon as 10 min after ammonia shock. The expression changed genes may be the candidate genes for the ammonia signal transmission or be involved in the nitrogen regulatory mechanism. First, P. stutzeri A1501 was treated with 0.1 mM ammonia and 0.5% Oxygen tension until the nitrogenase activity was detectable. Then the cells were sudden shifted from the nitrogen fixation conditions to the ammonia repression conditions by addition of 20 mM ammonia for 10min. Subsequently, the bacterium was collected and began the RNA extraction process. Thus, we compare the expression profilings in these two conditions in order to identify the candidate genes.

ORGANISM(S): Pseudomonas stutzeri A1501

SUBMITTER: yongliang yan

PROVIDER: E-GEOD-14775 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Nitrogen fixation is a highly energy-demanding process and highly regulated at multiple levels. The two major signals that regulate nitrogen fixation in most diazotrophs are oxygen and ammonia. In order to study the complex regulated mechanism and to highlight the complete nitrogen fixing system in genome level, here we present the transcriptional profiles of the nitrogen fixation genes of P.stutzeri A1501 in different growth conditions with a genome-wide DNA microarray. In this study, the three samples of "P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension","P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension-2" and "P.stutzeri A1501 treated with 0.1mM ammonia and 0.5% Oxygen tension-3" were three repeat experiments, while, the other three samples of "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-1", "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-2" and "P.stutzeri A1501 treated with 20mM ammonia and 0.5% Oxygen tension-3" were three repeat experiments. The gene expressions under these two growth phases were compared to investigate which genes' expression were effected by different ammonia concentrations. Keywords: nitrogen fixation, nitrogen repression P.stutzeri whole genome microarray, which was based on our sequenced strain A1501. The microarray used in this study featured 3988 of the 4145 ORFs identified in P.stutzeri strain A1501, because 157 ORFs were not on the microarray of the inability to amplify these products. ORFs were amplified with specific primer pairs (Invitrogen). To ensure that the elements of our array would detect specifically their corresponding genes and no others, the ORF coordinates fed into the primer program were circumscribed such that they would exclude regions of any ORF that contained significant similarity to any other ORF. Agarose gel electrophoresis was used to perform quality control on all PCR products. Oligonucleotides were removed from the PCR mix by PCR 96 cleanup plate (Millipore). DNA was resuspended in 12 Î¼l of spotting solution containing 50% dimethyl sulfoxide. PCR products were spotted onto gamma amino propylsilan coated GAPII slides (Corning) with a OmniGrid â¢ microarrayer (GeneMachines).The negative control genes were from the type III secretion system genes of shigella flexneri 2a and the commercial Arabidopsis genes (SpotReportâ¢ cDNA Array Validation System, Stratagene). The positive control genes were from the 16S rRNA of P.stutzeri A1501. We used Cy3-labeled genomic DNA and Cy5 dye-labeled cDNA, for hybridization. The cDNA was synthesized by reverse transcription of 5 Î¼g of total RNA. The labeled cDNA and genomic DNA samples were mixed and hybridized at 65â for 16 hours. Genomic DNA was used as a universal internal control for the quality of the microarray and also allowed for the comparison of results across multiple experiments. Each experiment was repeated three times from the beginning of inoculation.

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Expression profiling of P. stutzeri A1501 treated with 20mM ammonia shock for 10 min

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