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Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris

ABSTRACT: To address the question of how photosynthetic bacterium Rhodopseudomonas palustris differentially regulates gene expression of three nitrogenase isozymes (Mo, V, and Fe nitrogenases), we constructed Mo strain (Mo nitrogenase only strain), V strain (V nitrogenase only strain), and Fe strain (Fe nitrogenase only strain), and analyzed the whole genome transcriptome profiles of each mutant and wild-type cells grown under nitrogen-fixing conditions. RNA was isolated from various Rhodopseudomonas palustris strains that were grown to the mid-logarithmic phase of growth. Fluorescently labeled cDNA was prepared by direct incorporation of either Cy3-dCTP or Cy5-dCTP during a first-strand reverse transcription reaction. The hybridization mixtures containing the two labeled cDNA samples to be compared were applied to microarray slides that had been covered with Lifterslips (Erie Scientific Company, Portsmouth, NH). The slides were assembled with hybridization chambers (Corning, Corning, NY) and submerged in a 65ÂºC water bath. After 14-16 h of hybridization, the slides were washed and scanned with a ScanArray 4000XL scanner (PerkinElmer, Boston, MA). Images (Cy3 and Cy5) were captured as TIFF files and were analyzed with the image processing software ImaGene version 5.6 (BioDiscovery, Inc., El Segundo, CA). The software package lcDNA was used for data normalization and assessment of the statistical confidence intervals of gene expression. Duplicate calibration experiments and three comparative experiments using RNA from three separately grown cultures (three biological replicates) with duplicate slides for each (10 slides in total) were used to generate each data set.

ORGANISM(S): Rhodopseudomonas palustris

SUBMITTER: Federico Rey

PROVIDER: E-GEOD-3030 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris.

Oda Yasuhiro Y Samanta Sudip K SK Rey Federico E FE Wu Liyou L Liu Xiudan X Yan Tingfen T Zhou Jizhong J Harwood Caroline S CS

Journal of bacteriology 20051101 22

The photosynthetic bacterium Rhodopseudomonas palustris is one of just a few prokaryotes described so far that has vnf and anf genes for alternative vanadium cofactor (V) and iron cofactor (Fe) nitrogenases in addition to nif genes for a molybdenum cofactor (Mo) nitrogenase. Transcriptome data indicated that the 32 genes in the nif gene cluster, but not the anf or vnf genes, were induced in wild-type and Mo nitrogenase-expressing strains grown under nitrogen-fixing conditions in Mo-containing me ...[more]

PMID: 16267302

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Project description:The data in this series forms the basis for the analysis presented in the above named paper. In this study, we used cDNA microarrays to ask if changes in gene expression are observed in response to a dietary shift in Drosophila melanogaster, a dietary generalist. Methods: Flies used in this study were derived from a multifemale collection at Iraklion, Crete, Greece, in August 2002 and reared in the laboratory for several generations. Three replicates were performed. At the start of each replicate, we collected 300 newly eclosed adults, separated them by sex, and stored them at low density in shell vials with standard cornmeal media for 3 days. Sexually mature males and females were placed together and allowed to mate. Mated females were allowed to oviposit overnight in chambers containing cornmeal medium filled Petri dishes (http://flyfood.arl.arizona.edu/cornmeal.php3). Hatching first instar larvae were reared for 36 hours on cornmeal medium in Petri dishes and then transferred to one of two conditions: 1) a control Petri dish consisting of cornmeal medium, or 2) an experimental Petri dish containing pure mashed banana. After 24 hours, larvae were plucked from these dishes, washed, transferred to microcentrifuge tubes in groups of 20, and flash frozen in liquid nitrogen. All samples were stored at -80C until RNA extraction. RNA was extracted from each tube using a Qiagen RNeasy kit (Qiagen, Inc., standard protocol). We used cDNA microarrays printed by the Genetic Analysis and Technology Core Custom Microarray Facility at the University of Arizona to assess expression levels. Printing material was amplified from BDGP Unigene library v1.0 containing 6K ESTs. Products were purified using Millipore Multiscreen filtration plates and re-suspended in 50%DMSO solution. Each product was printed in triplicate on Corning GAPSII aminosilane slides using a Virtek Chipwriter PRO microarray spotter. Between 10-15 micrograms of RNA were used for each reaction. RNA was reverse transcribed and labeled with one of two fluorescent dyes, Cy3 or Cy5 (details of protocol at http://gatc.arl.arizona.edu/Services/Microarray/aminoallyllabelling.html). Hybridization was completed on a Genomic Solutions Gentac automated hybridization station. Labeled material was allowed to hybridize to the array for 16 hours at 60Â°C. Wash conditions were: 1) low stringency wash twice at 50Â°C for 40seconds (1X SSC/ 0.2% SDS), 2) medium stringency wash twice at 42Â°C for 40 seconds ( 0.1X SSC/ 0.2% SDS), 3) high stringency wash twice at 42Â°C for 40 seconds (0.1X SSC). Hybridized arrays were scanned using the Applied Precision ArrayworxE slide scanner. We performed three independent hybridizations and three dye flips for a total of six arrays (2 treatments x 2 dyes x 3 replications = 12 samples, for 6 arrays). Each replicate consisted of an independent biological sample isolated on separate days. Identification of series: There are 18 series in this experiment. The second number in each series record represents the slide number (there were six slides used in this experiment). The third number in each series record represents the set on a given slide. For instance, for series record 331_2_1, these data correspond to slide 2, set 1. Each slide has 3 series numbers assoicated with it, representing set 1,2, or 3 on the slide. Each slide was paired with another in a "dye flip" experiment; thus, slides 2 and 3 were paired, slides 25 and 28 were paired, and slides 29 and 30 were paired. Keywords = dietary shifts Keywords = ecological genomics

Dataset Information

Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris

Publications

Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris.

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