CGH of S. meliloti DnaA and Hda overproducing strains
Ontology highlight
ABSTRACT:
ORGANISM(S): Sinorhizobium meliloti
PROVIDER: E-MTAB-3924 | BioStudies |
REPOSITORIES: biostudies
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Project description:Assays 1-4: Effects of DnaA activity on S. meliloti chromosome, pSymA and pSymB replicon bias. Sheared DNA extracts of IPTG-induced vs uninduced cultures of DnaA (Assays 1,2) or Hda (Assays 3,4) overexpressors were labeled with Cy3 or Cy5 and hybridized applying dye-swap replicate design. Assay 5: Hybridization of sheared DNA of logarithmic (Cy3-labeled) vs stationary phase (Cy5-labeled) culture.
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Project description:Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N2-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (?pSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ?pSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism's niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).
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Project description:Sinorhizobium meliloti 1021 carries two megaplasmids, pSymA of 1,354 kb and pSymB of 1,683 kb, which are essential in establishing symbiosis with its legume hosts and important for bacterial fitness in the rhizosphere. We have previously shown that pSymA is self-transmissible and that its conjugal functions are regulated by the transcriptional repressor RctA. Here, we show conjugal transfer of pSymB as an in trans mobilization event that requires the type IV secretion system encoded by pSymA. pSymB carries a functional oriT and an adjacent relaxase gene, traA2, that is also transcriptionally repressed by rctA. Both symbiotic megaplasmids would require the relaxase genes in cis with their respective oriTs to achieve the highest transfer efficiencies.
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Project description:Differential gene expression in mice kidney after Amodiaquine and Sulfodoxine-Pyrimethamine anti-malaria treatments were evaluated. Dosages were decided based on standard malaria therapy in human. Mice (Mus musculus) belonging to control and Amodiaquine and Sulfodoxine-Pyrimethamine administered groups (n=4) were sacrificed. The kidneys were snap frozen in liquid nitrogen, and subsequently stored at -80M-BM-:C till further use. Kidney samples were homogenized and total RNA was extracted with TRI reagent. 25 ug of total RNA was converted into labeled cDNA using CyScribe first strand cDNA labeling kit. The Cy5 and Cy3 labeled cDNAs were resuspended in Cyscribe Hyb buffer containing 10ug/ml sheared Salmon sperm DNA and 10ug/ml Yeast tRNA. The labeled samples were hybridized to the arrays and incubated for 16-18hrs at 42M-BM-:C. Fluorescent array images were collected for both Cy3 and Cy5 with Molecular Dynamics III scanner supported with ImageQuant v5.0. Image intensity data were extracted and analyzed with ArrayVision v8.0 analysis software. Background-corrected data was LOWESS normalized and log ratios were calculated using Avadis v4. Further, mean log ratios were calculated for duplicate spots. Duplicate experiments were carried out for all the dosages.
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Project description:In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.
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Project description:DNA methylation in wild type bolting plants, wild type seedlings, and ddm1 seedlings. The purpose of the McrBC methylation microarray assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thanliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3. Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray. Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. This SuperSeries is composed of the following subset Series: GSE1329: DNA methylation in wild-type bolting Arabidopsis thaliana plants GSE1330: DNA methylation in ddm1 seedling Arabidopsis thaliana plants GSE1331: VC133+137, DNA methylation in ddm1 seedling Arabidopsis thaliana plants GSE1332: VC134+136, DNA methylation in wild-type seedling Arabidopsis thaliana plants Refer to individual Series
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Project description:The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.
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