Project description:BackgroundHorizontal gene transfer, the transfer and incorporation of genetic material between different species of organisms, has an important but poorly quantified role in the adaptation of microbes to their environment. Previous work has shown that genome size and the number of horizontally transferred genes are strongly correlated. Here we consider how genome size confuses the quantification of horizontal gene transfer because the number of genes an organism accumulates over time depends on its evolutionary history and ecological context (e.g., the nutrient regime for which it is adapted).ResultsWe investigated horizontal gene transfer between archaea and bacteria by first counting reciprocal BLAST hits among 448 bacterial and 57 archaeal genomes to find shared genes. Then we used the DarkHorse algorithm, a probability-based, lineage-weighted method (Podell & Gaasterland, 2007), to identify potential horizontally transferred genes among these shared genes. By removing the effect of genome size in the bacteria, we have identified bacteria with unusually large numbers of shared genes with archaea for their genome size. Interestingly, archaea and bacteria that live in anaerobic and/or high temperature conditions are more likely to share unusually large numbers of genes. However, high salt was not found to significantly affect the numbers of shared genes. Numbers of shared (genome size-corrected, reciprocal BLAST hits) and transferred genes (identified by DarkHorse) were strongly correlated. Thus archaea and bacteria that live in anaerobic and/or high temperature conditions are more likely to share horizontally transferred genes. These horizontally transferred genes are over-represented by genes involved in energy conversion as well as the transport and metabolism of inorganic ions and amino acids.ConclusionsAnaerobic and thermophilic bacteria share unusually large numbers of genes with archaea. This is mainly due to horizontal gene transfer of genes from the archaea to the bacteria. In general, these transfers are from archaea that live in similar oxygen and temperature conditions as the bacteria that receive the genes. Potential hotspots of horizontal gene transfer between archaea and bacteria include hot springs, marine sediments, and oil wells. Cold spots for horizontal transfer included dilute, aerobic, mesophilic environments such as marine and freshwater surface waters.
Project description:Previous work identified the winged helix-turn-helix DNA binding transcription factor (TF) RosR (encoded by VNG0258H gene), which dynamically regulates expression of more than 300 genes in response to oxidative stress in Halobacterium salinarum (Sharma 2012). RosR is required for survival of oxidants from multiple sources (e.g. H2O2 and paraquat), as deletion mutants are impaired for ROS outgrowth. Genes directly and indirectly controlled by RosR in response to ROS encode macromolecular repair functions. In the current study, we ask which of these genes are direct targets of RosR regulation. Dynamic chromatin immunoprecipitation combined with microarray (ChIP-chip) analysis validates that genes encoding these functions are direct targets of RosR binding and control. In addition, new RosR direct target genes are identified, including those encoding central cellular functions and a surprisingly high number of other TFs. The majority of the 252 sites throughout the genome are RosR-bound in the absence of stress and cleared of RosR binding in the presence of H2O2. However, binding is dynamic, with promoter-specific differences in the timing of RosR-DNA release and re-binding relative to ROS exposure.
Project description:Previous work identified the winged helix-turn-helix DNA binding transcription factor (TF) RosR (encoded by VNG0258H gene), which dynamically regulates expression of more than 300 genes in response to oxidative stress in Halobacterium salinarum (Sharma 2012). RosR is required for survival of oxidants from multiple sources (e.g. H2O2 and paraquat), as deletion mutants are impaired for ROS outgrowth. Genes directly and indirectly controlled by RosR in response to ROS encode macromolecular repair functions. In the current study, we ask which of these genes are direct targets of RosR regulation. Dynamic chromatin immunoprecipitation combined with microarray (ChIP-chip) analysis validates that genes encoding these functions are direct targets of RosR binding and control. In addition, new RosR direct target genes are identified, including those encoding central cellular functions and a surprisingly high number of other TFs. The majority of the 252 sites throughout the genome are RosR-bound in the absence of stress and cleared of RosR binding in the presence of H2O2. However, binding is dynamic, with promoter-specific differences in the timing of RosR-DNA release and re-binding relative to ROS exposure. Halobacterium salinarum harboring VNG0258H(rosR)::myc was grown to mid-logarithmic phase (OD600 ~ 0.2 - 0.4) and either left untreated or exposed to 25 mM H2O2 for 10, 20, and 60 minutes. Transcription factor-chromatin complexes from each treated time point and untreated cultures were then cross-linked in vivo with 1% formaldehyde for 30 min at room temperature and subjected to immunoprecipitation (IP) by virtue of the myc epitope tag as described previously (Schmid et al. 2011). One M-BM-5g of each IP sample was hybridized against matched, mock-treated controls on a custom 2 x 105,000 feature 60-mer oligonucleotide microarray (Agilent Technologies, AMADID 026819). On this high-resolution array, the entire H. salinarum genome was tiled every 30 bp in triplicate. Randomly selected regions of the genome were spotted in quadruplicate. Dye swaps were conducted to correct for bias in incorporation. Seven biological replicate experiments were conducted for VNG0258::myc in the absence of H2O2 and three in the presence of H2O2. Four biological replicate experiments in an H. salinarum strain harboring the empty vector control plasmid were conducted to detect background peaks resulting from technical aspects of the protocol. This yielded a total of at least 18 replicate intensity data points per 30-bp genomic region per condition. DNA fragments were directly labeled with Cy3 and Cy5 dyes (Kreatech) as described previously (Facciotti et al., 2007). Microarray slide hybridization and washing protocols were conducted according to the manufacturerM-bM-^@M-^Ys instructions (Agilent technologies) with the exception that hybridization was conducted in the presence of 37.5% formamide at 68M-KM-^ZC to ensure proper stringency.