Northern hybridization was performed using the DIG DNA Labeling and Detection kit (Roche Applied Science, IN, USA). The RNeasy Midi kit (Qiagen, CA, USA) was used for RNA Selonsertib molecular weight extraction. Total RNA was isolated from D. hafniense DCB-2 grown with 3-chloro-4-hydroxybenzoate,
3,5-dichlorophenol or ortho-bromophenol. Samples of 20 μg of RNA were loaded in triplicates on a 1% agarose gel containing 2.2 M formaldehyde. After electrophoresis, the RNA was transferred to a nylon membrane (Hybond-N, GE Healthcare Biosciences, NJ, USA) and each replicate on the membrane was hybridized with the DIG-labeled probes that were designed specifically for targeting the rdhA2, rdhA3, or rdhA6 genes. Hybridization Staurosporine manufacturer was performed for 16 h at 42°C and positive fragments were detected by chemiluminescence as described in the manufacturer’s manual. The
microarray data is deposited at GEO-NCBI with the accession numbers GSE33988 and GPL14935 for the raw data and platform, respectively. Genome sequencing and annotation The genome of D. hafniense DCB-2 was sequenced by the Joint Genome Institute (JGI). All general aspects of library construction and sequencing performed at the Joint Genome Institute are described at http://www.jgi.doe.gov/. Genome drafts were annotated by the automated pipeline of the Oak Ridge National Laboratory’s Computational Genomics Group, and the completed genome sequence of D. hafniense DCB-2 has been annotated and curated by the Integrated Microbial Genomes (IMG, http://img.jgi.doe.gov/cgi-bin/w/main.cgi) JAK inhibitors in development [87]. Comparative analysis
Comparative analysis of the microbial genomes and their individual genes were performed with analysis tools and sequence data available at IMG. Topology predictions for signal peptides, transmembrane proteins, and twin-arginine (Tat) signal peptides were performed by using SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/), TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and TatP 1.0 Server (http://www.cbs.dtu.dk/services/TatP/), respectively. Alignment of the two D. hafniense genomes was performed by using Mauve v 2.3.1 [88] with a view of 24 LCBs (locally collinear blocks) and their GC profiles were obtained by using the GC-Profile program next (http://tubic.tju.edu.cn/GC-Profile/), [88, 89]. Much of information on metabolic pathways, enzyme reactions, and chemicals were reassured with reference to MetaCyc [90]. Phylogenetic analysis Phylogenetic trees of selected proteins were constructed using MEGA 4.1 [91] based on the alignments generated by CLUSTALW algorithm and the neighbor-joining method with 500 bootstrap replications. Nucleotide sequence accession number The sequence data of D. hafniense DCB-2 can be accessed using GenBank accession number CP001336. Acknowledgements and funding We are grateful to the DOE Joint Genome Institute for selecting and sequencing D.