A single colony of this species was transferred to fresh medium and used for all subsequent experiments. The culture was confirmed as axenic by microscopy, colony morphology and 16S rRNA cloning and analyses. To explore the ability of the isolate to metabolize
a range of electron acceptors, nitrate, Fe(III)-NTA, Fe(III)-oxyhydroxide or Fe(III)-citrate was added (20 mM) to minimal medium with either acetate or glycerol (10 mM) as an electron donor. Electron donor utilization was tested using Fe(III)-citrate (20 mM) as the electron acceptor and lactate, formate, ethanol, glucose, yeast extract, benzoate, acetate or glycerol (10 mM) as potential electron donors. The pH tolerance was assessed using Fe(III)-citrate medium (20 mM) with glycerol (10 mM) as the electron donor at pH ranging from 3.5 to 10. The pH of the medium was adjusted http://www.selleckchem.com/products/ganetespib-sta-9090.html with NaOH or HCl prior to inoculation. The 16S–23S rRNA intergenic spacer region from the bacterial RNA operon was amplified as described previously using primers ITSF and ITSReub (Cardinale et al., 2004). The amplified
products were separated by electrophoresis in Tris-acetate–EDTA gel. DNA was stained with ethidium bromide and viewed under short-wave UV light. Positive microbial community changes identified by the Ribosomal Intergenic Spacer Analysis (RISA) justified further investigation by DNA sequencing of 16S rRNA gene clone libraries. PCR products were purified using a QIAquick PCR purification kit (Qiagen, UK) and ligated directly into a cloning vector containing topoisomerase I-charged vector arms (Agilent Technologies, UK) prior Selleckchem Roxadustat to transformation into Escherichia coli-competent cells expressing Cre recombinase (Agilent Technologies). White transformants that grew on LB agar containing ampicillin and X-Gal were screened for an insert using PCR. Primers were complementary to the flanking regions of the PCR insertion site of the cloning vector. The conditions for PCR method were as follows: an initial denaturation at
94 °C for 4 min, melting at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, followed by a final extension step at 72 °C for 5 min. The resulting PCR products were purified using an ExoSap protocol, and 2 μL of ExoSap mix (0.058 μL exonuclease I, 0.5 μL Shrimp alkaline buy Gefitinib phosphatase and 1.442 μL QH2O) was added to 5 μL of PCR product and incubated at 37 °C for 30 min followed by 80 °C for 15 min. Nucleotide sequences were determined by the dideoxynucleotide method (Sanger et al., 1977). An ABI Prism BigDye Terminator Cycle Sequencing kit was used in combination with an ABI Prism 877 Integrated Thermal Cycler and ABI Prism 377 DNA Sequencer (Perkin Elmer Applied Biosystems, UK). Sequences (typically 900 base pairs in length) were analysed against the NCBI (USA) database using the blast program packages and matched to known 16S rRNA gene sequences.