The proteins were transferred to nitrocellulose membranes and probed with an anti-6His antibody (Qiagen). Detection was carried out by chemiluminescence as described by the manufacturer (Applied Biosystems). To this date, the genome sequence of five strains of R. sphaeroides is available, additionally the genome sequences of two other Rhodobacter species have been reported (Rhodobacter capsulatus and Rhodobacter sp. SW2). To obtain additional

sequences of rpoN genes present in closely related species of R. sphaeroides, total DNA from R. azotoformans, R. blasticus, R. veldkampii, and Rv. sulfidophilum was used as template in a PCR using degenerated oligonucleotides. These oligonucleotides were designed to hybridize to highly conserved regions Regorafenib in vitro of rpoN. One primer targets a small section within the region encoding the domain known to bind the RNA polymerase core, whereas the second primer targets the DNA sequence encoding the highly conserved motif known as RpoN-box (see Fig. S1). The amplification reaction was carried out using an

alignment temperature gradient that ranged from 55 to 62 °C. A ABT-888 prominent band of the approximate expected size of 900 nt was detected in the lower temperatures of the gradient (55–57 °C) in all samples except R. veldkampii. The band obtained at 57 °C for each sample was gel-purified and cloned. As a first step to detect clones with different rpoN sequences, 30 independent clones of each sample were digested with HinfI to detect polymorphisms. Independently of the number of restriction patterns, 10 different clones were sequenced for each sample. Consistent with a single restriction pattern detected for the clones from R. blasticus and Rv. sulfidophilum, only one sequence corresponding to rpoN was obtained from all the sequenced clones. In contrast, three different restriction patterns were observed among the clones obtained from R. azotoformans. The sequence of these clones allowed us to identify three different rpoN genes. To obtain the full sequence of the identified rpoN genes, the sequences corresponding

to the terminal ends of each gene were amplified by restriction-site PCR (Sarkar et al., 1993) as described in Materials and Methods. To take advantage of the already sequenced genomes of other Rhodobacter strains, we added the rpoN genes present in these genomes to the database used in this work for Atorvastatin further analysis. A single copy of rpoN is present in R. capsulatus; two copies are present in Rhodobacter sp. SW2; R. sphaeroides ATCC17025 has three, and R. sphaeroides 2.4.1, WS8, ATCC17029, and KD131 have four copies of this gene. Our results suggest that R. blasticus has a single copy of rpoN and that R. azotoformans has three. The complete RpoN sequences from these bacteria were aligned. We included the well-characterized RpoN from E. coli to make the identification of functionally relevant regions easier. As occurs with RpoNs from R. sphaeroides and R.