1 mm, and a few beads of 2 mm diameter) three times for 45 s at 6.5 m s−1. Samples were centrifuged, filtered (0.22 μm), diluted 1 : 20 with 70% MeOH, and infused at 120 μL h−1. ICR-FT/MS was externally calibrated on clusters of arginine (10 ppm in 70% MeOH). A time domain transient of 2 megawords was used, and 300 scans were accumulated for one spectrum. Spectra were internally calibrated

with an error of ≤ 0.1 ppm, exported with a signal-to-noise ratio of 3, and aligned within a 1 ppm window. Putative metabolites were annotated using MassTRIX (Wägele et al., 2012). Only masses found in all replicates were considered and analyzed in Genedata Expressionist for MS 7.6 (Genedata, Martinsried). Promoters were searched by bprom (Softberry Inc., New York) and terminators by webgester db (Mitra et al., 2011). Microarray HSP inhibitor clinical trial data were accessed from the Gene Expression Database (genexpdb, http://genexpdb.ou.edu/index.php, see Table 1). Sequences were searched with learn more blastp or tblastn (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi, default parameters) using YaaW (Z0011) as query (Table S2). The evolutionary history of all species was inferred using the software package mega5 with a concatemer of 16s rRNA gene, atpD, adk, gyrB, purA, and recA by Minimum Evolution using p-distance. The bootstrap consensus was inferred from 1000 replicates

(Tamura et al., 2011). For some strains, not all sequences were available, and thus close relatives were used as surrogate, for example, some genes of Comamonas testosteroni CNB-2 were used for the yaaW-bearing strain ATCC 11996. The presence of htgA was detected using pairwise blastp

alignments with htgA (Z0012) as query (starting from the first GTG). htgA/yaaW sequences were examined for their nonsynonymous over synonymous rate ratio ω as described (Sabath et al., 2008; Sabath & Graur, 2010) including correction for multiple testing according to Benjamini & Hochberg (1995), after omitting alignment gaps (Tamura et al., 2011). 5′-RACE determined the major 5′-end of the + 1 transcription start of htgA to be 135 bp upstream. However, minor sites might be present, since Missiakas Metalloexopeptidase et al. (1993) found a site 82 bp upstream; others were predicted 98 (BProm) or 114 bp (Tutukina et al., 2007) upstream of the CTG-start codon of htgA. The upstream region of htgA was successfully tested for promoter activity using a promoterless gfp reporter. No terminator could be detected directly downstream of htgA but was detected downstream of dnaK (Fig. 1). Recently, strand-specific transcriptome sequencing showed that htgA is transcribed, albeit weakly, at some nonlaboratory growth conditions only (R. Landstorfer, S. Simon, S. Schober, D. Keim, S. Scherer & K. Neuhaus, unpublished data). The 5′-RACE major transcription start site of yaaW is 32 bp upstream of yaaI, but a minor site, 107 bp upstream of yaaW, was also detected.