For mixed-strain competitions, hatchlings were exposed to an inoculum containing an ∼1 : 1 ratio of wild type and mutant. At 48-h postinoculation,
individual squid were homogenized and dilution plated on LBS. The resulting colonies were patched onto LBS with added trimethoprim to determine the ratio of strains in each animal. Inocula were similarly plated and patched to determine the starting ratio. The relative competitiveness index (RCI) was determined by dividing the mutant to wild-type ratio in each animal by the ratio of these strains in the inoculum. The Ibrutinib mean RCI was calculated from log-transformed data. blast searches (Altschul et al., 1990) of the V. fischeri ES114 genome revealed the similarity of ORFs VF1308 and VF1309 to the N and C termini of E. coli FNR, respectively (Fig. 1a). We MAPK Inhibitor Library nmr suspected that a sequencing error had led
to the misannotation of fnr as two genes, and we therefore cloned and sequenced the region spanning VF1308 and VF1309. We found five errors in the genome database, leading to an erroneously predicted truncation of VF1308, which we corrected in GenBank (Mandel et al., 2008). In the revised sequence, VF1308 encodes a protein that is the same length as, and shares 84% identity with, E. coli FNR. This ES114 FNR is identical to the previously deposited V. fischeri MJ1 FNR (accession no. CAE47558). Importantly, the residues necessary for interactions with RNA Molecular motor polymerase (Williams et al., 1997; Lonetto et al., 1998; Blake et al., 2002; Lamberg et al., 2002), 4Fe–4S center assembly (Spiro & Guest, 1988; Kiley & Beinert, 1998), and DNA recognition (Spiro et al., 1990) in E. coli are conserved in V. fischeri FNR. Using TransTermHP (Kingsford et al., 2007), we also found a likely Rho-independent transcriptional terminator downstream of fnr (Fig. 1a and b). Given the 142-bp spacing and strong putative terminator between fnr and VF1310 (Fig. 1b), it seems likely that these are expressed on separate transcripts. Using quantitative RT-PCR, we found that the fnr∷tmpR allele in mutants described
below did not affect the transcript levels for VF1310. We next generated mutants disrupted in the putative fnr in V. fischeri ES114 and MJ1. We did not observe any attenuation of these strains under aerobic growth conditions, consistent with the role of FNR in other bacteria. Escherichia coli fnr mutants do not grow anaerobically with nitrate or fumarate as an electron acceptor (Lambden & Guest, 1976), and we found that V. fischeri fnr mutants were similarly attenuated. Specifically, when grown with minimal medium under anaerobic conditions, ES114 and MJ1 displayed nitrate- or fumarate-dependent growth on a nonfermentable carbon source (glycerol) that was lacking in the fnr mutants (e.g. Fig. 1c).