5-kbp product. The PCR product was sequenced to complete the sequence of the fbpA promoter region (Fig. 4). The nucleotide sequences upstream of fbpA and lktC were examined for motifs typical of NarP-binding sites using consensus sequences from NarP-regulated promoters in E. coli (Constantinidou et al., 2006). Several NarP-binding sequences were identified in the fpbA promoter region (Fig. 4). On the other hand, there were no apparent NarP-binding sequences in the lktC promoter. The lktC promoter has been reported to be quite unique and its regulation may involve several

regulatory factors (Uhlich et al., 2000). It is possible that expression of one of such factor is regulated by NarP. Total proteins from SH1217 and MhΔNarP7 grown in BHIB were examined by Western immunoblot to determine the relative Lkt levels. Lkt is one of the most important virulence CH5424802 cell line factors produced by M. haemolytica A1 and has been shown to attack bovine macrophages and neutrophils during an infection (Shewen & Wilkie, 1982; Clinkenbeard et al., 1989). The results in Fig. 5a showed that there is a higher level of Lkt accumulation from SH1217 grown in the presence of NaNO3, suggesting a response to nitrate and increased expression

of the lkt genes. On the other hand, MhΔNarP7 exhibited the same high level of Lkt accumulation even in the absence of NaNO3. The loss of NarP, resulting in increased expression of Lkt, suggests Y-27632 molecular weight that NarP functions either directly or indirectly to repress ADP ribosylation factor lkt expression. The relative levels of lkt mRNA was examined by RT-PCR using primers specific for lktA. The results in Fig. 5b showed an elevated amplification of the 177-bp product in total RNA extracted from SH1217 grown in BHIB+NaNO3 compared

with RNA from SH1217 grown in unsupplemented BHIB. In MhΔNarP7, the level of lkt mRNA was always elevated regardless of the presence or absence of additional NaNO3. The blast analysis identified five complete pairs of TCSs in the M. haemolytica A1 genome, which corresponds to the results of the genome project (Gioia et al., 2006). Analysis of the M. haemolytica A2 genome sequence (Lawrence et al., 2010) identified four TCS systems with amino acid identities of 99% to those in the A1 genome. The only TCS system absent in the A2 genome is CpxA/R. This is a relatively small number; for example, over 30 pairs of TCSs have been found in the E. coli genome (Mizuno, 1997; Oshima et al., 2002; Yamamoto et al., 2005). The small number is likely a result of the specific growth niches of this microorganism. As a commensal organism primarily found in the respiratory tract, M. haemolytica A1 (and A2) probably only needs to sense and respond to limited environmental signals and therefore do not possess an extensive array of TCSs. Similar observations have been made in Haemophilus influenzae and Actinobacillus pleuropneumoniae where four sensors and five regulators, and five putative TCS pairs are present, respectively (Mizuno, 1998; Foote et al., 2008).