, 1980). It has also been reported that the calf lungs become increasingly anaerobic during an infection (Jensen et al., 1976), and therefore the utilization of nitrate for anaerobic

respiration may be important. Typically, NarQ/P regulates genes whose products are involved in utilization of nitrate/nitrite as a terminal electron acceptor in anaerobic respiration (Stewart & Rabin, 1995). In E. coli, two pairs of proteins, NarQ/P and NarX/L, are involved in this function. Similar to M. haemolytica A1, H. influenzae, Pasteurella multocida and A. pleuropneumoniae also possess only NarQ/P (Stewart, 2003; Foote et al., 2008). In E. coli, some Nar-regulated genes are coregulated by the global anaerobic regulator Fnr (Choe & Reznikoff, 1993). Interestingly, FnrP, the Fnr homologue in M. haemolytica A1, has been shown to be involved in the regulation of leukotoxin (Lkt) expression (Uhlich et al., 2000), NVP-LDE225 which suggests a possible coregulation of Lkt by FnrP and the NarQ/P system. Multiple sequence alignments showed that the M. haemolytica A1 NarQ and NarP proteins

have features typical of the homologous proteins from E. coli and other related microorganisms. The high similarities were expected as these proteins sense and respond to the same environmental signal. The perfect alignment of M. haemolytica A1 NarP to the crystal Gemcitabine concentration structure of E. coli NarL suggests that M. selleck chemicals haemolytica NarP most likely functions as a transcriptional activator, with a C-terminal helix–turn–helix DNA-binding motif. narP knock-out mutant was constructed and was found to have lost its ability to respond to the addition of nitrate in the growth media. The slight change in growth kinetics and the characteristic drop in the final OD600 nm reading for SH1217 in nitrate-supplemented BHIB was not observed for MhΔNarP7. SDS-PAGE analysis showed that MhΔNarP7 has lost its ability

to alter its protein profile in response to additional nitrate. MS analysis of the 35-kDa protein that had lost its regulation in MhΔNarP7 revealed it to be FbpA. FbpA is a periplasmic protein involved in iron acquisition (Shouldice et al., 2003). This protein receives iron from the outer membrane transferring-binding proteins TbpA and TbpB, and then delivers it to the inner membrane-bound ferric transporters FbpB/C (Ogunnariwo & Schryvers, 1990; Tam & Saier, 1993). Very little is known about the regulation of this operon. Several studies have reported that this operon is iron regulated (Forng et al., 1997; Paustian et al., 2001), but its regulation in response to nitrate levels via NarP has never been reported. We have sequenced and reconstructed the missing fbpABC promoter. Analysis of the fbp promoter region identified several motifs typical for NarP-binding sequences.