Over 300 TCSs are known to date regulating various activities such as metabolism, respiration, stress response and chemotaxis. Some TCSs have
been shown to be involved in virulence of some bacteria, such as the BvgA/S system in Bordetella pertussis and Bordetella bronchiseptica (Cotter & Miller, 1997; Williams & Cotter, 2007) and the OmpR–EnvZ system in Shigella flexneri (Bernardini et al., 1990). In this study, we carried out blast searches against the M. haemolytica A1 genome to identify its TCS(s). Out of the five putative TCSs, the NarQ/P system was chosen for further investigation. Mannheimia VX 809 haemolytica A1 strain SH1217 was obtained from Dr Sarah Highlander, Baylor College of Medicine. Escherichia coli strain DH5 is from our laboratory collection.
Mannheimia haemolytica A1 was cultured in brain heart infusion broth (BHIB) at 37 °C, supplemented with chloramphenicol (5 mg L−1), ampicillin (25 mg L−1), or streptomycin (100 mg L−1) where necessary. To examine the response to the addition of nitrate, BHIB was supplemented with 2.5 mM NaNO3. This concentration was chosen through a titration experiment in search for the minimum concentration Epigenetics Compound Library in vitro of NaNO3 to elicit a response in SH1217 (data not shown). When necessary, M. haemolytica was grown under a semi-anaerobic condition by growing the liquid cultures in a sealed container without shaking (modified from Browning et al., 2006). Escherichia coli cultures were grown in Luria–Bertani + thymidine (50 mg L−1) broth at 37 °C, supplemented with ampicillin (100 mg L−1) where necessary. Multiple bioinformatics tools were used to search for putative TCS homologs in the M. haemolytica
A1 genome (accession number: AASA00000000). A consensus sequence of the RR regulatory domain (GAADY) was used to perform a blastp search against the M. haemolytica A1 genome sequence at Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu). The nucleotide sequences of the contigs that contain significant hits were retrieved and analyzed with the ‘sequence analysis’ software (Informagen Biotechnology Information Resource; http://www.informagen.com/SA/) to identify the ORFs containing the putative RRs. Amino acid sequences Ribonucleotide reductase of the putative RRs were then analyzed by a blastp search against the NCBI sequence database. The putative RR homologs were used to perform blastp against the M. haemolytica A1 genome to search for more RR homologs. Multiple rounds of the analyses were performed until no more additional hits were obtained. After the putative RR homologs were identified, consensus sequences for their cognate HKs were retrieved from the NCBI database. These HK sequences were used to perform blastp against the M. haemolytica A1 genome. The search results were analyzed as above. Again, multiple rounds of searches and analyses were performed until no new hits were obtained.