Amino acid sequences for the homologous proteins were obtained from NCBI and TIGR databases [National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the Institute of Genomic Research (http://www.tigr.org)]. Multiple sequence alignments were generated using the clustalw web-based program with default parameters [European Bioinformatics institute (http://www.ebi.ac.uk/clustalw)]. A model of putative NarP protein was made based on the crystal structure of E. coli NarL (Baikalov et al., 1996). After the putative M. haemolytica
NarP and E. coli NarL was aligned, the amino acids of the E. coli NarL was substituted with buy Bortezomib the corresponding one of the M. haemolytica NarP using deepview/swiss-pdbviewer (http://www.expasy.org/spdbv/; version 3.7). After the model was optimized with the same software, it was visualized using macpymol PLX 4720 (DeLano Scientific LLC; http://delanoscientific.com/; version 0.98). The construction of narP mutants was carried out as described in McKerral
& Lo (2002) and the narP mutants were selected according to the protocol of Fedorova & Highlander (1997b) (see Supporting Information). The growth characteristics of MhΔNarP7 in comparison with the parent SH1217 and their response to nitrate were examined. An overnight culture of SH1217 or MhΔNarP7 was diluted 1/100 into BHIB, with or without NaNO3 supplementation. Five-milliliter aliquots of this culture were added to 15 test tubes and grown semi-anaerobically at 37 °C. The OD600 nm of the cultures were determined acetylcholine over 8 h at 2-h intervals, taking measurements from three test tubes at each interval. The OD600 nm values of different strains/culturing conditions were compared using an unpaired, two-tailed t-test (P<0.005). SH1217 and MhΔNarP7 were grown in 5 mL BHIB with or without NaNO3 supplement, semi-anaerobically at 37 °C. The cells were harvested at OD600 nm of 0.5. Total protein preparations were prepared by adding equal volume of 2 × sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) loading buffer with the cell suspension and examined by SDS-PAGE and Western immunoblot according to Lo & Mellors (1996). After SDS-PAGE, the proteins were stained with Commassie brilliant blue. For Western immunoblot, the proteins were transferred to a nitrocellulose membrane as described (Lo et al., 1991), and blocked by immersion in a 3% gelatin solution in Tris-HCl-buffered saline containing 0.05% Tween 20 (TTBS). The Lkt neutralizing monoclonal antibody 601 (Gentry & Srikumaran, 1991) was used at a dilution of 1/2000 in antibody solution (1% gelatin in TTBS). The secondary antibody goat anti-mouse alkaline phosphates conjugate (Jackson Laboratories) was used at a dilution of 1/5000 in antibody solution. The membranes were developed using 5-bromo-4-chloro-3-indoyl-phosphate and nitroblue tetrazolium.