The Pasteurella multocida nrfE gene is upregulated during infection and is essential for nitrite reduction but not for virulence

Abstract

Pasteurella multocida is the causative agent of a range of diseases with economic importance in production animals. Many systems have been employed to identify virulence factors of P. multocida, including in vivo expression technology (IVET), signature-tagged mutagenesis, and whole-genome expression profiling. In a previous study in which IVET was used with P. multocida, nrfE was identified as a gene that is preferentially expressed in vivo. In Escherichia coli, nrfE is part of the formate-dependent nitrite reductase system involved in utilizing available nitrite as an electron accepter during growth under anaerobic conditions. In this study, we constructed an isogenic P. multocida strain that was unable to reduce nitrite under either aerobic or anaerobic conditions, thereby demonstrating that P. multocida nrfE is essential for nitrite reduction. However, the nrfE mutant was still virulent in mice. Real-time reverse transcription-PCR analysis indicated that nrfE was regulated independently of nrfABCD by an independent promoter that is likely to be upregulated in vivo.

FIG. 1.

Schematic diagram of the P. multocida nrfE mutagenesis construct and confirmation of the nrfE mutant by PCR. (A) A single 1.8-kb fragment containing nrfE was amplified by PCR by using primers 1914 and 1915 (indicated by the arrows labeled 1914 and 1915). This fragment was digested with BamHI to obtain two 900-bp fragments that were then ligated to either end of tet(M) to produce the mutagenesis cassette used for allelic exchange. (B) Schematic diagram of the genome organization around nrfE after insertion of the tet(M) cassette. The labeled arrows indicate primers used for PCR. (C) The genotype of AL362 was investigated by PCR. Genomic DNA from AL362 (lanes 3, 5, and 7) was compared to genomic DNA from wild-type strain X-73 (lanes 2, 4, and 6). Lanes 2 and 3, amplification with 1914 and 1915; lanes 4 and 5, amplification with 2277 and 2278; lanes 6 and 7, amplification with tet(M) primer 683 together with genomic primer 2278. Lane 1 contained λ DNA digested with HindIII.

FIG. 2.

Genomic organization of the formate-dependent nitrite reduction (nrf) locus in selected gram-negative bacteria. Genomic organizations shown are for the following strains: P. multocida Pm70 (GenBank accession number NC_002663), H. influenzae Rd (GenBank accession number NC_000907), and E. coli K-12 (GenBank accession number NC_000913). Open reading frames are indicated by labeled boxes. Cross-hatched boxes indicate predicted orthologs, and the percentage above each box indicates the level of protein sequence identity to the P. multocida Pm70 protein. The superscript a indicates homology to nrfF_2, and the superscript b indicates homology to nrfF_1 and nrfF_2.

FIG. 3.

Nitrite reduction by P. multocida strains grown either aerobically or anaerobically. Strains were grown in nitrate/nitrite CDM, and culture supernatants were tested for the presence of nitrite after 18 h (aerobic) or 72 h (anaerobic). The values are the means ± standard deviations for triplicate cultures. When grown both aerobically and anaerobically, wild-type strain X-73 reduced nitrite, whereas mutant strain AL362 was unable to reduce nitrite. Additionally, under both growth conditions, the nrfE-complemented strain AL466 was able to reduce nitrite at a level that was not significantly different from the level of nitrite reduction of wild-type strain X-73 (P > 0.05) but was significantly different from the levels of nitrite reduction of AL362 and AL465 (P < 0.05). The levels of nitrite in the vector control AL465 culture and the uninoculated medium control were significantly different from the levels for all of the other strains (P < 0.001) but not from each other in the aerobically grown cultures. In the anaerobic cultures, the medium control contained smaller amounts of nitrite than the amounts observed during the aerobic nitrite assays and was not significantly different from the wild-type X-73, AL362, and AL466 cultures (P > 0.05), but it was significantly different from AL465 cultures (P < 0.01). OD 530nm, optical density at 530 nm.

FIG. 4.

Schematic diagram of the positions of primers used in real-time RT-PCRs to assess the levels of transcripts at different points between nrfA and nrfE. The PCR sets were designated A, D, E, AA, AB, PA, and PB. The arrows and the numbers 2123, 2124, 2363, 2364, 2404, 2405, 2403, 2442, 2407, 2398, and 2399 indicate primers 2123, 2124, 2363, 2364, 2404, 2405, 2403, 2442, 2407, 2398, and 2399, respectively.

FIG. 5.

Relative levels of nrfA, nrfD, and nrfE expression as determined by real-time RT-PCR (normalized with gyrB) during anaerobic growth of P. multocida X-73 and AL362. Cultures of X-73 and AL362 were grown simultaneously in conditions optimal for nitrite reduction. The values are means ± standard deviations for relative expression determined from a minimum of three reactions.

FIG. 6.

Locations of real-time PCR primers within nrfD and nrfE from Pm70 (GenBank accession number NC_002663). Primer sites and directions are indicated by arrows below the sequence. Translational start and stop codons are indicated by boldface type.