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Members of the family Pasteurellaceae are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene, nadV, whose presence allows NAD-independent growth of Haemophilus influenzae and Actinobacillus pleuropneumoniae. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in Haemophilus ducreyi. When transformed into A. pleuropneumoniae, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida, and Actinobacillus actinomycetemcomitans. P. multocida and A. actinomycetemcomitans are among the NAD-independent members of the Pasteurellaceae. Homologues of NadV were not found in the sequenced genome of H. influenzae, an NAD-dependent member of the Pasteurellaceae, or in species known to utilize a different pathway for synthesis of NAD, such as Escherichia coli. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the Pasteurellaceae should help to elucidate the role of its homologues in other species.NAD is a critical cofactor required for energy metabolism and many oxidation-reduction reactions in both prokaryotic and eukaryotic cells. In many bacterial species, synthesis of NAD occurs de novo via quinolinic acid (3, 4). NAD can also be synthesized by a pyridine nucleotide salvage pathway via nicotinic acid (3, 4). Members of the family Pasteurellaceae do not possess either of these pathways for NAD biosynthesis. These bacterial species must acquire this essential nutrient from their environment either as NAD directly or from a limited number of precursors (18,19). This pyridine nucleotide requirement has been historically important in the identification and classification of members of the Pasteurellaceae, with species requiring an NAD supplement for growth in vitro described as V-factor dependent (12, 13). In V-factor-dependent species, the pyridine nucleotide source must possess an intact pyridine-ribose bond and the pyridine-carbonyl group must be amidated; therefore, nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can function as V-factors, but quinolinic acid, nicotinic acid, nicotinic acid mononucleotide, and nicotinamide (NAm) cannot (3,19). The ability to use NAm as a precursor for NAD biosynthesi...

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Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a disease characterized by pulmonary necrosis and hemorrhage caused in part by neutrophil degranulation. In an effort to understand the pathogenesis of this disease, we have developed an in vivo expression technology (IVET) system to identify genes that are specifically up-regulated during infection. One of the genes that we have identified as being induced in vivo is ohr, encoding organic hydroperoxide reductase, an enzyme that could play a role in detoxification of organic hydroperoxides generated during infection. Among the 12 serotypes of A. pleuropneumoniae, ohr was found in only serotypes 1, 9, and 11. This distribution correlated with increased resistance to cumene hydroperoxide, an organic hydroperoxide, but not to hydrogen peroxide or to paraquat, a superoxide generator. Functional assays of Ohr activity demonstrated that A. pleuropneumoniae serotype 1 cultures, but not serotype 5 cultures, were able to degrade cumene hydroperoxide. In A. pleuropneumoniae serotype 1, expression of ohr was induced by cumene hydroperoxide, but not by either hydrogen peroxide or paraquat. In contrast, an ohr gene from serotype 1 cloned into A. pleuropneumoniae serotype 5 was not induced by cumene hydroperoxide or by other forms of oxidative stress, suggesting the presence of a serotype-specific positive regulator of ohr in A. pleuropneumoniae serotype 1.

The nucleotide sequence of pNAD1, a plasmid from Haemophilus ducreyi identified on the basis of its ability to confer NAD independence on Actinobacillus pleuropneumoniae and H. influenzae, has been determined. In addition to containing the nadV gene, the plasmid contains homologues of the rstR and rstA genes, genes encoding repressor and replication proteins, respectively, in the Vibrio CTX and the Vibrio RS1 element, suggesting a single-stranded bacteriophage origin for pNAD1. Tandem copies of the plasmid are integrated into the H. ducreyi 35000HP genome.Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted ulcerative disease. H. ducreyi, a member of the Pasteurellaceae family, requires exogenous heme for growth but does not require NAD (V factor). V-factor-independent strains among the Pasteurellaceae can be differentiated from V-factor-dependent strains by the ability to utilize nicotinamide (NAm) as a precursor for NAD biosynthesis (12). H. haemoglobinophilus, which is V factor independent, synthesizes the enzyme nicotinamide phosphoribosyltransferase, which converts NAm to nicotinamide mononucleotide and allows the use of NAm as a source of pyridine nucleotide (7).For many species of Pasteurellaceae defined as V factor dependent, V-factor-independent variants have been identified. These include strains of Actinobacillus pleuropneumoniae, which causes pleuropneumonia in swine (17); H. paragallinarum, which causes fowl coryza (1, 10); and H. parainfluenzae, which can cause pneumonia and meningitis in humans (4). In H. parainfluenzae, H. paragallinarum, and certain H. ducreyi strains, the gene encoding V-factor independence has been shown to be present on a plasmid (1,19,20).Martin et al. identified a plasmid (designated pNAD1) that conferred NAD independence on H. influenzae strain KW20 and A. pleuropneumoniae (8). One gene (designated nadV) carried on this plasmid was shown to encode nicotinamide phosphoribosyltransferase. In the present study, we determined the sequence of the remainder of pNAD1 and determined that it encodes homologues of bacteriophage genes responsible for the replication of the Vibrio CTX phage and the RS1 element. Further, we demonstrated that the plasmid is integrated in tandem copies in the H. ducreyi 35000HP genome (GenBank accession no. NC_002940).Bacterial strains and culture conditions. H. ducreyi strains 35000HP and ATCC 27722 were grown at 35°C with 5% CO 2 on chocolate agar (Becton Dickinson). Chocolate agar plates supplemented with kanamycin at 20 g/ml and chocolate plates lacking NAD were prepared as previously described (14). Escherichia coli strains were grown on Luria-Bertani (LB) plates or in LB broth supplemented with appropriate antibiotics. Where appropriate, kanamycin was used at 10 or 20 g/ml and ampicillin was used at 50 g/ml.Recombinant DNA techniques. All restriction enzymes were purchased from New England Biolabs. Calf intestine alkaline phosphatase and T4 DNA ligase were purchased from Gibco BRL. Plasmid isolations were performed using ...