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Comparative Study
. 2001 Feb;183(4):1168-74.
doi: 10.1128/JB.183.4.1168-1174.2001.

Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence

P R Martin  1 , R J Shea, M H Mulks
Affiliations
Comparative Study

Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence

P R Martin et al. J Bacteriol. 2001 Feb.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Biochemical pathway for the biosynthesis of NAD as found in the family Pasteurellaceae. NAD-dependent species lack the ability to convert NAm to NMN.
FIG. 2
FIG. 2
Subclones of pNAD1 constructed in the E. coli–A. pleuropneumoniae shuttle vector pGZRS18 (25). The location of the nadV gene is indicated with an arrow. Plasmids pGZNAD1, pGZNAD7, and pGZNAD8 were constructed using the restriction sites shown. Plasmid pGZNAD9 was constructed using synthetic primers to PCR amplify the nadV gene. The ability of these clones to confer NAD independence on A. pleuropneumoniae is indicated in the right-hand column. Restriction sites: A, AvaI; B, BamHI; E, EcoRI; and P, PstI.
FIG. 3
FIG. 3
Alignment of the predicted NadV amino acid sequence with homologues found in other species. Black shaded regions indicate residues that are identical in the majority of species. Gray shaded regions indicate residues that are functionally conserved in the majority of species. Species abbreviations: aact, Actinobacillus actinomycetemcomitans; pmul, Pasteurella multocida; syn, Synechocystis; drad, Deinococcus radiodurans; mgen, Mycoplasma genitalium; mpne, M. pneumoniae; sput, Shewanella putrefaciens; hduc, Haemophilus ducreyi; hum, human PBEF. The alignment was obtained using the Pileup program from the Genetics Computer Group package (6).
FIG. 4
FIG. 4
Incorporation of [14C]NAm into NMN. NAm-PRTase assays were performed with [14C]NAm as the substrate, and incorporation of radiolabel into NMN monitored over time. Dark bars, A. pleuropneumoniae/pGZNAD9; light bars, A. pleuropneumoniae/pGZRS18.
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