Haemophilus ducreyi Strain ATCC 27722 Contains a Genetic Element with Homology to the Vibrio RS1 Element That Can Replicate as a Plasmid and Confer NAD Independence on Haemophilus influenzae

The nucleotide sequence of pNAD1, a plasmid from Haemophilusducreyi identified on the basis of its ability to confer NADindependence 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 ${phi}$ and the Vibrio RS1 element, suggesting a single-strandedbacteriophage origin for pNAD1. Tandem copies of the plasmidare integrated into the H. ducreyi 35000HP genome.

INTRODUCTION

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Introduction

Haemophilus ducreyi is the causative agent of chancroid, a sexuallytransmitted ulcerative disease. H. ducreyi, a member of thePasteurellaceae family, requires exogenous heme for growth butdoes not require NAD (V factor). V-factor-independent strainsamong the Pasteurellaceae can be differentiated from V-factor-dependentstrains by the ability to utilize nicotinamide (NAm) as a precursorfor NAD biosynthesis (12). H. haemoglobinophilus, which is Vfactor independent, synthesizes the enzyme nicotinamide phosphoribosyltransferase,which converts NAm to nicotinamide mononucleotide and allowsthe 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 includestrains of Actinobacillus pleuropneumoniae, which causes pleuropneumoniain swine (17); H. paragallinarum, which causes fowl coryza (1,10); and H. parainfluenzae, which can cause pneumonia and meningitisin humans (4). In H. parainfluenzae, H. paragallinarum, andcertain H. ducreyi strains, the gene encoding V-factor independencehas been shown to be present on a plasmid (1, 19, 20).

Martin et al. identified a plasmid (designated pNAD1) that conferredNAD independence on H. influenzae strain KW20 and A. pleuropneumoniae(8). One gene (designated nadV) carried on this plasmid wasshown to encode nicotinamide phosphoribosyltransferase. In thepresent study, we determined the sequence of the remainder ofpNAD1 and determined that it encodes homologues of bacteriophagegenes responsible for the replication of the Vibrio CTX phageand the RS1 element. Further, we demonstrated that the plasmidis integrated in tandem copies in the H. ducreyi 35000HP genome(GenBank accession no. NC_002940).

Bacterial strains and culture conditions.

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Bacterial strains and culture...

H. ducreyi strains 35000HP and ATCC 27722 were grown at 35°Cwith 5% CO₂ on chocolate agar (Becton Dickinson). Chocolateagar plates supplemented with kanamycin at 20 µg/ml andchocolate 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 µg/mland ampicillin was used at 50 µg/ml.

Recombinant DNA techniques.

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Recombinant dna techniques.

All restriction enzymes were purchased from New England Biolabs.Calf intestine alkaline phosphatase and T4 DNA ligase were purchasedfrom Gibco BRL. Plasmid isolations were performed using Qiagenpurification kits (Qiagen, Valencia, Calif.). Electroporationof E. coli and H. ducreyi was performed as described previously(13). Standard recombinant DNA methods were performed as describedpreviously (13). Southern blotting was performed by the ECLmethod according to the instructions of the manufacturer (AmershamPharmacia Biotech, Piscataway, N.J.).

Plasmids were prepared (using a Qiagen Miniprep kit) from H.ducreyi ATCC 27722 and 35000HP. No plasmid DNA was observedon ethidium bromide-stained agarose gels or SYBR-Gold stainedagarose gels, but plasmid DNA was detected in plasmid preparationsby Southern hybridization (data not shown). Although the plasmidwas present at very low concentrations, NAD-independent clonesof A. pleuropneumoniae or H. influenzae strain Rd were readilyidentified after transformation with the Miniprep DNA preparedfrom H. ducreyi ATCC 27722. A plasmid containing the nadV genewas isolated (using a Qiagen Miniprep procedure) from theseNAD-independent transformants. The plasmid isolated from anH. influenzae strain Rd transformant was sequenced using anABI 3100 DNA automated sequencer and dye terminator chemistries.Sequencing primers were generated on the basis of the previouslydetermined sequence of the nadV gene (GenBank accession no.AF273842), and additional primers were generated as necessaryto sequence both strands of the complete plasmid. Contig assemblyand sequence analysis were performed with DNASTAR software (Madison,Wis.) and with GCG software (Genetics Computer Group, Madison,Wis.). Blast homologies were identified using the National Centerfor Biotechnology Information server (http://www.ncbi.nlm.nih.gov/BLAST/).

Characterization of pNAD1.

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Characterization of pnad1.

Plasmid pNAD1 is a circular 5,568-bp plasmid (Fig. 1). In additionto containing the previously reported nadV gene, the sequencecontains several potential coding regions. One open readingframe (ORF) (designated plpR [phage-like protein]) encodes aputative protein that is 55% identical to the product of therstR gene of the cholera toxin phage (CTX ${phi}$ ) from Vibrio choleraeO139 Calcutta, a repressor that regulates the transcriptionof the rstA gene (Fig. 2A) (3, 11). Transcribed in the oppositedirection is a putative gene (designated plpA) whose productis 51% identical to ORF320 of bacteriophage If1 and 36% identicalto the rstA gene product, a protein required for replicationof CTX ${phi}$ DNA (Fig. 2B) (18). Downstream of plpA is a small ORFdesignated plpD that is 31% identical to the product of geneV of enterobacterial phage I2-2, a putative single-strandedDNA binding protein (accession no. NP_039617). A fourth ORF(designated ORF167) has homology to several hypothetical proteins.Several other small ORFs have been noted but are of unknownsignificance, as they have no homologues in the GenBank database(Fig. 1). The Vibrio RS1 element has a third gene (designatedrstB) (18). The product of the rstB gene is thought to playa role in integration of the RS1 element. No homologue of thisgene is present in pNAD1. The sequence of pNAD1 has been depositedin GenBank (accession no. AY434675).

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FIG. 1. Circular map of pNAD1. The nadV gene encodes nicotinamide phosphoribosyl transferase as described by Martin and coworkers (GenBank accession no. AF273842) (8). The plpR and plpA genes, as well as the gene encoding a single-stranded DNA binding protein (designated plpD), are shown. Other ORFs are designated by the number of residues in the putative protein products. Plasmid pNAD1 has been assigned GenBank accession no. AY434675.

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FIG. 2. (A) ClustalW alignment of the derived amino acid sequences of the plpR gene and the rstR gene from V. cholerae O139 Calcutta (accession no. AAF07851 and AAF43270). (B) ClustalW alignment of the derived amino acid sequences of the plpA gene, the rstA1 gene from V. cholerae strain SCE264 (accession no. AAK07627) (11), and ORF320 from bacteriophage If1 (accession no. NP_047361).

The pNAD1 element replicates as a plasmid in H. influenzae Rd.We were interested in determining whether it would replicatein E. coli. The ${Omega}$ Kan-2 interposon (16) was cloned into a uniqueBglII site in the nadV gene, and the ligation mixture was transformedinto H. influenzae. Clones were selected on chocolate agar containingkanamycin, and a plasmid with the correct restriction map wassaved as pNAD1:: ${Omega}$ Kan-2. Plasmid pNAD1:: ${Omega}$ Kan-2 was transformedinto E. coli DH5 ${alpha}$ , and clones were selected on LB agar containingkanamycin at 10 µg/ml. Transformants were not obtained,indicating that pNAD1:: ${Omega}$ Kan-2 could not become established inE. coli (data not shown).

H. ducreyi strain 35000HP has tandem copies of pNAD1 integrated into the chromosome.

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H. ducreyi strain 35000hp...

Strain 35000HP is the prototype strain used by most investigators.This strain is thought to be plasmid free. The sequence of thegenome of this strain was recently completed; again, there wasno indication that a plasmid was present. When a Qiagen Miniprepwas prepared and used to transform H. influenzae, however, aplasmid conferring NAD independence on H. influenzae was readilyidentified. This plasmid had the same restriction map as pNAD1.When the genome sequence was probed with the nadV gene, it wasapparent that there were tandem integrated copies of pNAD1 inthe H. ducreyi genome. The genomic region from strain 35000HPis shown in Fig. 3. The ompP2A and ompP2B genes encode porin-likeproteins with homology to the P2 protein of H. influenzae. Downstreamof the ompP2B gene are a number of putative genes, several ofwhich have homology to putative phage proteins. Hd1436 is mostclosely related to a putative phage-related secreted proteinfrom Yersinia pestis CO92 (YPO2280) (accession no. NP_405817;48% identical over a 356-amino-acid [aa] overlap) (15). Hd1437is 40% identical to a putative Y. pestis CO92 phage-relatedmembrane protein (YPO2278) (accession no. NP_405815) (15). TheC-terminal 17 aa of Hd1439 are 76% identical to YPO2277 (accessionno. NP_405814) (15), but overall, the putative proteins arenot closely related. The 5' portion of the Hd1439 gene (Fig.3) is located within the pNAD1 plasmid. The second copy of theplasmid contains only the 5' portion of Hd1439 (Fig. 3) (annotatedas ORF80b in Fig. 1). Downstream of the nadV2 gene is Hd1456,the ackA gene that encodes acetate kinase. The first 51 basesof the acetate kinase gene are located in the pNAD1 plasmid(ORF17) (Fig. 1).

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FIG. 3. Map of the chromosomal region of the H. ducreyi 35000HP genome containing pNAD1. The pNAD1 genes are color coded as shown in Fig. 1 for easy reference.

A gene with homology to the nadV gene is present in a numberof organisms (8). The ORFs with the highest homology are fromShewanella oneidensis MR-1 (5), Cytophaga hutchinsonii (accessionno. ZP_00118436), and Mycoplasma pneumoniae M129 (2, 6). Amongthe Pasteurellaceae, the gene is absent from H. influenzae butpresent in Pasteurella multocida and A. actinomycetemcomitans(9). In the S. oneidensis, P. multocida, and M. pneumoniae genomes,the gene order does not resemble that seen in H. ducreyi.

To experimentally demonstrate that pNAD1 was integrated intothe H. ducreyi 35000HP genome, Southern hybridization analysiswas performed using an enhanced chemiluminescence methodology.Genomic DNA from strain 35000HP was digested with BglII (cutsonce in the pNAD1 plasmid) and BstEII, an enzyme that does notcut pNAD1. The positions of the restriction enzyme sites areshown in Fig. 3. The Southern blot was probed with pNAD1 (Fig.4). BglII fragments of the anticipated sizes (12.8, 5.6, and2.4 kb) were observed. Similarly, the expected 16.8-kb genomicBstEII fragment was observed, indicating that there are twotandem copies of pNAD1 integrated into the H. ducreyi chromosome.

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FIG. 4. Chromosomal DNA was prepared from H. ducreyi strain 35000HP and digested with BglII or BstEII, and fragments were separated on an agarose gel. After transfer, the blot was hybridized to a PCR product containing the majority of pNAD1 that had been labeled by an enhanced chemiluminescence procedure. The size of the restriction fragments confirms the chromosomal localization of pNAD1. The size of the BstEII genomic fragment is consistent with two tandem copies of pNAD1.

Miller and coworkers recently identified a T4-like bacteriophage(designated KVP40) that infects V. cholerae and other Vibriospecies (E. S. Miller, Abstr. 103rd Gen. Meet. Am. Soc. Microbiol.,abstr. M-020, 2003, and M. Pineda, B. Szczypinski, E. S. Miller,and D. M. Hinton, Abstr. 103rd Gen. Meet. Am. Soc. Microbiol.,abstr. M-008, 2003). The genome of this bacteriophage encodesa complete NAD scavenging system including the nadV gene. PlasmidpNAD1 appears (by sequence homology) to have evolved from asingle-stranded DNA CTX ${phi}$ -like bacteriophage that acquired thenadV gene, possibly from a KVP40-like phage, and then underwenta duplication event. Phage-like sequences are recognizable adjacentto one copy of pNAD1 in the strain 35000HP genome, but onlythe 5' portion of the Hd1439 gene is present in pNAD1. The existenceof plasmid-mediated NAD independence in other members of thePasteurellaceae suggests that pNAD1, or a related plasmid, hasbeen horizontally transferred among members of this group oforganisms.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health(grants R01 AI45091 and R01 HD-96-003).

FOOTNOTES

* Corresponding author. Mailing address: Children's Research Institute, Room W502, 700 Children's Dr., Columbus, OH 43205. Phone: (614) 722-2680. Fax: (614) 722-3273. E-mail: munsonr{at}pediatrics.ohio-state.edu.

Editor: V. J. DiRita

REFERENCES

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