Charactt[sic]erization of a plasmid conferring NAD independence in Haemophilus paragallinarum

[摘要] English: Members of the family Pasteurellaceae are classified in part by whether or not they require NAD+ supplement for growth on laboratory media. It is known that this phenotype is determined by a plasmid whose presence allows NAD+-independent growth of Haemophilus paragallinarum.In this study, this 6-kb plasmid, which was previously shown to be responsible for NAD+ independent growth of H. paragallinarum on defined media, was isolated. Isolated plasmid DNA was shredded by sonification and subcloned into vector PGEM-T easy. The recombinant plasmid was transformed into E.coli the transformants isolated were sequenced.Sequence analysis revealed one open reading frame of 1119bp that is predicted to encode a protein with a molecular mass of 43kD. Compared with the sequence databases, this protein was found to have significant sequence homology to Quinolinate phosphoribosyltransferase of Bacillus anthracis this enzyme is responsible for the production of nicotinic acid mononucleotide (NAMN) from nicotinate and quinolinate. A 3284bp nucleotide fragment of the plasmid revealed four additional open reading frames. Proteins encoded on this fragment of the plasmid all have significant homology to proteins from H. influenzae of which all have functions related to production and immunity of the bacteriocin haemocim. This bacteriocin produced by most type b-encapsulated strains of H. influenzae, is toxic to virtually all non-type b strains of H. influenzae idependent of encapsulation. This bacteriocin is thought to inhibit DNA synthesis, of susceptible strains. Purification of this bacteriocin and testing its toxicity to other pathogens as a possible antimicrobial drug might form the bases of a future study.Previous work has indicated that plasmid bearing strains of H. paragallinarum are less virulent, thus creating the possibility that more virulent wild type strains can be transformed and used as live vaccines. The influence of transformation with this plasmid on other members of the family Pasteurellaceae and the possibility of creating live vaccines should be further investigatedSince species of the genus Haemophilus cannot easily be transformed with plasmid, this naturally occurring plasmid could be modified to create a vector, which has specific application in the transformation of Haemophilus species.

[发布机构] University of the Free State