A gene encoding for bacteriocin of Prevotella nigrescens

Background: Nigrescin is a bacteriocin produced by Prevotella nigrescens ATCC 25261. This protein has bactericidal activity against gram-negative anaerobic bacteria which are related to periodontal diseases including Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia. Our previous study reported that nigrescin is encoded by the nig gene cluster containing 4 open reading frames (ORFs), designated as nigA nigB nigC and nigD. Objectives: To search for the gene encodes for nigrescin protein. Methods: Each ORF of nig gene cluster was constructed into expression vector. The recombinant protein of NigA, NigB, NigC and NigD were purified and assayed for bacteriocin activity against Porphyromonas gingivalis. Results: The ORFs of nig gene cluster in Pr. nigrescens ATCC 25261 were re-analyzed, it was revealed that the position of nig ORFs similar to previously designated location, except the start codon of nigC was reassigned. The new nigC gene is initiate at nucleotide base positon of 2,454 and stop at position of 3,608 (position designated in relative to the first nucleotide base of the nig locus) and putatively encodes a protein with molecular mass of 41.9 kDa. The N-terminal 6xHistidine-tag recombinant protein of NigA, NigB, NigC and NigD were over-expressed in E. coli BL21star (DE3) and purified by Ni-NTA resin. Only recombinant NigC showed inhibitory activity against P. gingivalis strain A244 with minimal inhibition concentration (MIC) 40 ug/ml. Conclusion: The result indicates that nigC is the gene that encodes nigrescin.

This study was supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand