THE HEMAGGLUTININ GENES HAGB AND HAGC OF PORPHYROMONAS-GINGIVALIS ARETRANSCRIBED IN-VIVO AS SHOWN BY USE OF A NEW EXPRESSION VECTOR

The hemagglutinin genes hagB and hagC of Porphyromonas gingivalis, a p utative periodontopathic microorganism, have been cloned, sequenced, a nd characterized. However, the roles of these putative virulence genes have not yet been determined, in this study, an in vivo expression te chnology vector termed pPGIVET was constructed and used to determine i f hagB and hagC were expressed during an infectious process, We constr ucted pPGIVET as a conjugative suicide plasmid containing a multiple-c loning site (MCS) upstream of two tandem promoterless reporter genes t hat encode tetracycline resistance [tetA(Q)2] and galactokinase (galK) . The promoter and a portion of the open reading frame (ORF) of hagB w ere inserted into the MCS in both a positive and a negative orientatio n relative to the reporter genes. These constructs were conjugated int o P. gingivalis 381, Southern blot analysis of different transconjugan ts indicated that Campbell insertions had occurred at the chromosomal hagB locus and also at the hagC locus, which has high (99%) homology t o the ORF of hagB. pPGIVET-labeled clones in which the hag promoters w ere positively oriented relative to the reporter genes expressed tetra cycline resistance and galactokinase activity in vitro and in vivo at significantly higher levels than did the wild-type strain or clones in which the hag promoters were negatively oriented, Expression of tetra cycline resistance allowed substantial enrichment of heterodiploids ov er wild-type cells during a mixed infection in the mouse abscess model , These results indicate that hagB and hagC are transcriptionally acti ve in vivo and suggested that pPGIVET may be used to isolate P. gingiv alis genes expressed only during an infectious process.