The hemagglutinin genes hagB and hagC of Porphyromonas gingivalis are transcribed in vivo as shown by use of a new expression vector.
The hemagglutinin genes hagB and hagC of Porphyromonas gingivalis, a putative periodontopathic microorganism, have been cloned, sequenced, and characterized. However, the roles of these putative virulence genes have not yet been determined. In this study, an in vivo expression technology vector termed pPGIVET was constructed and used to determine if hagB and hagC were expressed during an infectious process. We constructed pPGIVET as a conjugative suicide plasmid containing a multiple-cloning site (MCS) upstream of two tandem promoterless reporter genes that encode tetracycline resistance [tetA(Q)2] and galactokinase (galK). The promoter and a portion of the open reading frame (ORF) of hagB were inserted into the MCS in both a positive and a negative orientation relative to the reporter genes. These constructs were conjugated into P. gingivalis 381. Southern blot analysis of different transconjugants indicated that Campbell insertions had occurred at the chromosomal hagB locus and also at the hagC locus, which has high (99%) homology to the ORF of hagB. pPGIVET-labeled clones in which the hag promoters were positively oriented relative to the reporter genes expressed tetracycline 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 tetracycline resistance allowed substantial enrichment of heterodiploids over wild-type cells during a mixed infection in the mouse abscess model. These results indicate that hagB and hagC are transcriptionally active in vivo and suggested that pPGIVET may be used to isolate P. gingivalis genes expressed only during an infectious process.
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville 32610, USA.
S W
SW
Lee
J D
JD
Hillman
A
A
Progulske-Fox
D000038:Abscess / Q000382:microbiology; D018829:Adhesins, Bacterial; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D016866:Bacteroidaceae Infections / Q000382:microbiology*; D003227:Conjugation, Genetic; D005260:Female; D005686:Galactokinase / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D006388:Hemagglutinins / Q000235:genetics*; D037102:Lectins; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000472:pathogenicity; D011401:Promoter Regions, Genetic; D013753:Tetracycline Resistance / Q000235:genetics; D014158:Transcription, Genetic; D014774:Virulence
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; C098582:HagB protein, Porphyromonas gingivalis; D006388:Hemagglutinins; D037102:Lectins; D005686:Galactokinase