The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5 end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study ( showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola.

A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, al-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50°C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(O-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction. * Corresponding author.35405 that has the ability to hydrolyze many functionally important serum and tissue proteins. MATERIALS AND METHODSBacterial strains and culture conditions. T. denticola ATCC

Ninety-six subjects were assigned to one of four groups according to severity of gingival inflammation and bacterial plaque accumulation on the teeth. Following a standardized toothbrushing procedure, blood specimens from a vein in the antecubital fossa were cultured under aerobic and stringent anaerobic conditions. The percentage of positive cultures increased significantly with increasing severity of gingival inflammation, as did the number of species of organisms isolated. Thirty different microbial species indigenous to the oral cavity, including many strict anaerobes, were recovered. The study has implications for standards of oral health which might be considered necessary in patients with congenital or acquired endocardial defects or cardiovascular prostheses.

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MCBRIDE AND WOLFEthen the simplest explanation for the poly(U) binding to the 60S subunit fraction is that it is nonspecific binding as indicated by its relative insensitivity to inhibition by ATA.