The binding activity of the Porphyromonas gingivalis envelope and hemoglobin was examined over a wide range of pH values from 4.5 to 9.0. The binding activity in low-pH buffers was much higher than that at high pH; the optimum pHs for the binding were found to be 4.5 and 5.0. Since the hemoglobin bound to the envelope was found to dissociate in the pH 8.5 and 9.0 buffers, the binding is reversible. We hypothesized that hemoglobin-binding protein (HbBP), responsible for the binding to hemoglobin, exists in the envelope and confirmed its presence by dot blot determination with peroxidase-conjugated hemoglobin. Then we attempted to isolate HbBP from the solubilized (by a detergent) materials of the envelope by affinity chromatography. The molecular mass of HbBP was 19 kDa, and the isoelectric point was 4.3.

Sixteen homologs of multidrug resistance efflux pump operons of the resistance-nodulation-cell division (RND) family were found in the Bacteroides fragilis genome sequence by homology searches. Disruption mutants were made to the mexB homologs of the four genes most similar to Pseudomonas aeruginosa mexB. Reverse transcription-PCR was conducted and indicated that the genes were transcribed in a polycistronic fashion and that the promoter was upstream of bmeA (the mexA homolog). One of these disruption mutants (in bmeB, the mexB homolog) was more susceptible than the parental strain to certain cephems, polypeptide antibiotics, fusidic acid, novobiocin, and puromycin. The gene for this homolog and the adjacent upstream gene, bmeA, were cloned in a hypersensitive Escherichia coli host. The resultant transformants carrying B. fragilis bmeAB were more resistant to certain agents; these agents also had lower MICs for the B. fragilis bmeB disruption mutants than for the parental strain. The putative efflux pump operon is composed of bmeA, bmeB, and bmeC (a putative outer membrane channel protein homologous with OprM). Addition of the efflux pump inhibitors, carbonyl cyanide m-chlorophenylhydrazone (a proton conductor that eliminates the energy source) and Phe-Arg ␤-naphthylamide (MC-207,110) (the first specific inhibitor described for RND pumps in P. aeruginosa), resulted in lowered MICs in the parental strain but not in the bmeB disruption mutant, indicating that the bmeB pump is affected by these inhibitors. This is the first description of RND type pumps in the genus Bacteroides.Bacteroides fragilis, an anaerobic gram-negative rod, is an opportunistic pathogen that can cause significant mortality in infections resulting from abdominal trauma or surgery (7,19). Although it accounts for only 0.5% of the enteric flora, it is the Bacteroides species most frequently isolated from patients with intra-abdominal infections and/or bacteremia (in which mortality reached 45% if inactive therapy was given). It often presents a serious problem for therapy, since it is resistant to many antibiotics, including most of the penicillins, cephalosporins, and the quinolones (1,3,19,20,25).Gram-negative bacteria including B. fragilis are usually more resistant to a large number of antibiotics and other noxious agents than are gram-positive bacteria. Clinically significant levels of antibiotic resistance are caused by interplay between the efficient outer membrane (OM) permeability barrier, ubiquitous periplasmic ␤-lactamases, and recently recognized multidrug resistance (MDR) efflux pumps (17). These pumps have broad substrate specificity and may act synergistically with the permeability barrier to result in significant intrinsic resistance to many antimicrobials. These pumps expel the antimicrobial from the cell into the surrounding space, and the antimicrobials then have to pass through the OM permeability barrier to regain entry to the cell (18). Thus, the MDR pumps can effect significant resistance even when their transporter activi...

A peptidase hydrolyzed X-Pro-p-nitroanilide was purified from the cell extract of Prevotella intermedia ATCC 25611 by ion-exchange chromatography and hydrophobic interaction chromatography. The purified enzyme exhibited a molecular size of 74 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the maximum enzyme activity was found between pH 7.0 and pH 7.5. This peptidase was a serine enzyme and hydrolyzed Lys-Pro-p-nitroanilide, Arg-Pro-p-nitroanilide, and Ala-Pro-p-nitroanilide, but Lys-Ala-p-nitroanilide was not split. The enzyme may be classified as a dipeptidyl peptidase IV.

A lysine-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion-exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and the pI value was 7.3. The enzyme hydrolysed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins.

Heparinase was purified to homogeneity from the cell extract of an oral bacterium, Prevotella heparinolytica, by a combination of anion exchange chromatography, gel filtration chromatography, and hydroxyapatite chromatography. Properties of the purified P. heparinolytica heparinase (P. heparinase) were investigated. The enzyme exhibited a maximum activity in 50 mM Tris-HCl buffer, pH 7.5-8.0, containing 75 mM sodium acetate, 0.1 M NaCl, and 1 mM CaCl2. Optimum conditions for the maximum activity of P. heparinase were similar to those of the heparinase from Flavobacterium heparinum (F. heparinase). The two enzymes also yielded similar digestion profiles of various glycosaminoglycans and heparin tetrasaccharides, suggesting that they have a similar substrate specificity. Kinetic study of the P. heparinase reaction using porcine intestinal heparin as substrate gave a Km value of 3.8 x 10(-5) M and a Vmax value of 11.4 micromol/min x mg protein. The Michaelis constant of P. heparinase was slightly larger than but not significantly different from that of F. heparinase. The amino acid composition of P. heparinase was also similar to that of F. heparinase, but its N-terminal sequence of 20 amino acid residues was different and hitherto unreported. These results together indicate that these heparinases are different proteins with closely similar enzymatic properties. Since F. heparinum produces not only heparinase but also heparitinase II, which has a broad substrate specificity, F. heparinase may be contaminated with this enzyme. In contrast, P. heparinolytica does not produce heparitinase II, and P. heparinase should prove a useful tool for degrading heparin without the risk of contamination with heparitinase II.