SummaryThe obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene (rgp1) and was also present in genes for lysine-specific cysteine proteinases (prtP and kgp) and a haemagglutinin (hagA) of P. gingivalis. The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis.

A protease was purified from Bacteroides gingivalis ATCC 33277 culture fluid by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, and isoelectric focusing. The enzyme was active against benzoyl-L-arginine-p-nitroanilide, carbobenzoxy-L-phenylalanyl-L-valyl-L-argininep-nitroanilide, azoalbumin, azocasein, azocoll, and p-tosyl-L-arginine methyl ester. The molecular weight of the enzyme was about 300,000 as determined by gel filtration. Its isoelectric point was 5.0. The maximum activity was found at pH 7.5, and the optimum temperature for activity was between 40 and 45°C. The apparent Km

From the culture supernatant of Bacteroides gingivalis ATCC 33277, a thiol protease was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and isoelectric focusing. Its molecular weight was 43 kDa and showed similar enzymatic properties to a 300-kDa protease that was previously characterized.

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...