Yukinaga Shibata

Sixteen Homologs of the Mex-Type Multidrug Resistance Efflux Pump in Bacteroides fragilis

ABSTRACT 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 β-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.

Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis

BackgroundPorphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation.MethodsTo elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining.ResultsComparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation.ConclusionThese results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

Porphyromonas gingivalis mutant defective in a putative extracytoplasmic function sigma factor shows a mutator phenotype.

INTRODUCTION Porphyromonas gingivalis is implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis must possess the ability to tolerate stress signals outside the cytoplasmic membrane by transcriptional activation of genes encoding proteins involved in defense or repair processes. Some bacteria utilize a distinct subfamily of sigma factors to regulate extracytoplasmic function (hence termed the ECF subfamily). METHODS To elucidate their role in P. gingivalis, a chromosomal mutant carrying a disruption of an ECF sigma factor PG1318-encoding gene was constructed. Hemagglutination and proteolytic activities were measured in the PG1318-defective mutant. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and southern blot analysis were used to assess transcription of kgp in the PG1318-defective mutant. Frequency of spontaneous mutation that conferred resistance to l-trifluoromethionine was measured in the PG1318-defective mutant. RESULTS The PG1318-defective mutant formed non-pigmented colonies on blood agar plates at a relatively high frequency. Arginine-specific and lysine-specific proteinase activities of the non-pigmented variants were remarkably decreased compared with those of the parent strain and the pigmented variants. RT-PCR analysis showed that kgp was not transcribed in some non-pigmented variants and southern blot analysis revealed that there was a deletion in their kgp region. Frequency of mutation conferring resistance to l-trifluoromethionine was significantly higher in the PG1318-defective mutant than in the wild-type. CONCLUSION These results suggest that PG1318 plays a role in the regulation of mutation frequency in the bacterium.

Isolation and properties of a tripeptidyl peptidase from a periodontal pathogen Prevotella nigrescens

Prolyltripeptidyl amino peptidase activity was found in a crude extract of Prevotella nigrescens and this enzyme was purified by procedures including concentration with ammonium sulfate, ion exchange chromatography, gel filtration, and isoelectric focusing. This peptidase hydrolyzed Ala-Ala-Pro-p-nitroanilide as well as Ala-Phe-Pro-p-nitroanilide. Furthermore, several p-nitroanilide derivatives of dipeptides with a proline residue in the second position from the amino-terminal end (Xaa-Pro) were also cleaved detectably. The molecular mass of this tripeptidase was calculated as 56 kDa and its isoelectric point was 5.8. The enzyme was inactivated completely by heating at 60 degrees C for 5 min and inhibited significantly by specific serine enzyme inhibitors.

Cellular locations of proteinases and association with vesicles in porphyromonas gingivalis

We found that locations of arginine-specific gingipain (RGP) in the cellular fractions in the crude extract, envelope, vesicles, and culture supernatants were 48%, 16%, 17%, and 31%, respectively, and the corresponding values of lysine-specific gingipain (KGP) were 47%, 10%, 7%, and 36%, respectively. Although the molecular mass of RGP in the culture supernatant had been determined as 43 kDa, and that of KGP had been as 48 kDa, molecular masses of both proteinases solubilized from the vesicles were estimated to be over 1,500 kDa, since they eluted in the void volume of the column in the gel filtration on Sephacryl S-300. There was no reduction of molecular size by the following treatment with SDS, high-concentration NaCl, or urea. Interestingly, the occurrence of the macromolecular forms could not observed in other enzymes tested such as monopeptidyl, dipeptidyl, and tripeptidyl peptidases, as well as alkaline phosphatase. Therefore, occurrence of the macromolecular forms may be restricted to the proteinases. When the vesicle and culture supernatants containing free RGP and KGP were mixed and incubated, neither RGP nor KGP seemed to bind to vesicles. RGP bound to the vesicle was found to be more stable to heat treatment than the free form, suggesting that association of RGP with the vesicle caused heat stability of this enzyme.

Research articleSome binding properties of the envelope of Porphyromonas gingivalis to hemoglobin

Porphyromonas gingivalis was found to bind to hemoproteins (hemoglobin, myoglobin, catalase, cytochrome c) and the binding properties of the envelope of P. gingivalis to hemoglobin were investigated. Maximum amount of hemoglobin bound to 1 mg of the envelope was 58 μg. No significant binding was observed at 4°C and the binding was inhibited strongly by tosyl-l-lysine chloromethyl ketone, Leupeptin, EDTA and partially by meta-periodate. Heating of the envelope at 70°C for 15 min resulted in complete loss of the binding activity. The binding activity of the envelope was not influenced by the treatment with the endogenous proteases. The envelope saturated with hemoglobin could no longer bind to other hemoproteins tested, indicating that binding site for these hemoproteins are common.

Hemoglobin binding activity and hemoglobin-binding protein of Prevotella nigrescens

Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelope was 51.2 μg. Kd for the reaction at pH 5.0 was 2.1 × 10-10M (210 pM). From the dot blot assay, hemoglobin could bind to a protein solubilized from the envelope by a detergent, referred to as hemoglobin-binding protein (HbBP), then it was purified by the sequential procedures of ion exchange chromatography, affinity chromatography and isoelectric focusing. Molecular weight and isoelectric point of the HbBP were 46 kDa and 6.1, respectively.