The capacity to form volatile sulfur compounds was tested in bacteria isolated from subgingival microbiotas and in a representative number of reference strains. A majority of the 75 tested oral bacterial species and 7 unnamed bacterial taxa formed significant amounts of hydrogen sulfide from L-cysteine. The most active bacteria were found in the genera Peptostreptococcus, Eubacterium, Selenomonas, Centipeda, Bacteroides and Fusobacterium. Methyl mercaptan from L-methionine was formed by some members of the genera Fusobacterium, Bacteroides, Porphyromonas and Eubacterium. When incubated in serum for 7 d, the most potent producers of hydrogen sulfide were Treponema denticola and the black-pigmented species, Bacteroides intermedius, Bacteroides loescheii, Porphyromonas endodontalis and Porphyromonas gingivalis. P. endodontalis and P. gingivalis also produced significant amounts of methyl mercaptan in serum. No other volatile sulfur compound was detected in serum or in the presence of L-cysteine and L-methionine. These findings significantly increase the list of oral bacteria known to produce volatile sulfur compounds.

In 12 species of Fusobacterium the following characteristics were studied; the desulfhydration of L-cysteine and L-methionine by resting cell suspensions, the formation of alpha-keto-acids from L-cysteine, D-cysteine and L-methionine by cell extracts, and the formation of hydrogen sulfide from L-cysteine, D-cysteine and L-cysteine by cell extracts separated by polyacrylamide gel electrophoresis. Multiple forms of L-cysteine desulfhydrase activity were found in most of the species. In some of them also D-cysteine desulfhydrase activity was demonstrated. Seven of the species had high L-methionine gamma-lyase activity. L-cysteine activity was present in 5 of the species.

There are high amounts of hydrogen sulfide in deep periodontal pockets. This volatile sulfur compound may be formed from L-cysteine, but only low levels of this amino acid can be expected to be present in periodontal pockets. Glutathione, L-gamma-glutamyl-L-cysteinylglycine, is in high concentration in most tissue cells, and this tripeptide may be more readily available as a source of hydrogen sulfide formation in the pockets. The ability of 37 different species of oral bacteria to utilize glutathione in hydrogen sulfide formation was studied. Of these species, only 2 species of Peptostreptococcus and 5 species of Fusobacterium formed high amounts of hydrogen sulfide from glutathione within 24 h. Since the initial rate of hydrogen sulfide formation was more than 5 times higher in Peptostreptococcus micros than in any of the other bacterial species, the kinetics of sulfide formation from glutathione by P. micros was further elucidated. The formation of sulfide followed quite closely hyperbolic Michaelis-Menten kinetics. The maximal initial rate of sulfide formation (Vmax) was 163 +/- 2 nmol sulfide per minute per milligram of cellular protein. Half maximal initial rate (Km) was obtained at 7.4 +/- 0.8 microM glutathione. The initial rate of sulfide formation from L-cysteine was much slower and was almost proportional to L-cysteine concentration. This difference in kinetics of sulfide formation between glutathione and L-cysteine strongly suggested that glutathione was actively transported into the cell, whereas the transport of L-cysteine was more or less controlled by diffusion. The sulfide formation from the dipeptide L-cysteinylglycine also followed quite closely hyperbolic Michaelis-Menten kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptostreptococcus anaerobius VPI 4330-1 was exposed to atmospheric oxygen in a dilution blank (0.2% gelatin, salts, resazurin) solution. The organisms were rapidly killed when the solution contained cysteine. The organisms were effectively protected by catalase and horseradish peroxidase as well as by the metal ion-chelating agents 8-hydroxyquinoline and 2,2'-bipyridine. Superoxide dismutase increased the rate of killing of the organisms, whereas singlet oxygen quenchers and scavengers of hydroxyl free radicals did not protect the organisms from the toxic effect of cysteine. Hydrogen peroxide was formed when cysteine was exposed to oxygen in the dilution blank solution, and the reaction was inhibited by metal ion-chelating agents. The organisms were rapidly killed by 20 ,uM hydrogen peroxide in anaerobic dilution blank solution. The toxic effect of hydrogen peroxide was completely abolished by catalase and metal ion-chelating agents. These results indicated that hydrogen peroxide was formed in the dilution blank solution in a metal ion-catalyzed autoxidation of cysteine and that hydrogen peroxide was toxic to P. anaerobius VPI 4330-1 in a reaction also catalyzed by metal ions. Cysteine may be toxic to or may inhibit growth of Escherichia coli (11, 25, 46), Bacillus subtilis (52), yeasts (7, 33, 36), and fungi (4, 49). Cysteine appears to inhibit growth by two different mechanisms in E. coli. It may interfere with biosynthesis of leucine, isoleucine, threonine, and valine, or it may interact with the function of membrane-bound enzymes (25). The growth-inhibiting effect of cysteine in yeasts has been ascribed to its ability to chelate metal ions necessary for the activities of various enzymes (7, 36). Cysteine is routinely used in many media for the cultivation of anaerobic bacteria (22). In a study on the bactericidal effects of various culture media exposed to atmospheric oxygen (13), it was observed that cysteine was toxic to Peptostreptococcus anaerobius VPI 4330-1. We now report that hydrogen peroxide is formed from cysteine in the presence of oxygen in a metal ion-catalyzed reaction and that hydrogen peroxide is toxic to P. anaerobius VPI 4330-1 in a reaction also catalyzed by metal ions. MATERIALS AND METHODS Microorganisms. P. anaerobius strain VPI 4330-1 (ATCC 27337) was used as the test strain (23). It was kept on blood agar plates at 4°C under strictly anaerobic conditions in an anaerobic box with an atmosphere of 10% H2 and 5% CO2 in nitrogen (54).

We recently studied the utilization of glutathione (L-gamma-glutamyl-L-cysteinylglycine), L-cysteinylglycine and L-cysteine by anaerobic bacteria. The rate of hydrogen sulfide formation from these compounds was determined and it was concluded that Peptostreptococcus micros and Fusobacterium nucleatum subsp. nucleatum had an active transport of small peptides. In the present study it is shown that methyl mercaptan formation from L-methionine and L-methionyl-containing peptides can also be used to study peptide utilization. There were differences among the periodontal bacteria P. micros, F. nucleatum subsp. nucleatum, and Porphyromonas gingivalis in their capacity to use L-cysteine and L-methionine and peptides containing these amino acids. The peptides were used more efficiently by P. micros and F. nucleatum subsp. nucleatum than by P. gingivalis. All three species used the peptides more efficiently than the free amino acids. The efficiency in utilizing various amino acids and peptides may be among the key determinants of the periodontal microbial ecology.