Correlation between oral malodor and periodontal bacteria

Volatile sulfur compounds (VSCs), including hydrogen sulfide, methyl mercaptan, and dimethyl sulfide, are primarily responsible for oral malodor. Recently, the mgl gene encoding L-methionine-alpha-deamino-gamma-mercaptomethane-lyase, which produces methyl mercaptan, was cloned from Porphyromonas gingivalis. This article discusses the mechanism and pathogenic role of the formation of VSCs by oral bacteria.     

Chromatographic separation of methionine,methionine sulphoxide,methionine sulphone,and their products of oral microbial metabolism

The metabolic pathways of methionine sulphoxide and methionine sulphone were investigated employing a combination of gas chromatography, thin-layer chromatography, paper chromatography, and radioactive methods of analyses. Gas chromatographic analysis demonstrated that methionine, methionine sulphoxide, and methionine sulphone all yielded qualitatively similar volatile sulphur compounds, namely, methyl mercaptan, dimethyl disulphide, and small amounts of dimethyl sulphide. The study indicated that the principal pathway of methionine sulphoxide and methionine sulphone metabolism is mediated via methionine which gives rise to methyl mercaptan, part of which is oxidized to dimethyl disulphide. Whereas methionine sulphoxide was readily reduced to methionine, the reduction of methionine sulphone proceeded at a considerably slower rate.     

3-Chloro-DL-alanine resistance by L-methionine-alpha-deamino-gamma-mercaptomethane-lyase activity

The antibacterial agent 3-chloro-DL-alanine (3CA) is an inhibitor of peptidoglycan synthesis. Fusobacterium nucleatum and Porphyromonas gingivalis, the bacteria responsible for oral malodor, are shown to be resistant to 1 mM 3CA, whereas Streptococcus mutans and Escherichia coli are sensitive to this antibacterial agent at the same concentration. We isolated the 3CA resistance gene from F. nucleatum and showed that the gene encodes an L-methionine-alpha-deamino-gamma-mercaptomethane-lyase that catalyzes the alpha,gamma-elimination of L-methionine to produce methyl mercaptan. The enzyme also exhibits 3CA chloride-lyase (deaminating) activity. This antibacterial agent is expected to be useful for specific selection of malodorous oral bacteria producing high amounts of methyl mercaptan.     

TRPM7 regulates polarized cell movements

TFM (L-trifluoromethionine), a potential prodrug, was reported to be toxic towards human pathogens that express MGL (L-methionine γ-lyase; EC 4.4.1.11), a pyridoxal phosphate-containing enzyme that converts L-methionine into α-oxobutyrate, ammonia and methyl mercaptan. It has been hypothesized that the extremely reactive thiocarbonyl difluoride is produced when the enzyme acts upon TFM, resulting in cellular toxicity. The potential application of the fluorinated thiomethyl group in other areas of biochemistry and medicinal chemistry requires additional studies. Therefore a detailed investigation of the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF?S? and CF?HS?) has been undertaken to trap and identify chemical intermediates produced by enzyme processing of molecules containing these fluorinated moieties. TvMGL (MGL from Trichomonas vaginalis) and a chemical model system of the reaction were utilized in order to investigate the cofactor-dependent activation of TFM and previously uninvestigated DFM (L-difluoromethionine). The differences in toxicity between TFM and DFM were evaluated against Escherichia coli expressing TvMGL1, as well as the intact human pathogen T. vaginalis. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogues and their cellular toxicity are discussed.     

High production of methyl mercaptan by L-methionine-alpha-deamino-gamma-mercaptomethane lyase from Treponema denticola

Methyl mercaptan is derived from l-methionine by the action of l-methionine-alpha-deamino-gamma-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola, a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l-methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K(m) value for l-methionine (0.55mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.     

Chemical reactivity of metallic copper in a model system containing biological metabolites

Chemical reactivity of metallic copper in a model system containing biological metabolites is described. Methionine, methional, and propanal produced ethylene when exposed to metallic copper in the presence of oxygen. It may be that metallic copper in this system serves as the '1 electron reducing agent' in the proposed chemical model system (Kumamoto et al). The requirement for oxygen was verified by removing this electron acceptor and observing the reduced ethylene production. Preliminary studies have shown that other reaction products of the reaction of copper metal with methionine include dimethyl sulfide and dimethyl disulfide or methyl mercaptan or both. These data further suggest that these chemicals are liberated from methionine when copper comes in contact with methionine-containing biological fluids.     

Inhibitory effect of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> on periodontopathic and cariogenic bacteria

The interaction between Lactobacillus reuteri, a probiotic bacterium, and oral pathogenic bacteria have not been studied adequately. This study examined the effects of L. reuteri on the proliferation of periodontopathic bacteria including Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Tannerella forsythia, and on the formation of Streptococcus mutans biofilms. Human-derived L. reuteri strains (KCTC 3594 and KCTC 3678) and rat-derived L. reuteri KCTC 3679 were used. All strains exhibited significant inhibitory effects on the growth of periodontopathic bacteria and the formation of S. mutans biofilms. These antibacterial activities of L. reuteri were attributed to the production of organic acids, hydrogen peroxide, and a bacteriocin-like compound. Reuterin, an antimicrobial factor, was produced only by L. reuteri KCTC 3594. In addition, L. reuteri inhibited the production of methyl mercaptan by F. nucleatum and P. gingivalis. Overall, these results suggest that L. reuteri may be useful as a probiotic agent for improving oral health.     

Screening test for deodorizing substances from marine algae and identification of phlorotannins as the effective ingredients in Eisenia bicyclis

Aqueous extracts from 33 species of marine algae were assessed for their methyl mercaptan-trapping activity by gas chromatography to search for novel natural oral deodorants. Brown algae belonging to the Laminariales such as Eisenia bicyclis, Ecklonia cava and Ecklonia kurome were found to show remarkable deodorizing action against methyl mercaptan. The effective components in Eisenia bicyclis were identified as a phlorotannin, a group of molecules which are characteristic components of Laminariales. In addition phlorotannins extracted from E. bicyclis were more effective at reducing methyl mercaptan than conventional natural deodorants such as chlorophyll and sodium copper chlorophyllin.Author for Correspondence     

Effect of methionine and its related compounds on rumen bacterial activity

The effect of several methionine sources (L‐methionine = L‐MET; DL‐ methionine = DL‐MET, DL‐S‐methyl‐methionine‐sulphonium‐chloride = SMM; N‐hydroxymethyl‐DL‐methionine‐Ca = NHM; methionine‐hydroxy‐analog free acid=MHA; methionine‐sulphoxyde=MSO) on rumen bacterial growth was studied using a new methodical approach which utilises a methionine free assay medium (Bacto Methionine Assay Media, Difco) supplemented by increasing quantities of the methionine sources and inoculated with one drop of diluted rumen bacteria. The optical density was measured after 18 h incubation on 39 °C.

L‐ and DL‐MET promoted the highest growth response, while SMM and NHM exerted significantly (p < 0.05) lower optical densities. MHA and MSO showed no growth response.

The methodical approach and the possible bacterial strains, which might have contributed to the growth response have been discussed.     

Role of Polysulfides in Reduction of Elemental Sulfur by the Hyperthermophilic Archaebacterium Pyrococcus furiosus

Polysulfides formed through the breakdown of elemental sulfur or other sulfur compounds were found to be reduced to H₂S by the hyperthermophilic archaebacterium Pyrococcus furiosus during growth. Metabolism of polysulfides by the organism was dissimilatory, as no incorporation of ³⁵S-labeled elemental sulfur was detected. However, [³⁵S]cysteine and [³⁵S]methionine were incorporated into cellular protein. Contact between the organism and elemental sulfur is not necessary for metabolism. The sulfide generated from metabolic reduction of polysulfides dissociates to a strong nucleophile, HS⁻, which in turn opens up the S₈ elemental sulfur ring. In addition to H₂S, P. furiosus cultures produced methyl mercaptan in a growth-associated fashion.     

Chemotaxis of Pseudomonas aeruginosa: involvement of methylation.

The involvement of a protein methyl transfer system in the chemotaxis of Pseudomonas aeruginosa was investigated. When a methionine auxotroph of P. aeruginosa was starved for methionine, chemotaxis toward serine, measured by a quantitative capillary assay, was reduced 80%, whereas background motility was unaffected or increased. When unstarved bacteria were labeled with L-[methyl-3H]methionine, a labeled species of 73,000 molecular weight which was methylated in response to stimulation by L-serine was identified. Under appropriate electrophoretic conditions, the 73,000 molecular weight species was resolved into two bands, both of which responded to stimulation by L-serine, L-arginine, and alpha-aminoisobutyrate (AIB) with an increased incorporation of methyl label. Arginine, which elicited the strongest chemotactic response in the capillary assay, also stimulated the greatest methylation response. Methylation of the 73,000 molecular weight species reached a maximum 10 min after stimulation by AIB and returned to the unstimulated level upon removal of the AIB. In vitro labeling of cell extracts with S-adenosyl[methyl-3H]methionine indicated that the 73,000 molecular weight species are methylated by an S-adenosylmethionine-mediated reaction. These results indicate that chemotaxis of P. aeruginosa toward amino acids is mediated by dynamic methylation and demethylation of methyl-accepting chemotaxis proteins analogous to those of the enteric bacteria.     

Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H(2)S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth.     

Isolation and purification of methyl mercaptan oxidase fromRhodococcus rhodochrous for mercaptan detection

Methyl mercaptan oxidase was successfully induced fromRhodococcus rhodochrous IGTS8 using methyl mercaptan gas and purified to homogeneity for the detection of mecrcaptans. The purification procedure involved DEAE-Sephacel and Superose 12 column chromatography with recovery yields of 85.8 and 83.3%, and a specific activity of 92.7 and 303.4 units/mg-protein, respectively. The molecular weight of purified methyl mercaptan, oxidase was determined to be 64.5 kDa by SDS-PAGE. The extract from gel filtration chromatography oxidizes methyl mercaptan to produce formaldehyde, which can be easily detected by the purpald-coloring method. Optimum temperature for activity was achieved at 60°C. This enzyme was inhibited by both K₂SO₄ and NaCl at concentration of less than 100 mM and recovered to original activity at concentration of 200 mM. In the presence of methanol, the activity decreased by 33%.     

Recent advances in elucidation of biological corrinoid functions

Abstract: Eleven adenosylcorrinoid-dependent rearrangements and elimination reactions have been described during the last four decades of vitamin B₁₂ research. In contrast, only the cobamide-dependent methionine synthase was well established as a corrinoid-dependent methyl transfer reaction. Yet, investigations during the last few years revealed nine additional corrinoid-dependent methyltransferases. Many of these reactions are catalyzed by bacteria which possess a distinct C₁ metabolism. Notably acetogenic and methanogenic bacteria carry out such methyl transfers in their anabolism and catabolism. Tetrahydrofolate or a similar pterine derivative is a key intermediate in these reactions. It functions as methyl acceptor and the methylated tetrahydrofolate serves as a methyl donor.     

Four families of folate-independent methionine synthases

Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.     

Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein.

When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the standard 20-amino acid repertoire. Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophysical probes. Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are observed when TFM is incorporated into LaL. To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance. Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances associated with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107. In addition, 19F and [1H-13C]-HMQC NMR experiments have been utilized with paramagnetic line broadening and K2PtCl4 reactivity experiments to obtain information about the probable spatial position of each Met in the protein. These results are compared with the recently determined crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure.     

Kinetic characterization of methionine gamma-lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis

Methionine gamma-lyase (MGL) (EC 4.4.1.11), which is present in certain lineages of bacteria, plants, and protozoa but missing in mammals, catalyzes the single-step degradation of sulfur-containing amino acids (SAAs) to alpha-keto acids, ammonia, and thiol compounds. In contrast to other organisms possessing MGL, anaerobic parasitic protists, namely Entamoeba histolytica and Trichomonas vaginalis, harbor a pair of MGL isozymes. The enteric protozoon En. histolytica shows various unique aspects in its metabolism, particularly degradation of SAAs. Trifluoromethionine (TFM), a halogenated analog of Met, has been exploited as a therapeutic agent against cancer as well as against infections by protozoan organisms and periodontal bacteria. However, its mechanism of action remains poorly understood. In addition, the physiological significance of the presence of two MGL isozymes in these protists remains unclear. In this study, we compared kinetic parameters of the wild-type and mutants, engineered by site-directed mutagenesis, of the two MGL isotypes from En. histolytica (EhMGL1 and EhMGL2) for various potential substrates and TFM. Intracellular concentrations of l-Met and l-Cys suggested that these SAAs are predominantly metabolized by EhMGL1, not by EhMGL2. It is unlikely that O-acetyl-l-serine is decomposed by EhMGLs, given the kinetic parameters of cysteine synthase reported previously. Comparison of the wild-type and mutants revealed that the contributions of several amino acids implicated in catalysis differ between the two isozymes, and that the degradation of TFM is less sensitive to alterations of these residues than is the degradation of physiological substrates. These results support the use of TFM to target MGL.     

Synthesis of Unmethylated Deoxyribonucleic Acid in a dnaB Temperature-Sensitive Mutant of Escherichia coli Starved for Methionine

Strain CRT 266, a polyauxotrophic dnaB temperature-sensitive mutant of Escherichia coli K, was investigated for residual deoxyribonucleic acid (DNA) synthesis when returned to the permissive temperature in the absence of protein synthesis. In the presence of methionine, a delayed extra-initiation occurs as well as an erratic long-lasting synthesis. In the absence of methionine, there is no evidence for extra-initiation, whereas the long-lasting synthesis is only slightly depressed. A direct role of methionine independent of protein synthesis in the extra-initiation process is postulated. The largest residual syntheses, with or without methionine, are obtained when (i) bacteria are first grown in a rich medium, (ii) bacteria are shifted to the nonpermissive temperature for 2 h in the same medium, and (iii) bacteria are then starved for aminoacid for 20 h at the permissive temperature. Under these conditions, DNA extracted from methionine-starved cells appears to be a mixture of half-methylated and unmethylated products. The possibility of the occurence of a few methyl groups on the so-called unmethylated DNA is discussed.     

Carbon-13 nuclear-magnetic-resonance spectroscopy of whole cells and of cytochrome C from Neurospora crass grown with (S-Me-13C)methionine.

Neurospora crassa cytochrome C biosynthetically labelled with [S-Me-13C]methionine was prepared and analysed by 13C nuclear-magnetic-resonance spectroscopy. The methyl group of methionine is extensively incorporated into an N-trimethyl-lysine-72 residue arise from S-adenosylmethionine transmethylation, and that the methyl carbons of methionine residues are sufficiently close to the haem centre to experience chemical shifts from the ring currents of the tetrapyrrole pi electrons and broadening due to binding of methionine-80 with the haem, as well as interaction of the S-E113C]methyl groups with the paramagnetic iron centre. Although whole cells of the labelled Neurospora produced a 13C resonance at the expected position for the methionyl methyl group most of the methyl label was diverted into N-tetra-alkyl ammonium compounds. After an active state of growth these labelled N-methyl compounds appear, in the main, to be low-molecular-weight derivatives of choline which, if associated with membrane, are in a sufficiently fluid environment to have short rotational correlation times. During a subsequent dormant growth period these compounds become associated to some extent with relatively more immobile phases as a result of membrane binding or an increase in membrane rigidity.     

Methyl-Deficient Transfer Ribonucleic Acid and Macromolecular Synthesis in Methionine-Starved Saccharomyces cerevisiae

Haploid methionine auxotrophs of Saccharomyces cerevisiae continue to multiply for several hours after withdrawal of a required amino acid from the medium. Macro-molecular synthesis continues during this period of residual growth, although the net ribonucleic acid (RNA) and protein content is constant during the later part of this period. In this study, growth after withdrawal of methionine was in some cases accompanied by accumulation of transfer RNA (tRNA), which was shown by methylation in vitro to be deficient in methyl groups. This phenomenon was shown by only four of nine methionine auxotrophs tested, but no evidence could be found that these four strains had "relaxed" control of RNA synthesis. The nine methionine-requiring strains represent mutations in five different positions in the methionine biosynthesis pathway, and only mutants blocked at two of these five positions accumulated methyl-deficient tRNA. This accumulation therefore appears to be correlated with the position of the strain's block in the pathway of methionine biosynthesis.