1887

Abstract

produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of HS production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from ATCC 25586 contained three major HS-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce HS from -cysteine (∼30 %) than Fn1220. The Fn0625 protein degraded a variety of substrates containing C–S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from -cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of -cysteine with Fn1220 produced HS and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except -cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the gene showed several-fold higher expression than and housekeeping genes in exponential-phase cultures of . Thus, we conclude that Fn0625 and Fn1220 produce HS in distinct manners: Fn0625 carries out -elimination of -cysteine to produce HS, pyruvate and ammonia, whereas Fn1220 catalyses the -replacement of -cysteine to produce HS and lanthionine, the latter of which may be used for peptidoglycan formation in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.039180-0
2010-07-01
2020-07-10
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/7/2260.html?itemId=/content/journal/micro/10.1099/mic.0.039180-0&mimeType=html&fmt=ahah

References

  1. Alting A. C., Engels W. J. M., van Schalkwijk S., Exterkate F. A.. 1995; Purification and characterizaiton of cystathionine β-lyase from Lactococcus lactis subsp. cremoris B78 and its possible role in favor development in cheese. Appl Environ Microbiol61:4037–4042
    [Google Scholar]
  2. Awano N., Wada M., Mori H., Nakamori S., Takagi H.. 2005; Identification and functional analysis of Escherichia coli cysteine desulfhydrases. Appl Environ Microbiol71:4149–4152
    [Google Scholar]
  3. Becker M. A., Kredich N. M., Tomkins G. M.. 1969; The purification and characterization of O-acetylserine sulfhydrylase-A from Salmonella typhimurium. J Biol Chem244:2418–2427
    [Google Scholar]
  4. Boronat A., Britton P., Jones-Mortimer M. C., Kornberg H. L., Lee L. G., Murfitt D., Parra F.. 1984; Location on the Escherichia coli genome of a gene specifying O-acetylserine (thiol)-lyase. J Gen Microbiol130:673–685
    [Google Scholar]
  5. Chen X., Jhee K. H., Kruger W. D.. 2004; Production of the neuromodulator H2S by cystathionine β-synthase via the condensation of cysteine and homocysteine. J Biol Chem279:52082–52086
    [Google Scholar]
  6. Chiku T., Padovani D., Zhu W., Singh S., Vitvitsky V., Banerjee R.. 2009; H2S biogenesis by human cystathionine γ-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia. J Biol Chem284:11601–11612
    [Google Scholar]
  7. Chu L., Burgum A., Kolodrubetz D., Holt S. C.. 1995; The 46-kilodalton-hemolysin gene from Treponema denticola encodes a novel hemolysin homologous to aminotransferases. Infect Immun63:4448–4455
    [Google Scholar]
  8. Chu L., Ebersole J. L., Kurzban G. P., Holt S. C.. 1997; Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities. Infect Immun65:3231–3238
    [Google Scholar]
  9. Claesson R., Edlund M. B., Persson S., Carlsson J.. 1990; Production of volatile sulfur compounds by various Fusobacterium species. Oral Microbiol Immunol5:137–142
    [Google Scholar]
  10. Fukamachi H., Nakano Y., Yoshimura M., Koga T.. 2002; Cloning and characterization of the l-cysteine desulfhydrase gene of Fusobacterium nucleatum. FEMS Microbiol Lett215:75–80
    [Google Scholar]
  11. Ito S., Nagamune H., Tamura H., Yoshida Y.. 2008; Identification and molecular analysis of βC–S lyase producing hydrogen sulfide in Streptococcus intermedius. J Med Microbiol57:1411–1419
    [Google Scholar]
  12. Kapatral V., Anderson I., Ivanova N., Reznik G., Los T., Lykidis A., Bhattacharyya A., Bartman A., Gardner W.. other authors 2002; Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. J Bacteriol184:2005–2018
    [Google Scholar]
  13. Kato K., Umemoto T., Sagawa H., Kotani S.. 1979; Lanthionine as an essential constituent of cell wall peptidoglycan of Fusobacterium nucleatum. Curr Microbiol3:147–151
    [Google Scholar]
  14. Kolenbrander P. E., Andersen R. N., Blehert D. S., Egland P. G., Foster J. S., Palmer R. J. Jr. 2002; Communication among oral bacteria. Microbiol Mol Biol Rev66:486–505
    [Google Scholar]
  15. Kredich N. M., Tomkins G. M.. 1966; The enzymic synthesis of l-cysteine in Escherichia coli and Salmonella typhimurium. J Biol Chem241:4955–4965
    [Google Scholar]
  16. Kurzban G. P., Chu L., Ebersole J. L., Holt S. C.. 1999; Sulfhemoglobin formation in human erythrocytes by cystalysin, an l-cysteine desulfhydrase from Treponema denticola. Oral Microbiol Immunol14:153–164
    [Google Scholar]
  17. Li L., Bhatia M., Zhu Y. Z., Zhu Y. C., Ramnath R. D., Wang Z. J., Anuar F. B., Whiteman M., Salto-Tellez M., Moore P. K.. 2005; Hydrogen sulfide is a novel mediator of lipopolysaccharide-induced inflammation in the mouse. FASEB J19:1196–1198
    [Google Scholar]
  18. Martinez-Cuesta M. C., Pelaez C., Eagles J., Gasson M. J., Requena T., Hanniffy S. B.. 2006; YtjE from Lactococcus lactis IL1403 Is a C–S lyase with α, γ-elimination activity toward methionine. Appl Environ Microbiol72:4878–4884
    [Google Scholar]
  19. Moore W. E., Moore L. V.. 1994; The bacteria of periodontal diseases. Periodontol 2000;5:66–77
    [Google Scholar]
  20. Morhart R. E., Mata L. J., Sinskey A. J., Harris R. S.. 1970; A microbiological and biochemical study of gingival crevice debris obtained from Guatemalan Mayan Indians. J Periodontol41:644–649
    [Google Scholar]
  21. Ng W., Tonzetich J.. 1984; Effect of hydrogen sulfide and methyl mercaptan on the permeability of oral mucosa. J Dent Res63:994–997
    [Google Scholar]
  22. Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T.. 1995; How to measure and predict the molar absorption coefficient of a protein. Protein Sci4:2411–2423
    [Google Scholar]
  23. Persson S.. 1992; Hydrogen sulfide and methyl mercaptan in periodontal pockets. Oral Microbiol Immunol7:378–379
    [Google Scholar]
  24. Persson S., Edlund M. B., Claesson R., Carlsson J.. 1990; The formation of hydrogen sulfide and methyl mercaptan by oral bacteria. Oral Microbiol Immunol5:195–201
    [Google Scholar]
  25. Pianotti R., Lachette S., Dills S.. 1986; Desulfuration of cysteine and methionine by Fusobacterium nucleatum. J Dent Res65:913–917
    [Google Scholar]
  26. Qi M., Nelson K. E., Daugherty S. C., Nelson W. C., Hance I. R., Morrison M., Forsberg C. W.. 2005; Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization. J Bacteriol187:3739–3751
    [Google Scholar]
  27. Ratcliff P. A., Johnson P. W.. 1999; The relationship between oral malodor, gingivitis, and periodontitis. A review. J Periodontol70:485–489
    [Google Scholar]
  28. Schmidt A.. 1987; d-Cysteine desulfhydrase from spinach. Methods Enzymol143:449–451
    [Google Scholar]
  29. Singh S., Padovani D., Leslie R. A., Chiku T., Banerjee R.. 2009; Relative contributions of cystathionine β-synthase and γ-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions. J Biol Chem284:22457–22466
    [Google Scholar]
  30. Soda K.. 1968; Microdetermination of d-amino acids and d-amino acid oxidase activity with 3-methyl-2-benzothiazolone hydrazone hydrochloride. Anal Biochem25:228–235
    [Google Scholar]
  31. Sperandio B., Polard P., Ehrlich D. S., Renault P., Guedon E.. 2005; Sulfur amino acid metabolism and its control in Lactococcus lactis IL1403. J Bacteriol187:3762–3778
    [Google Scholar]
  32. Tapuhi Y., Schmidt D. E., Lindner W., Karger B. L.. 1981; Dansylation of amino acids for high-performance liquid chromatography analysis. Anal Biochem115:123–129
    [Google Scholar]
  33. Tonzetich J.. 1971; Direct gas chromatographic analysis of sulphur compounds in mouth air in man. Arch Oral Biol16:587–597
    [Google Scholar]
  34. Vasstrand E. N., Jensen H. B., Miron T., Hofstad T.. 1982; Composition of peptidoglycans in Bacteroidaceae: determination and distribution of lanthionine. Infect Immun36:114–122
    [Google Scholar]
  35. Washio J., Sato T., Koseki T., Takahashi N.. 2005; Hydrogens sulfide-producing bacteria in tongue biofilm and their relationship with oral malodour. J Med Microbiol54:889–895
    [Google Scholar]
  36. Yaegaki K., Qian W., Murata T., Imai T., Sato T., Tanaka T., Kamoda T.. 2008; Oral malodorous compound causes apoptosis and genomic DNA damage in human gingival fibroblasts. J Periodontal Res43:391–399
    [Google Scholar]
  37. Yang G., Sun X., Wang R.. 2004; Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3. FASEB J18:1782–1784
    [Google Scholar]
  38. Yoshida Y., Nakano Y., Amano A., Yoshimura M., Fukamachi H., Oho T., Koga T.. 2002; lcd from Streptoccus anginosus encodes a C–S lyase with α, β-elimination activity that degarades L-cysteine. Microbiology148:3961–3970
    [Google Scholar]
  39. Yoshida Y., Negishi M., Amano A., Oho T., Nakano Y.. 2003; Differences in the βC–S lyase activities of viridans group streptococci. Biochem Biophys Res Commun300:55–60
    [Google Scholar]
  40. Yoshida Y., Ito S., Sasaki T., Kishi M., Kurota M., Suwabe A., Kunimatsu K., Kato H.. 2008; Molecular and enzymatic characterization of βC–S lyase in Streptococcus constellatus. Oral Microbiol Immunol23:245–253
    [Google Scholar]
  41. Yoshida Y., Ito S., Tamura H., Kunimatsu K.. 2010; Use of a novel assay to evaluate enzymes that produce hydrogen sulfide in Fusobacterium nucleatum. J Microbiol Methods80:313–315
    [Google Scholar]
  42. Yoshimura M., Nakano Y., Yamashita Y., Oho T., Saito T., Koga T.. 2000; Formation of methyl mercaptan from l-methionine by Porphyromonas gingivalis. Infect Immun68:6912–6916
    [Google Scholar]
  43. Zdych E., Peist R., Reidl J., Boos W.. 1995; MalY of Escherichia coli is an enzyme with the activity of a βC–S lyase (cystathionase. J Bacteriol177:5035–5039
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.039180-0
Loading
/content/journal/micro/10.1099/mic.0.039180-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error