Abstract
A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity. Compared with the wild-type parent strain, the isolated mutants possessed 1/10 of the enzyme activity responsible for cleavage of glycine-arginine-4-methoxy-beta-naphthylamide (Gly-Arg-MNA). A concomitant loss in activity against arginine-arginine-4-methoxy-beta-naphthylamide (Arg-Arg-MNA) was also observed. Both activities were similarly affected by various proteinase inhibitors, suggesting that the same enzyme is responsible for the Arg-Arg-MNA peptidase and Gly-Arg-MNA peptidase activities. Growth rates of wild-type and mutant strains grown in batch culture with various nitrogen sources did not differ. However, a role for the Gly-Arg-MNA peptidase activity was demonstrated in coculture experiments with gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production were reduced by approximately 25% in cocultures containing the mutants when compared with that of wild-type-containing cultures. These reductions could not be accounted for simply by the decrease in ammonia production by the mutant strain alone. To our knowledge, this paper reports the first successful use of chemical mutagenesis with ruminal microorganisms.
Full Text
The Full Text of this article is available as a PDF (274.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Champion K. M., Helaszek C. T., White B. A. Analysis of antibiotic susceptibility and extrachromosomal DNA content of Ruminococcus albus and Ruminococcus flavefaciens. Can J Microbiol. 1988 Oct;34(10):1109–1115. doi: 10.1139/m88-196. [DOI] [PubMed] [Google Scholar]
- Chen G., Russell J. B. More monensin-sensitive, ammonia-producing bacteria from the rumen. Appl Environ Microbiol. 1989 May;55(5):1052–1057. doi: 10.1128/aem.55.5.1052-1057.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen Z., Potempa J., Polanowski A., Wikstrom M., Travis J. Purification and characterization of a 50-kDa cysteine proteinase (gingipain) from Porphyromonas gingivalis. J Biol Chem. 1992 Sep 15;267(26):18896–18901. [PubMed] [Google Scholar]
- Gardner R. G., Russell J. B., Wilson D. B., Wang G. R., Shoemaker N. B. Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4. Appl Environ Microbiol. 1996 Jan;62(1):196–202. doi: 10.1128/aem.62.1.196-202.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kadowaki T., Yoneda M., Okamoto K., Maeda K., Yamamoto K. Purification and characterization of a novel arginine-specific cysteine proteinase (argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphyromonas gingivalis. J Biol Chem. 1994 Aug 19;269(33):21371–21378. [PubMed] [Google Scholar]
- Konings W. N., Poolman B., Driessen A. J. Bioenergetics and solute transport in lactococci. Crit Rev Microbiol. 1989;16(6):419–476. doi: 10.3109/10408418909104474. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Lantz M. S., Allen R. D., Ciborowski P., Holt S. C. Purification and immunolocalization of a cysteine protease from Porphyromonas gingivalis. J Periodontal Res. 1993 Nov;28(6 Pt 2):467–469. doi: 10.1111/j.1600-0765.1993.tb02104.x. [DOI] [PubMed] [Google Scholar]
- McDonald J. K., Zeitman B. B., Reilly T. J., Ellis S. New observations on the substrate specificity of cathepsin C (dipeptidyl aminopeptidase I). Including the degradation of beta-corticotropin and other peptide hormones. J Biol Chem. 1969 May 25;244(10):2693–2709. [PubMed] [Google Scholar]
- Newbold C. J., Wallace R. J., Watt N. D. Properties of ionophore-resistant Bacteroides ruminicola enriched by cultivation in the presence of tetronasin. J Appl Bacteriol. 1992 Jan;72(1):65–70. doi: 10.1111/j.1365-2672.1992.tb04883.x. [DOI] [PubMed] [Google Scholar]
- Okamoto K., Misumi Y., Kadowaki T., Yoneda M., Yamamoto K., Ikehara Y. Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis. Arch Biochem Biophys. 1995 Feb 1;316(2):917–925. doi: 10.1006/abbi.1995.1123. [DOI] [PubMed] [Google Scholar]
- PITTMAN K. A., BRYANT M. P. PEPTIDES AND OTHER NITROGEN SOURCES FOR GROWTH OF BACTEROIDES RUMINICOLA. J Bacteriol. 1964 Aug;88:401–410. doi: 10.1128/jb.88.2.401-410.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Russell J. B., Strobel H. J., Chen G. J. Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production. Appl Environ Microbiol. 1988 Apr;54(4):872–877. doi: 10.1128/aem.54.4.872-877.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shoemaker N. B., Anderson K. L., Smithson S. L., Wang G. R., Salyers A. A. Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. Appl Environ Microbiol. 1991 Aug;57(8):2114–2120. doi: 10.1128/aem.57.8.2114-2120.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wallace R. J., McKain N. A survey of peptidase activity in rumen bacteria. J Gen Microbiol. 1991 Sep;137(9):2259–2264. doi: 10.1099/00221287-137-9-2259. [DOI] [PubMed] [Google Scholar]
- Wallace R. J., McKain N. Analysis of peptide metabolism by ruminal microorganisms. Appl Environ Microbiol. 1989 Sep;55(9):2372–2376. doi: 10.1128/aem.55.9.2372-2376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]