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Peptide utilization by Prevotella spp. and its impact on ruminal nitrogen metabolism
Abstract
Two aspects of peptide metabolism in Prevotella spp., uptake and degradation, were investigated. Oligopeptides containing the amino acid analogs oxalysine and ethionine inhibited growth of several Prevotella species, and the inhibition could be reversed by the inclusion of nutritional peptides in the growth medium. Based on the mode of action of the toxic amino acid mimetics in other microorganisms, these results demonstrate the use of the smugglin concept in rumen bacteria: inhibitory compounds incorporated into larger molecules that are normally transported were used to inhibit growth of Prevotella spp. The characterization of the mode of action of protamine, a 32-amino acid polycationic peptide, on Prevotella spp. was conducted to determine its applicability as a smugglin. Protamine exerted its toxic effects by disrupting the outer membrane, which was demonstrated by: (i) an increased sensitivity to hydrophobic antibiotics (novobiocin and monensin), and; (ii) release of the periplasmic enzyme alkaline phosphatase following short-term exposure to protamine. As such, it is unlikely that protamine could be used as a smugglin. Studies of a dipeptidyl peptidase activity from Prevotella bryantii strain B$\sb1$4 using enzyme inhibitors revealed that the enzyme responsible for much of the Gly-Arg-MNAse (PrtA) activity in cell extracts is similar to gingipain R, member of a novel family of calcium-dependent cysteine proteases isolated from various strains of Porphyromonas gingivalis. Chemical mutagenesis was used to generate two mutant strains defective in PrtA. An ecological role for the PrtA activity was demonstrated in co-culture experiments with Gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production was reduced by approximately 25% in co-cultures containing the mutants when compared with wild-type-containing cultures. PrtA enrichment studies were undertaken, and multiple peaks of Gly-Arg-MNAse activity following ion-exchange chromatography of the cytoplasmic/periplasmic fraction of P. bryantii B$\sb1$4 were obtained. Peaks of activity possessed different sensitivities to oxygen, and this result led to a further characterization of the PrtA activities. Studies with Triton X-100 and the effect of aerobiosis indicated that at least two Gly-Arg-MNAse activities are present in P. bryantii B$\sb1$4: PrtA is the predominant, intracellular, O$\sb2$-sensitive enzyme, whereas PrtB is a membrane-associated activity that O$\sb2$-insensitive.
Subject Area
Livestock|Microbiology|Anatomy & physiology|Animals
Recommended Citation
Madeira, Humberto M. F, "Peptide utilization by Prevotella spp. and its impact on ruminal nitrogen metabolism" (1998). ETD collection for University of Nebraska-Lincoln. AAI9829526.
https://digitalcommons.unl.edu/dissertations/AAI9829526