Peptide metabolism in the lactococci and its regulation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
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Aspects of peptide metabolism in the lactococci have been investigated to increase the understanding of how these nutritionally fastidious bacteria, which have a central role in the dairy manufacturing industry, are able to grow in a complex medium such as milk. Peptide metabolism by lactococci in milk encompasses the processes by which large oligopeptides, produced from milk-caseins by the extracellular activity of the cell wall-associated proteinase, are converted into an intracellular pool of metabolisable amino acids. This involves the activities of both membrane-bound transport systems and peptidases. Early research into lactococcal peptide utilisation has proposed significant differences between Lactococcus lactis strains with respect to the mechanisms by which these bacteria utilise peptides in their environment. More recent studies of the lactococcal peptide carrier systems, based on intensive studies of only a single strain, have proposed a major role for a carrier system capable of transporting di- and tripeptides, and a subsidiary role for another system transporting oligopeptides containing four or more residues. Yet to date, peptidases with an extracellular location capable of degrading the large casein-peptides into smaller peptides have not been isolated. This current study has attempted to investigate more fully the in vivo activity of the oligopeptide transport system, and to assess whether it may have a more fundamental role in peptide utilisation than previous work has suggested. For this study a model series of homologous peptides of increasing size from the dipeptide Val-Gly to the octapeptide Val-Gly7 , all based on the essential amino acid valine, was used. The larger peptides in this series, Val-Gly3 , Val-Gly4 and Val-Gly7 , were synthesised for this work. The ability of Lactococcus lactis subsp. cremoris E8 8 to transport these peptides, and to grow in a chemically defined medium where they constitute the sole source of the essential amino acid valine, was studied . Preliminary peptide uptake studies were also performed using oligopeptides derived from a proteolytic cleavage of β-casein. The collective results of these studies suggest that the upper size limit, and the relative activity of this transport system, may be sufficient to permit this strain to utilise relatively large casein-derived oligopeptides without the need for hydrolysis into smaller peptides and free amino acids. A comparative study of peptide transport by a number of Lactococcus lactis strains was undertaken to investigate previously published observations indicating significant differences in the mechanisms of peptide uptake between lactococcal strains. While the results of this comparative study are consistent with the general model proposing two separate peptide carrier systems, they have revealed that significant differences can exist between strains in the relative activities and possible substrate specificities of these transport systems consistent with previous work that the lactococci have only two peptide carrier systems. These observations imply the need for caution in extrapolating the results obtained from the study of a single strain to lactococci as a whole. In contrast to the finding of significant strain differences with respect to the relative rates of peptide transport, a comparative study of the relative activity of six different intracellular peptidases showed relatively few differences in peptidase activity between strains. An investigation was also carried out to assess whether the peptidases and transport systems involved in the utilisation of peptides were nutritionally regulated. No clear evidence was obtained for the significant induction of either the intracellular peptidase complement or the di-/tripeptide transport system. An attempt was also made to isolate a mutant of Lactococcus lactis subsp. cremoris E8 unable to utilise dipeptides, to assess whether the di-/tripeptide transport system or the intracellular dipeptidase of this strain were essential to casein utilisation. This attempt was not successful.
Lactococcus, Lactic acid bacteria, Peptides Metabolism