Abstract
BackgroundIn industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products.ResultsIn the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transfered by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus.ConclusionWe have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.
Highlights
Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of dairy products due to their efficient utilisation of milk constituents, principally lactose and caseins
Casein breakdown is initiated by the cell-envelope proteinases (CEPs) and the resulting oligopeptides are transported into the cell where they are further hydrolysed by a set of various intracellular peptidases [2,3]
Fast acidifying strains contain the prtS genomic island and a full-length srtA gene The strategy chosen to optimise the acidification rate of S. thermophilus strains consists of transferring the minimal genetic requirements associated with the fast acidifying phenotype
Summary
Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of dairy products due to their efficient utilisation of milk constituents, principally lactose and caseins Their capacity to produce lactic acid as the main metabolic end-product of lactose fermentation is of major economic importance, since acidification inhibits the growth of spoilage organisms. Similar to many surface proteins such as adhesins, CEPs generally contain a C-terminal LPXTG motif and are anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) in Gram-positive bacteria [3,7]. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and optimise the fermentation process of dairy products
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