Variability and molecular typing of Streptococcus thermophilus strains displaying different proteolytic and acidifying properties Wessam Galia, Clarisse Perrin, Magali Genay, Annie Dary * Unite´ de Recherche sur l’Animal et les Fonctionnalite ´s des Produits Animaux, U.C. Institut National de la Recherche Agronomique 340, Nancy-Universite´,Vandoeuvre-le `s-Nancy, France article info Article history: Received 6 May 2008 Received in revised form 11 August 2008 Accepted 12 August 2008 abstract Proteolytic and acidifying properties of Streptococcus thermophilus strains isolated from yoghurt or cheeses were evaluated. Among 30 strains tested, 12 exhibited cell envelope-associated proteinase activity (PrtS þ ), three displayed a slight PrtS activity (PrtS þ/ ) and 15 were PrtS  , despite the presence of the corresponding gene (prtS) in eight of them. Sequencing of the prtS gene in four PrtS  and one PrtS þ strains revealed that the absence of PrtS activity in the PrtS  strain probably results from an alteration of the prtS regulation. The strains displaying the highest acidifying capacities were all PrtS þ . All but one PrtS þ strains were phylogenetically close, as shown by the sequencing of their rDNA internal transcribed spacer (ITS) 16S-23S. More specifically, the high proteolytic and acidifying capacities are associated with the presence of a type II-ITS. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Lactic acid bacteria (LAB) require an exogenous source of amino acids or peptides for optimal growth since they are unable to synthesize several amino acids. Since the concentrations of amino acids or peptides in milk are not sufficient to ensure growth at high cell density, LAB need to utilise caseins as an amino acid source. Caseins are hydrolyzed to peptides and amino acids by cell envelope-associated proteinases (CEP) and intracellular peptidases. The CEP of lactococci have been extensively studied. They are inhibited by serine protease inhibitors and the mechanism of secretion, maturation and attachment to the cell wall has been well characterized (Savijoki et al., 2006). Several other LAB, such as Lactobacillus delbrueckii subsp. bulgaricus or Lactobacillus helveticus, have been reported to possess a CEP (Gilbert et al., 1996; Pederson et al., 1999). The thermophilic LAB Streptococcus thermophilus is widely used as a starter to produce fermented milk products because of its capacity to produce lactic acid by catabolising lactose, so contrib- uting to milk acidification and to its organoleptic properties. Some S. thermophilus strains are unable to decrease the pH to a value lower than 5.2 and therefore are commonly mixed with other starter bacteria. Among a collection of 97 S. thermophilus strains, the three strains displaying the highest acidifying capacities also showed the highest proteolytic activities (Shahbal et al., 1991). The presence of high proteinase activities allowed these strains to grow and produce acid more rapidly in milk than the other 94 strains could (Shahbal et al., 1993). The CEP (called PrtS) of one of these three strains (strain CNRZ385) has been characterized. It belongs to the subtilisin-like serine protease family and is covalently associated to the cell wall via its LPXTG amino acid motif (Fernandez-Espla et al., 2000; Shahbal et al., 1993). These results raise several questions that are addressed in the present work. First, does the low level or absence of CEP activity in 94 out of 97 strains (Shahbal et al., 1991) result from an absence or mutation of the corresponding gene? Indeed, among the three genomes that have been sequenced in the S. thermophilus species, only one (that of strain LMD-9) possesses a prtS gene. The second question concerns the variability of the gene encoding the CEP in S. thermophilus, and a third question concerns the phyloge- netic relationships existing between strains that display different acidifying and proteolytic potential. 2. Materials and methods 2.1. Bacterial strains and growth conditions S. thermophilus strains used in this work are listed in Table 1 . They were isolated in our laboratory either from yoghurt or from cheese (P. Bracquart, personal communication), or came from the CNRZ (Centre National de Recherches Zootechniques, INRA, Jouy- en-Josas, France) collection or ATCC (American type culture collection, Manassas, VA, USA). Strains were stored at 80  C in reconstituted skim milk (10%, w/v). Cultures were incubated over- night in skim milk at 42  C before each experiment. For DNA extraction or proteolytic and urease activity studies, S. thermophilus * Corresponding author. Tel.: þ33 83684267. E-mail address: Annie.Dary@scbiol.uhp-nancy.fr (A. Dary). Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2008.08.004 International Dairy Journal 19 (2009) 89–95