ORIGINAL ARTICLE CRISPR analysis of bacteriophage-insensitive mutants (BIMs) of industrial Streptococcus thermophilus – implications for starter design S. Mills 1,2 , C. Griffin 1,2 , A. Coffey 3 , W.C. Meijer 2 , B. Hafkamp 2 and R.P. Ross 1,4 1 Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland 2 CSK Food Enrichment, Ede, the Netherlands 3 Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland 4 Alimentary Pharmabiotic Centre, Cork, Ireland Introduction Streptococcus thermophilus is a universal thermophilic dairy starter used for the manufacture of yoghurt and cheese and is considered the second most important industrial starter strain after Lactococcus lactis (Rasic and Kurmann 1978; Fox 1993; Hols et al. 2005). The increased exploitation of Strep. thermophilus in products such as Mozzerella and yoghurt has simultaneously led to an increased incidence of bacteriophage-related problems. Keywords bacteriophage-insensitive mutants, CRISPR, phage resistance, starter culture, Streptococcus thermophilus. Correspondence Reynolds Paul Ross, Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland. E-mail: paul.ross@teagasc.ie 2009 ⁄ 0766: received 30 April 2009, revised 7 July 2009 and accepted 14 July 2009 doi:10.1111/j.1365-2672.2009.04486.x Abstract Aims: An efficient approach for generation of bacteriophage-insensitive mutants (BIMs) of Streptococcus thermophilus starters was described in our laboratory [Mills et al. (2007) J Microbiol Methods 70, 159–164]. The aim of this study was to analyse the phage resistance mechanism responsible for BIM formation. Methods and Results: Three clustered regularly interspaced short palindromic repeat (CRISPR) regions have been identified in Strep. thermophilus, and Strep. thermophilus can integrate novel spacers into these loci in response to phage attack. Characterization of three sets of BIMs indicated that two sets had altered CRISPR1 and ⁄ or CRISPR3 loci. A range of BIMs of yoghurt starter CSK938 were generated with the same phage in different phage challenge experiments, and each acquired unique spacer regions ranging between one and four new spacers in CRISPR1. In addition, the BIM that acquired only one new spacer in CRISPR1 also acquired an additional spacer in CRISPR3. A fourth BIM, generated with a different phage, had two spacers deleted from CRISPR1 but acquired two spacers in CRISPR3. Analysis of the Mozzarella starter CSK939 and its associated BIMs indicated that formation of second generation BIMs does not lead to increases in spacer number but to alterations in spacer regions. BIMs of an exopolysaccharide (EPS)-producing strain that lost the ability to produce EPS did not harbour an altered CRISPR, suggesting that phage sensitivity may be related to the EPS-producing phenotype. Conclusions: Acquisition ⁄ deletion of new spacers in CRISPR loci in response to phage attack generates distinctly individual variants. It also demonstrates that other modifications may be responsible for the phage resistance of Strep. thermophilus BIMs. Significance and Impact of the Study: Isolation of individual BIMs that have unique spacers towards the leader region of the CRISPR locus may be a very useful approach for rotation strategies with the same starter backbone. Upon phage infection, BIMs ‘in reserve’ can be slotted into the rotation scheme. Journal of Applied Microbiology ISSN 1364-5072 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 945–955 No claim to government works 945