Generation of bacteriophage-insensitive mutants of Streptococcus thermophilus using an antisense RNA CRISPR-Cas silencing approach

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McDonnell, Brian
Mahony, Jennifer
Hanemaaijer, Laurens
Kouwen, Thijs R.H.M.
van Sinderen, Douwe
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American Society for Microbiology
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Predation of starter lactic acid bacteria such as Streptococcus thermophilus by bacteriophages is a persistent and costly problem in the dairy industry. CRISPR-mediated Bacteriophage Insensitive Mutants (BIMs), while straightforward to generate and verify, can quickly be overcome by mutant phages. The aim of this study was to develop a tool allowing the generation of derivatives of commercial S. thermophilus strains which are resistant to phage attack through a non-CRISPR-mediated mechanism, with the objective of generating BIMs exhibiting stable resistance against a range of isolated lytic S. thermophilus phages. To achieve this, standard BIM generation was complemented by the use of the wild-type (WT) strain which had been transformed with an antisense mRNA-generating plasmid (targeting a crucial CRISPR-associated [cas] gene], in order to facilitate the generation of non-CRISPR-mediated BIMs. Phage sensitivity assays suggest that non-CRISPR-mediated BIMs exhibit some advantages compared to CRISPR-mediated BIMs derived from the same strain.Importance: The outlined approach reveals the presence of a powerful host-imposed barrier for phage infection in S. thermophilus. Considering the detrimental economic consequences of phage infection in the dairy processing environment, the developed methodology has widespread applications, particularly where other methods may not be practical or effective in obtaining robust, phage-tolerant S. thermophilus starter strains.
Bacteriophage , Resistance , Insensitive , Mutant , mRNA , Adsorption , Receptor, , Sedimentation , Streptococcus thermophilus , Dairy industry , Phage infection , Dairy processing
McDonnell, B., Mahony, J., Hanemaaijer, L., Kouwen, T. R. H. M. and van Sinderen, D. (2017) 'Generation of bacteriophage-insensitive mutants of Streptococcus thermophilus using an antisense RNA CRISPR-Cas silencing approach', Applied and Environmental Microbiology, 84(4), e01733-17 (14pp). doi: 10.1128/aem.01733-17
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