Generation of bacteriophage-insensitive mutants of Streptococcus thermophilus using an antisense RNA CRISPR-Cas silencing approach
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Accepted version
Date
2017-11-27
Authors
McDonnell, Brian
Mahony, Jennifer
Hanemaaijer, Laurens
Kouwen, Thijs R.H.M.
van Sinderen, Douwe
Journal Title
Journal ISSN
Volume Title
Publisher
American Society for Microbiology
Published Version
Abstract
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.
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Keywords
Bacteriophage , Resistance , Insensitive , Mutant , mRNA , Adsorption , Receptor, , Sedimentation , Streptococcus thermophilus , Dairy industry , Phage infection , Dairy processing
Citation
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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Copyright
© 2017 American Society for Microbiology. All Rights Reserved. This is the accepted manuscript version of an article published in Applied and Environmental Microbiology. To access the published work see http://dx.doi.org/10.1128/AEM.01733-17