Ectopic Spacer Acquisition in Streptococcus thermophilus CRISPR3 Array.

Rodrigo Achigar,Felipe Machado,Yuyu Shao,María Pía Campot,Martina Scarrone,Cécile Philippe,Valentina Duvós,María Julia Pianzzola,Geneviève M Rousseau,Moïra B Dion,Sylvain Moineau

doi:10.3390/microorganisms9030512

Rodrigo Achigar, Felipe Machado + Show 9 more

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https://doi.org/10.3390/microorganisms9030512

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Abstract

Streptococcus thermophilus relies heavily on two type II-A CRISPR-Cas systems, CRISPR1 and CRISPR3, to resist siphophage infections. One hallmark of these systems is the integration of a new spacer at the 5′ end of the CRISPR arrays following phage infection. However, we have previously shown that ectopic acquisition of spacers can occur within the CRISPR1 array. Here, we present evidence of the acquisition of new spacers within the array of CRISPR3 of S. thermophilus. The analysis of randomly selected bacteriophage-insensitive mutants of the strain Uy01 obtained after phage infection, as well as the comparison with other S. thermophilus strains with similar CRISPR3 content, showed that a specific spacer within the array could be responsible for misguiding the adaptation complex. These results also indicate that while the vast majority of new spacers are added at the 5′ end of the CRISPR array, ectopic spacer acquisition is a common feature of both CRISPR1 and CRISPR3 systems in S. thermophilus, and it can still provide phage resistance. Ectopic spacer acquisition also appears to have occurred naturally in some strains of Streptococcus pyogenes, suggesting that it is a general phenomenon, at least in type II-A systems.

Highlights

Streptococcus thermophilus is a lactic acid bacterium used extensively for the manufacture of several fermented dairy products, such as yogurt and several cheeses [1,2,3,4].Siphophage infections of these Gram-positive bacteria are the leading cause of milk fermentation failures worldwide [5,6,7,8]
A total of 39 (60.9%) CR3 loci were retrieved from publicly available S. thermophilus genomes and 45 (90%) CR3 loci were detected by PCR from the
Integration of new spacers at the 50 end of the clustered regularly interspaced short palindromic repeats (CRISPR) loci is one of the hallmark features of the CRISPR-Cas systems in S. thermophilus [42] and other bacterial species [3], ectopic spacer acquisition in CR1 was previously described in S. thermophilus [38]

Summary

Introduction

Streptococcus thermophilus is a lactic acid bacterium used extensively for the manufacture of several fermented dairy products, such as yogurt and several cheeses [1,2,3,4].Siphophage infections of these Gram-positive bacteria are the leading cause of milk fermentation failures worldwide [5,6,7,8]. It is well-documented that S. thermophilus strains rely on the CRISPR-Cas system (a prokaryotic adaptive immune system) to protect itself against phage attacks [9,10,11,12,13,14,15]. This system is composed of a clustered regularly interspaced short palindromic repeats (CRISPR) array and its associated cas genes [16,17,18].

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