Ectopic Spacer Acquisition in Streptococcus thermophilus CRISPR3 Array

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.

SpacerPlacer: Ancestral reconstruction of CRISPR arrays reveals the evolutionary dynamics of spacer deletions

“…The partial order within the spacer arrays encodes the information that is generated from polarized insertions [7], one of the most consistent properties of CRISPR arrays. This has been empirically confirmed for many Cas types, except for type II-A systems, where ectopic insertions have also been found [47–49].…”

Bacteriophage-host interactions in Streptococcus thermophilus and their impact on co-evolutionary processes

“…In dairy streptococci, the development of bacteriophage-insensitive mutants (BIMs) has traditionally been achieved through exposure of the starter culture (or individual strains) to whey containing phages that are problematic against the culture or strain. The generation of spontaneous BIMs of S. thermophilus is largely facilitated by the innate CRISPR–Cas systems and has been the approach of choice by many starter culture providers for decades as it is inexpensive, requires limited expertize and facilities, it generally has a limited, if not completely negligible, impact on technological properties of strains, and is acceptable to regulatory authorities as a natural process (Mills et al 2010 ; Chirico et al 2014 ; Achigar et al 2021 ). While this process allows companies to respond rapidly to emerging or persistent phage problems in a factory-specific manner, such CRISPR-derived BIMs may be rapidly overcome by evolved phages with single point mutations in the acquired spacer regions (which are typically ∼30 bp in length) (Deveau et al 2008 ).…”

Dairy streptococcal cell wall and exopolysaccharide genome diversity

“…S. thermophilus incorporates defence mechanisms to limit phage proliferation, through chromosomally-or plasmid-encoded R-M (restriction and modification) systems [25] and also CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems [26]. Strains of this bacterial species are known to harbour up to four CRISPR-Cas systems (CR1 to CR4), with CR1 and CR3 being the most active in spacer acquisition [27,28]. These systems have the capacity to evolve, adapt and acquire spacers in response to phage (or plasmid) exposure, which provides increased resistance to specific phages [29,30].…”