Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

Abstract

CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites by leveraging small non-coding RNAs for nuclease-dependent degradation of their nucleic acid targets. In contrast to all other types of CRISPR-Cas systems, the mechanisms and biological roles of type IV systems have remained largely overlooked.Here, we describe a previously uncharted diversity of type IV gene cassettes, distributed across diverse prokaryotic genome backgrounds, and propose their classification into subtypes and variants. Congruent with recent findings, type IV modules were primarily found on plasmid-like elements. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a yet unrecognised role in mediating conflicts between plasmids that compete to monopolize their hosts. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing an answer to the enigmatic absence of adaptation modules in these systems. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.

CRISPR-Cas System in Antibiotic Resistance Plasmids in Klebsiella pneumoniae

“…The CRISPR-positive plasmids we describe are also very large (200-430 kb; Table 1). Like the previously reported type IV CRISPR-Cas system (Makarova et al, 2015;Enas Newire et al, 2019;Pinilla-Redondo et al, 2019) homologs of cas region genes csf2, csf3, DinG helicase (csf4), cas6 (csf5) were present, the organization of the cas genes was very similar and the adaptation genes cas1 and cas3 were absent, all typical of the previously reported type IV CRISPR-Cas. The large subunit csf1 gene thought to be the signature gene for type IV system was absent in the CRISPR-Cas system reported here, but two additional cas genes (csx3 and cas10 homologs) and two genes of unknown functions were identified in the cas gene locus, any of which may compensate for the absent csf1.…”

“…The Cas1 protein of type IV CRISPR-Cas is a Zn-finger containing protein with a weak similarity to Zn-finger sequences of Cas10 and it has been suggested that Csf1 could be a highly divergent, inactivated and N-terminally truncated Cas10-like polymerase derivative (Makarova et al, 2011a). The presence of DinG helicase (csf4), only previously reported in type IV-A CRISPR-Cas (Koonin et al, 2017;Makarova et al, 2018;Pinilla-Redondo et al, 2019), further supports the designation of these plasmid systems as type IV-A CRISPR-Cas. A preliminary study identified putative type IV CRISPR-Cas system in the IncH1B/IncFIB plasmids of Enterobacteriaceae (Enas Newire et al, 2019).…”

“…For example, if a cell carries two incompatible plasmids and one plasmid encodes a TA system, then after segregation of these incompatible plasmids, only plasmid carrying TA system will be maintained into daughter cells and cells carrying the other plasmid are eliminated from the population. Similar to those systems, it was suggested that plasmid mediated type IV CRISPR-Cas system may involve in the competition between plasmids by acquiring spacers specifically targeting different plasmids (Pinilla-Redondo et al, 2019). Chromosomal CRISPR are known to acquire spacers against different MGEs (Samson et al, 2015) and many plasmid-borne CRISPR spacers we found were also directed against other plasmids, including three unique spacers targeting 100% identical sequences (the common and highly conserved traN, traH, and traL of conjugative plasmids) in more than 700 other plasmids in GenBank.…”

“…Type IV system can be classified as two sub-types, type IV-A and IV-B, based on the presence of DinG family helicase and type IV specific effector protein Csf5. Type IV-A encodes a DinG helicase (Csf4) and an effector protein Csf5 and whereas type IV-B lacks these proteins (Makarova et al, 2015(Makarova et al, , 2018Koonin et al, 2017;Hille et al, 2018;Pinilla-Redondo et al, 2019) and usually, the type IV-A system carries CRISPR-array.…”

The ecology of plasmid-coded antibiotic resistance: a basic framework for experimental research and modeling

“…Bacteria can be immune to an incoming plasmid due to clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems, which have been demonstrated to prevent conjugation [48] . In a recent study, Pinilla-Redondo et al [49] showed that these system are carried by plasmid-like elements and involved in competition between plasmids. These authors concluded that they are used to eliminate other plasmids with similar properties and lifestyles in order to monopolize the host environment.…”