Identification of a Type IV CRISPR-Cas system located exclusively on IncHI1B/IncFIB plasmids in Enterobacteriaceae

During an investigation of CRISPR carriage in clinical, multi-drug resistant , Klebsiella pneumoniae , a novel CRISPR-Cas system (which we have designated Type IV-B) was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002-2003) and a single K. pneumoniae isolate from the UK (isolated in 2017). Sequence analysis of other genomes available in GenBank revealed that this novel Type IV-B CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on the chromosome. Type IV-B is found exclusively on IncHI1B/IncFIB plasmids and is associated with multiple putative transposable elements. Type IV-B has a single repeat-spacer array (CRISPR1) upstream of the cas loci with some spacers matching regions of conjugal transfer genes of IncFIIK/IncFIB(K) plasmids suggesting a role in plasmid incompatibility. Expression of the cas loci was confirmed in available clinical isolates by RT-PCR; indicating the system is active. To our knowledge, this is the first report describing a new subtype within Type IV CRISPR-Cas systems exclusively associated with IncHI1B/IncFIB plasmids. Importance Here, we report the identification of a novel subtype of Type IV CRISPR-Cas that is expressed and exclusively carried by IncHI1B/IncFIB plasmids in Enterobacteriaceae , demonstrating unique evolutionarily juxtaposed connections between CRISPR-Cas and mobile genetic elements (MGEs). Type IV-B encodes a variety of spacers showing homology to DNA from various sources, including plasmid specific spacers and is therefore thought to provide specific immunity against plasmids of other incompatible groups (IncFIIK/IncFIB(K)) . The relationship between Type IV-B CRISPR-Cas and MGEs that surround and interrupt the system is likely to promote rearrangement and be responsible for the observed variability of this type. Finally, the Type IV-B CRISPR-Cas is likely to co-operate with other cas loci within the bacterial host genome during spacer acquisition.