CHARACTERIZATION OF A TYPE I-F CRISPR-CAS SYSTEM FROM THE CLINICAL ISOLATE Shewanella sp. Sh95

GISELA PARMECIANO DI NOTO; CECILIA QUIROGA; DANIELA CENTRON

San Miguel de Tucumán

Congreso; XII Congreso de Microbiología General; 2017

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (cas)are essential components of the adaptive immune system of bacteria, which provides protectionagainst bacteriophages and plasmids. CRISPRs are composed of short nucleotide palindromicrepeats interspaced by short segments of foreign DNA, called spacers. Shewanella sp. Sh95 is agram-negative rod isolated from an ocular infection. The Shewanella genus thrives in several aquaticenvironments and it is known for its potential in bioremediation and biocell fuels. More recently, theyhave been considered an emergent opportunistic pathogen. We previously sequenced the completegenome of strain Sh95, where a CRISPR array was found. The aim of this study was to characterizeand understand the evolution of CRISPR-cas system from Shewanella sp. Sh95. The CRISPR arraycomprised 152 repeats (28 bp-long) and 152 spacers (32 bp-long). Upstream of the array we identifiedthe cascade genes: cas1, cas2/3, csy1, csy2, csy3 and csy4. Analysis of these genes confirmed thatthe CRISPR-cas system of Shewanella sp. Sh95 belonged to the subtype I-F. Comparative analysiswith other Shewanella genomes revealed that this system is poorly conserved in the genus.Furthermore, the insertion site of this system occurred at the gene radC and downstream of the phagetranscriptional regulator alpA. Strain Sh95 system had two types of repeats with highly similarsequences. Analysis using the Mfold software showed that both types of repeats conserved thesecondary structure necessary for processing the CRISPRs RNA. On the other hand, spacer analysisshowed that some of these elements might interfere with the expression of essential genes of phagesfrom the families Lambda and Mu, providing infection immunity. Noteworthy, we found several spacersalong the CRISPR array interfering with the same genes, which revealed a chronology of theinfections caused by these phages. Experimental analysis confirmed that cas genes were expressedunder different nutrient and stress conditions by qRT-PCR assays which suggests that Sh95CRISPR-cas system is still active. Last, we evaluated plasmid loss upon transformation of pCR2.1,pACYC184 and pCR-SmaI2 into strain Sh95 to evaluate the ability of the CRISPR-cas system toeliminate exogenous material, which resulted in their complete loss after 5 days except for plasmidpCR-SmaI2 which was lost after 4 days. Taking together our results suggest that Shewanella sp. Sh95adapted to clinical setting by acquiring beneficial traits, such as a type I-F CRISPR-cas immunesystem. This feature will provide a clear advantage in order to survive in a nosocomial environment toavoid infection by different bacteriophages.