In silico prediction and in vitro detection of prophages and Crispr-cas in bacterial genomes of Antarctic environments

NAPOLITANO, NICOLÁS; QUIROGA, CECILIA; PELLIZA, LEONARDO; MAC CORMACK WALTER P.; ARAN, MARTÍN; LOPEZ, JOSÉ L

Buenos Aires

Congreso; Reunión Conjunta SAIB/Samige 2020; 2020

Sociedad Argentina de Inestigaciones Bioquímicas

Bacteriophages play akey role in microbial evolution, biogeochemical cycles and human diseases.Phages are genetically diverse, and the particular architecture of theirgenomes is the result of constant genetic exchange with other phages and thegenome of their hosts, which highlights their complex evolutionary history andgenerates a wide spectrum of genomic diversity. On the other hand, bacteriaencode in their genome defense mechanisms to avoid infection. Our hypothesis isthat the Antarctic environmental bacteria, scarcely explored in their contentof prophages and defense systems,could show a new genetic spectrum of viruses and defenses. Therefore, the aimof this work is to predict in silico, induce in vitro with Mitomycin C and characterize the presence of prophages and CRISPR-Cas systems in Antarcticbacteria. For this purpose, three isolates whose complete genome was obtainedin the laboratory were explored in silico in search of prophages (ACLAME Prophinder online version) and CRISPR-Cas components (CRISPRsfinder online version). B. argentinensis JUB59 contig 10 (Accession number: AFXZ01000009, 212842 bp) showed in silico a prophage (that could beinduced invitro) and also componentsof the CRISPR-Cas system(preliminarily identified as Type I). Rhodococcus Sp. strain ADHcontig_010 (Accession number: TMP_0005587, 227198bp) showed the prediction of aprophage (which could not beinduced yet) and putative proteins Cas3 (Cas-TypeI) and Csf2, Csf3, Csf4 (Cas-Type IV). Shewanella frigidimarina (NZ_LRDC00000000.1)did not show the prediction of prophages or CRISP-Cascomponents. On the other hand, a rich spectrum (512 sequences) of Cas3homologous sequences has been detected in sequences of 6 marine sediment metagenomes (IMG Genome ID:3300000129,3300000132, 3300000136,3300000135, 3300000119,3300000123) obtainedfrom Potter Cove, Carlini Base, Antarctica.The query sequences used for the search were those detected in the genomes of Bizionia argentinensis and Rhodococcus Sp.In other 7 bacterialisolates whose genomes are not available or when the in silico prediction does notgive positive results, the induction of prophages with Mitomycin C will be carried out, and in case of a positive resultthe search of Cas proteins by PCR willbe performed. For this PCR screening, generic primers will be designed from theexploration of the 6 marine metagenomes mentioned above. Although this work is in a preliminarystage, the sequences of the phages detected in silico and the one induced with Mitomycin C are not registered in the databases and their closesthomologues are members of the Caudovirales order. Similarly, the Casproteins detected in silicoare homologous with an average identity of 72.1% (±17.2) with the query (Cas3),which is a diverse group. Our working hypothesis has been partially confirmedand the results obtained provide new information on prophages and defense systemsthat have been little or no described until now at all.