Characterization of CRISPR RNA biogenesis and Cas6 cleavage-mediated inhibition of a provirus in the haloarchaeon Haloferax mediterranei

J Bacteriol. 2013 Feb;195(4):867-75. doi: 10.1128/JB.01688-12. Epub 2012 Dec 14.

Abstract

The adaptive immune system comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and cas (CRISPR-associated) genes has been discovered in a wide range of bacteria and archaea and has recently attracted comprehensive investigations. However, the subtype I-B CRISPR-Cas system in haloarchaea has been less characterized. Here, we investigated Cas6-mediated RNA processing in Haloferax mediterranei. The Cas6 cleavage site, as well as the CRISPR transcription start site, was experimentally determined, and processing of CRISPR transcripts was detected with a progressively increasing pattern from early log to stationary phase. With genetic approaches, we discovered that the lack of Cas1, Cas3, or Cas4 unexpectedly resulted in a decrease of CRISPR transcripts, while Cas5, Cas6, and Cas7 were found to be essential in stabilizing mature CRISPR RNA (crRNA). Intriguingly, we observed a CRISPR- and Cas3-independent inhibition of a defective provirus, in which the putative Cascade (CRISPR-associated complex for antiviral defense) proteins (Cas5, Cas6, Cas7, and Cas8b) were indispensably required. A sequence carried by a proviral transcript was found to be homologous to the CRISPR repeat RNA and vulnerable to Cas6-mediated cleavage, implying a distinct interference mechanism that may account for this unusual inhibition. These results provide fundamental information for the subtype I-B CRISPR-Cas system in halophilic archaea and suggest diversified mechanisms and multiple physiological functions for the CRISPR-Cas system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Base Sequence
  • Catalytic Domain
  • DNA, Archaeal / genetics
  • Gene Expression Regulation, Archaeal / physiology*
  • Genome, Archaeal
  • Haloferax mediterranei / metabolism
  • Haloferax mediterranei / virology*
  • Oligonucleotide Array Sequence Analysis
  • Proviruses / physiology*
  • RNA, Archaeal / genetics
  • RNA, Archaeal / metabolism*
  • Transcription Initiation Site

Substances

  • Archaeal Proteins
  • DNA, Archaeal
  • RNA, Archaeal