High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system

Abstract

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNAmediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.defense mechanism | phage-host interactions | positive selection T he clustered regularly interspaced short palindromic repeats (CRISPR) system is a significant defense mechanism of prokaryotes against viruses and horizontally transferred DNA (1-3) and RNA (4). It is found in ∼90% of archaeal genomes and ∼40% of bacterial genomes, and consists of a CRISPR array, usually preceded by a leader DNA sequence, located near a cluster of CRISPR-associated (cas) genes (5-7). RNA transcribed from the CRISPR array (crRNA) is processed by Cas proteins and directs interfering proteins to target DNA/RNA matching the sequences between the repeats. These sequences, called spacers, often originate from plasmids and phages; thus, the system adaptively targets these invaders. High variability is found among bacterial species in the number, sequence, and length of the CRISPR arrays; the sequence and length of the leader DNA; and the number and sequence of the associated proteins. The CRISPR arrays are composed of 2 to ∼250

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

“…For example, the thermosome subunit (PF1974), identified in Csa immunoprecipitations, is a heat shock protein (Hsp60 homolog) and could act as a chaperone in proper folding of some Cas proteins (Klumpp and Baumeister 1998;Shockley et al 2003). By analogy, previous work suggests that an Escherichia coli chaperonin protein (high temperature protein G) is essential for maintaining functional levels of Cas3 and thereby CRISPR-mediated silencing (Yosef et al 2011). Moreover, PF1563, a subunit of DNA-dependent RNA polymerase was also identified in Csa immunoprecipitations.…”

CRISPR decoys

“…Many of these studies are aimed at investigating the genetic requirements of CRISPR immunity and rely on the construction of mutants lacking vital elements of CRISPR-Cas loci, such as the repeat-spacer array. In spite of the wide distribution of CRISPR-Cas loci in bacteria and archaea, genetic studies have been limited to genetically tractable organisms such as Escherichia coli, 12,15 Streptococcus thermophilus, 11 Staphylococcus epidermidis, 16 cRiSPR loci consist of an array of short repeats separated by spacer sequences that match the genome of viruses and plasmids that infect prokaryotes. Transcription of the cRiSPR array generates small antisense RNAs that mediate immunity against these invaders.…”

The Bacterial CRISPR/Cas System as Analog of the Mammalian Adaptive Immune System

“…Our lab recently showed that another chaperone, HtpG, is essential for activity of the E. coli CRISPR/Cas system. 32 In its absence, the steady-state concentration of Cas3 in the cell is maintained at a nonfunctional level. This highlights HtpG's role as a helper molecule to the effector molecule, just like T-helper cells activate the cytotoxic T cells.…”

“…Nevertheless, the specificity and binding affinity of the two recognition modes are high in both cases. Certain regulatory proteins such as H-NS, LeuO, and the recently identified HtpG [28][29][30][31][32] are analogous to the T-helper cells, in the sense that they control the timing and strength of the response by regulating expression and steady-state levels of the effector proteins. For example, H-NS was shown in the E. coli CRISPR/Cas system to effectively shut down system activity by binding to the promoters regulating transcription of the system components.…”

“…28,30 We have recently shown that in E. coli, HtpG acts as a positive modulator of the CRISPR/ Cas system by increasing the steady-state level of an effector protein, Cas3. 32 Cas3 is analogous to the cytotoxic T cells in that it probably specifically destroys DNA marked by crRNAs produced by the system.…”