Full text loading...
Chapter 19 : Protection against Foreign DNA
Category: Microbial Genetics and Molecular Biology
Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase
This chapter briefly addresses the well-characterized restriction-modification system (R-M), non-sugar-specific nucleases (SNSN), and histone-like nucleoid structuring (H-NS). It more specifically elaborates on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR/CRISPR-associated (Cas), a recently described microbial system, provides acquired immunity against phages and plasmids by targeting nucleic acids in a sequence-specific manner. CRISPR features may be exploited for typing purposes, ecological and epidemiological studies, and also for enhancing phage resistance in bacteria. R-M systems commonly act as the first line of intracellular defense against foreign DNA. Some SNSN, such as Vvn from Vibrio vulnificus and EndoI from Escherichia coli, are periplasmic and thus prevent the uptake of foreign DNA. The ubiquitous and predatory nature of phages may explain the overwhelming representation of phage sequences in CRISPR spacers, but a recent report showed that CRISPR can dramatically impact the ability of plasmids to transfer genetic material in Staphylococcus epidermidis. Also, this study experimentally confirmed that CRISPR targets DNA directly in Staphylococcus. The CRISPR RNAs (crRNAs) seem to specifically guide the Cas defense apparatus toward foreign nucleic acid molecules that match the sequence of the spacers. This study also showed that Cas3, a predicted HD nuclease fused to a DEAD-box helicase, is required for the phage-resistance phenotype. The extent of the impact of CRISPR on phage genomes is perhaps best illustrated by extensive genome recombination events observed in environmental phage populations in response to CRISPR.
CRISPR/Cas systems in Streptococcus thermophilus DGCC7710. In this strain, at least CRISPR1 and CRISPR3 loci are able to acquire novel spacers following phage challenge (Barrangou et al., 2007 ; Deveau et al., 2008 ; Horvath et al., 2008 ). Although CRISPR1 and CRISPR3 belong to the same “Nmeni” subtype (Haft et al., 2005 ), CRISPR3 Cas enzymes are unable to complement CRISPR1 cas knock-outs. CRISPR2 belongs to the “Mtube” subtype, whereas CRISPR4 belongs to the “Ecoli” subtype. CRISPR1 to CRISPR4 systems may also be classified into families Sthe1, Sthe2, Sthe3, and Ldbu1, respectively (Horvath et al., 2009 ). For each system, the overall genetic organization of the CRISPR/Cas locus is shown on the top line, where cas genes are filled with diagonal hatching and CRISPRs are depicted as black rectangles. Downstream of CRISPR4, stars represent non-sens mutations in a pseudogene of unknown function. Below, the content of each CRISPR repeat-spacer array is detailed with diamonds (repeats) and rectangles (spacers). Spacers showing significant similarity to known S. thermophilus phage sequences are horizontally hatched (28 of 59 spacers); the spacer filled with dots is identical to a S. thermophilus plasmid sequence. The white letter T indicates that the terminal repeat is degenerated at the 3” end. The consensus sequence of the repeat is also indicated for each system. Underlined letters correspond to degenerate bases within the terminal repeat. L1, L2, L3, L4: CRISPR leaders. R = A or G; H = C, T, or A.
Diversity of CRISPR repeat sequences found in lactic acid bacteria genomes. CRISPR repeat sequences of lactic acid bacteria, including some Actinobacteria, cluster into families, mostly corresponding to subtypes (Haft et al., 2005 ; Horvath et al., 2009 ). Left, dendrogram deduced from the multiple alignment of Cas1 protein sequences. Right, nine CRISPR families can be identified that share similar characteristics such as repeat sequence and repeat and spacer length.
The acquisition of novel CRISPR spacers provides immunity against phages. Following phage challenge, certain S. thermophilus strains acquired novel repeat-spacer units at CRISPR1 (and/or CRISPR3, data not shown here). Novel spacer sequences are derived from the genome of the phage(s) used in the challenge and provide a high reduction of the efficiency of plaquing when there is 100% identity between spacer and proto-spacer. The native structure of S. thermophilus DGCC7710 CRISPR1 locus is shown at the top. In the middle, novel spacers (white rectangles) are acquired at the leader end of the locus (left), providing resistance against phage 858, or 2972, or both (right). On the bottom, the location of proto-spacers on the genome map of phages 858 and 2972 is shown; both phage DNA strands and all functional modules have been shown to be sources of spacers. Stars indicate differences between spacer and proto-spacer sequences: a single-base mutation allows the phage to escape from the CRISPR-based immunity.
Putative mechanism of the CRISPR/Cas immune system. (A) Immunization. Upon entry of invading (phage, plasmid) DNA into the bacterial cell, some cas gene products, by an unknown mechanism, catalyze the insertion of a short foreign sequence as a new spacer (downstream of a new repeat) at the leader end of the CRISPR locus. If the bacterium survives the invasion, this new genetic information, integrated within the chromosome, is transmitted to daughter cells. (B) Immunity. After transcription of the CRISPR locus as a full-length RNA, short CRISPR RNAs (crRNAs) are produced by endonucleolytic cleavage by Cas enzymes within the CRISPR repeat sequence. These short crRNAs, corresponding to spacer sequences flanked by partial repeat sequences, are sequestered and subsequently used by Cas proteins as guides that allow the recognition and cleavage of any invading DNA bearing an identical sequence (named proto-spacer) in the vicinity of the CRISPR motif. The absence of this proto-spacer-associated motif within the endogenous CRISPR array prevents autoimmunity.