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The Adaptive Immune System of Haloferax volcanii.

Maier LK, Dyall-Smith M, Marchfelder A - Life (Basel) (2015)

Bottom Line: To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated).A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system.For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology II, Ulm University, 89069 Ulm, Germany. lisa-katharina.maier@uni-ulm.de.

ABSTRACT
To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated). Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I-III) and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA) maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

No MeSH data available.


Motifs for protospacer acquisition and for target detection. In the adaptation step (upper panel) fewer motifs (SAM motifs) might be detected, while in the interference step more motifs are recognised and trigger degradation. Interference experiments using plasmid invaders show that six different motifs (TIMs) can trigger degradation in Haloferax.
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life-05-00521-f006: Motifs for protospacer acquisition and for target detection. In the adaptation step (upper panel) fewer motifs (SAM motifs) might be detected, while in the interference step more motifs are recognised and trigger degradation. Interference experiments using plasmid invaders show that six different motifs (TIMs) can trigger degradation in Haloferax.

Mentions: While the plasmid invader approach was able to identify the PAMs used by Haloferax in the defence stage, it does not reveal the motifs driving the acquisition process, which can so far only be inferred from sequence alignment data (of cognate spacers and target invaders). If the sequences of the invader elements present in the Dead Sea in 1975 were known then this would be relatively easy to determine, but these data are not available, so the alignments presented in Table 1 must be interpreted with caution. Some matching sequences display PAMs that are consistent with laboratory findings (TAT, TTC, CAC; shown in bold type in Table 1) while others do not. One of the latter, TAC, appears to be overly represented. The diversity seen in the PAM region of the alignments likely reflects the diverse origins and nature of the matching sequences, mostly genomic/metagenomic data (rather than metaviromes). In contrast, an in silico comparison of Haloquadratum walsbyi (type I-B) spacers and metavirome sequences identified a number of likely sources of spacer acquisition events, and the associated PAMs were almost always the same: TTC [20,52]. PAM sequences are assumed to be connected to repeat sequence and CRISPR subtype [13], and given the high conservation of haloarchaeal repeats [36] and the presence of a subtype I-B system in both of these organisms, coincident PAM requirements seem reasonable. Hence, only a subset of PAM sequences linked to the defence reaction appear to be active in the acquisition step. Such a constraint and more stringent PAM usage has now been demonstrated in several other organisms representing different subtypes [13], which has led to a subdivision of PAM sequences into motifs important for acquisition of new spacers (SAM—spacer acquisition motif), and those essential for the interference reaction (TIM—target interference motif) (Figure 6) [13].


The Adaptive Immune System of Haloferax volcanii.

Maier LK, Dyall-Smith M, Marchfelder A - Life (Basel) (2015)

Motifs for protospacer acquisition and for target detection. In the adaptation step (upper panel) fewer motifs (SAM motifs) might be detected, while in the interference step more motifs are recognised and trigger degradation. Interference experiments using plasmid invaders show that six different motifs (TIMs) can trigger degradation in Haloferax.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390866&req=5

life-05-00521-f006: Motifs for protospacer acquisition and for target detection. In the adaptation step (upper panel) fewer motifs (SAM motifs) might be detected, while in the interference step more motifs are recognised and trigger degradation. Interference experiments using plasmid invaders show that six different motifs (TIMs) can trigger degradation in Haloferax.
Mentions: While the plasmid invader approach was able to identify the PAMs used by Haloferax in the defence stage, it does not reveal the motifs driving the acquisition process, which can so far only be inferred from sequence alignment data (of cognate spacers and target invaders). If the sequences of the invader elements present in the Dead Sea in 1975 were known then this would be relatively easy to determine, but these data are not available, so the alignments presented in Table 1 must be interpreted with caution. Some matching sequences display PAMs that are consistent with laboratory findings (TAT, TTC, CAC; shown in bold type in Table 1) while others do not. One of the latter, TAC, appears to be overly represented. The diversity seen in the PAM region of the alignments likely reflects the diverse origins and nature of the matching sequences, mostly genomic/metagenomic data (rather than metaviromes). In contrast, an in silico comparison of Haloquadratum walsbyi (type I-B) spacers and metavirome sequences identified a number of likely sources of spacer acquisition events, and the associated PAMs were almost always the same: TTC [20,52]. PAM sequences are assumed to be connected to repeat sequence and CRISPR subtype [13], and given the high conservation of haloarchaeal repeats [36] and the presence of a subtype I-B system in both of these organisms, coincident PAM requirements seem reasonable. Hence, only a subset of PAM sequences linked to the defence reaction appear to be active in the acquisition step. Such a constraint and more stringent PAM usage has now been demonstrated in several other organisms representing different subtypes [13], which has led to a subdivision of PAM sequences into motifs important for acquisition of new spacers (SAM—spacer acquisition motif), and those essential for the interference reaction (TIM—target interference motif) (Figure 6) [13].

Bottom Line: To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated).A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system.For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology II, Ulm University, 89069 Ulm, Germany. lisa-katharina.maier@uni-ulm.de.

ABSTRACT
To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated). Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I-III) and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA) maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

No MeSH data available.