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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.


Potential Cascade complex composition of the Haloferax type I-B system modeled according to the published structure of the E. coli I-E system. Experimental data regarding the actual structure has not yet been reported. (A) According to the iBAQ analysis the Cascade complex in Haloferax contains 8.5 Cas7 proteins, 1 Cas6b and 1.7 Cas5 proteins. In addition we observed a loose association of Cas8b. (B) The minimal stable Cascade complex could consist of just Cas5 and Cas7 and the short crRNA. Cas8b is essential for the interference reaction but only loosely associated with the complex.
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life-05-00521-f003: Potential Cascade complex composition of the Haloferax type I-B system modeled according to the published structure of the E. coli I-E system. Experimental data regarding the actual structure has not yet been reported. (A) According to the iBAQ analysis the Cascade complex in Haloferax contains 8.5 Cas7 proteins, 1 Cas6b and 1.7 Cas5 proteins. In addition we observed a loose association of Cas8b. (B) The minimal stable Cascade complex could consist of just Cas5 and Cas7 and the short crRNA. Cas8b is essential for the interference reaction but only loosely associated with the complex.

Mentions: In Haloferax, deletion of the cas6b gene results in loss of crRNAs, confirming the essential role of the Cas6b protein in crRNA metabolism for the I-B system [34,35]. Further analysis showed that for a normal steady state concentration of crRNAs not only Cas6b but also Cas5 and Cas7 are required, suggesting that they protect the crRNA from degradation [34]. Haloferax cells with only Cas5, Cas6b and Cas8b (e.g., without Cas7) still contain crRNAs, but they are present at a significantly reduced level. While Cas8b can, to some extent, also account for crRNA level stabilization, crRNAs are most efficiently protected when Cas7 is present. A co-purification approach using FLAG-tagged Cas7 protein revealed that the Cascade-like complex of Haloferax contains Cas5, Cas6b and Cas7 subunits [34]. The Cas8b protein seems to be very loosely attached since it cannot be reproducibly co-purified, suggesting that the Haloferax I-B Cascade complex has a core of Cas5 and Cas7 and that Cas6b and Cas8b are more loosely associated (Figure 3). Using mass spectrometry and intensity-based absolute quantification (iBAQ) the components of the core complex were shown to occur in the following ratio 1.7:1:8.5 (Cas5:Cas6b:Cas7) [34]. This composition differs from the observed composition of the type I-E Cascade complex, which consists of 1 Cas5, 1 Cas6, 6 Cas7, 1 Cas8 and additionally contains two copies of the small subunit Cse2 [26,27,28]. The greater number of Cas7 proteins needed by Haloferax may be due to a difference in spacer length. In Haloferax, spacers are 34–39 nt in length whereas in E. coli they are only 32 nt. An additional Cas7 protein may be needed to cover the extra 2–7 nucleotides of spacer.


The Adaptive Immune System of Haloferax volcanii.

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

Potential Cascade complex composition of the Haloferax type I-B system modeled according to the published structure of the E. coli I-E system. Experimental data regarding the actual structure has not yet been reported. (A) According to the iBAQ analysis the Cascade complex in Haloferax contains 8.5 Cas7 proteins, 1 Cas6b and 1.7 Cas5 proteins. In addition we observed a loose association of Cas8b. (B) The minimal stable Cascade complex could consist of just Cas5 and Cas7 and the short crRNA. Cas8b is essential for the interference reaction but only loosely associated with the complex.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-00521-f003: Potential Cascade complex composition of the Haloferax type I-B system modeled according to the published structure of the E. coli I-E system. Experimental data regarding the actual structure has not yet been reported. (A) According to the iBAQ analysis the Cascade complex in Haloferax contains 8.5 Cas7 proteins, 1 Cas6b and 1.7 Cas5 proteins. In addition we observed a loose association of Cas8b. (B) The minimal stable Cascade complex could consist of just Cas5 and Cas7 and the short crRNA. Cas8b is essential for the interference reaction but only loosely associated with the complex.
Mentions: In Haloferax, deletion of the cas6b gene results in loss of crRNAs, confirming the essential role of the Cas6b protein in crRNA metabolism for the I-B system [34,35]. Further analysis showed that for a normal steady state concentration of crRNAs not only Cas6b but also Cas5 and Cas7 are required, suggesting that they protect the crRNA from degradation [34]. Haloferax cells with only Cas5, Cas6b and Cas8b (e.g., without Cas7) still contain crRNAs, but they are present at a significantly reduced level. While Cas8b can, to some extent, also account for crRNA level stabilization, crRNAs are most efficiently protected when Cas7 is present. A co-purification approach using FLAG-tagged Cas7 protein revealed that the Cascade-like complex of Haloferax contains Cas5, Cas6b and Cas7 subunits [34]. The Cas8b protein seems to be very loosely attached since it cannot be reproducibly co-purified, suggesting that the Haloferax I-B Cascade complex has a core of Cas5 and Cas7 and that Cas6b and Cas8b are more loosely associated (Figure 3). Using mass spectrometry and intensity-based absolute quantification (iBAQ) the components of the core complex were shown to occur in the following ratio 1.7:1:8.5 (Cas5:Cas6b:Cas7) [34]. This composition differs from the observed composition of the type I-E Cascade complex, which consists of 1 Cas5, 1 Cas6, 6 Cas7, 1 Cas8 and additionally contains two copies of the small subunit Cse2 [26,27,28]. The greater number of Cas7 proteins needed by Haloferax may be due to a difference in spacer length. In Haloferax, spacers are 34–39 nt in length whereas in E. coli they are only 32 nt. An additional Cas7 protein may be needed to cover the extra 2–7 nucleotides of spacer.

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.