Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.
Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.
Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Hessen D-35043, Germany.
Srivatsa
S
Dwarakanath
Institute for Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Hessen D-35392, Germany.
Susanne
S
Brenzinger
Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Hessen D-35043, Germany.
Daniel
D
Gleditzsch
Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Hessen D-35043, Germany.
André
A
Plagens
Plant Development, Department Biology I, Biocentre LMU Munich, Großhaderner Str. 2-4, Planegg-Martinsried D-82152, Germany.
Andreas
A
Klingl
Institute for Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Hessen D-35392, Germany.
Kai
K
Thormann
Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Hessen D-35043, Germany LOEWE Center for Synthetic Microbiology (Synmikro), Marburg, Hessen D-35043, Germany lennart.randau@mpi-marburg.mpg.de.
Lennart
L
Randau
D001426:Bacterial Proteins / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000378:metabolism; D059372:Nucleotide Motifs; D012329:RNA, Bacterial / Q000378:metabolism; D020614:Shewanella putrefaciens / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D004247:DNA