In Vivo Protein Interactions and Complex Formation in the Pectobacterium atrosepticum Subtype I-F CRISPR/Cas System

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism.

[1]  A. Savchenko,et al.  Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference , 2011, The EMBO journal.

[2]  Albert J R Heck,et al.  Structural basis for CRISPR RNA-guided DNA recognition by Cascade , 2011, Nature Structural &Molecular Biology.

[3]  Ibtissem Grissa,et al.  The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats , 2007, BMC Bioinformatics.

[4]  Peter C. Fineran,et al.  SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria* , 2012, The Journal of Biological Chemistry.

[5]  S. Mulepati,et al.  Structural and Biochemical Analysis of Nuclease Domain of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Protein 3 (Cas3)* , 2011, The Journal of Biological Chemistry.

[6]  Konstantin Severinov,et al.  CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. , 2012, Molecular cell.

[7]  Philippe Horvath,et al.  Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system , 2011, The EMBO journal.

[8]  Jennifer A. Doudna,et al.  Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease , 2010, Science.

[9]  S. Ehrlich,et al.  Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. , 2005, Microbiology.

[10]  M. Sternberg,et al.  Protein structure prediction on the Web: a case study using the Phyre server , 2009, Nature Protocols.

[11]  R. Garrett,et al.  Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms , 2012, Molecular microbiology.

[12]  Jennifer A. Doudna,et al.  Structures of the RNA-guided surveillance complex from a bacterial immune system , 2011, Nature.

[13]  J. Doudna,et al.  RNA-guided genetic silencing systems in bacteria and archaea , 2012, Nature.

[14]  Erik J. Sontheimer,et al.  Self vs. non-self discrimination during CRISPR RNA-directed immunity , 2009, Nature.

[15]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[16]  L. Marraffini,et al.  Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site , 2011, Proceedings of the National Academy of Sciences.

[17]  M. F. White,et al.  Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)* , 2011, The Journal of Biological Chemistry.

[18]  Stan J. J. Brouns,et al.  CRISPR Interference Directs Strand Specific Spacer Acquisition , 2012, PloS one.

[19]  U. Qimron,et al.  Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli , 2012, Nucleic acids research.

[20]  Marko Djordjevic,et al.  Transcription, processing and function of CRISPR cassettes in Escherichia coli , 2010, Molecular microbiology.

[21]  A. Fraser,et al.  Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Terns,et al.  Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. , 2008, RNA.

[23]  J. García-Martínez,et al.  Short motif sequences determine the targets of the prokaryotic CRISPR defence system. , 2009, Microbiology.

[24]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Sylvain Moineau,et al.  Bacteriophage resistance mechanisms , 2010, Nature Reviews Microbiology.

[26]  Albert J R Heck,et al.  RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions , 2011, Proceedings of the National Academy of Sciences.

[27]  N. Grishin,et al.  A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action , 2006, Biology Direct.

[28]  L. Marraffini,et al.  CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA , 2008, Science.

[29]  Stan J. J. Brouns,et al.  Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes , 2008, Science.

[30]  Samuel H Sternberg,et al.  Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. , 2012, RNA.

[31]  Jing Zhang,et al.  Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. , 2012, Molecular cell.

[32]  Peter C. Fineran,et al.  Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum , 2011, RNA biology.

[33]  M. DeLisa,et al.  Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein* , 2012, The Journal of Biological Chemistry.

[34]  George A. O'Toole,et al.  Non-Identity-Mediated CRISPR-Bacteriophage Interaction Mediated via the Csy and Cas3 Proteins , 2011, Journal of bacteriology.

[35]  K. Zhou,et al.  Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. , 2009, Structure.

[36]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[37]  R. Terns,et al.  Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. , 2008, Genes & development.

[38]  Stan J. J. Brouns,et al.  Evolution and classification of the CRISPR–Cas systems , 2011, Nature Reviews Microbiology.

[39]  R. Barrangou,et al.  CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes , 2007, Science.

[40]  S. Shuman,et al.  Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. , 2010, Acta crystallographica. Section F, Structural biology and crystallization communications.

[41]  E. Koonin,et al.  A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats* , 2008, Journal of Biological Chemistry.

[42]  Hongwei Wang,et al.  Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. , 2012, Structure.

[43]  Peter C. Fineran,et al.  Biotechnological exploitation of bacteriophage research. , 2007, Trends in biotechnology.

[44]  Philippe Horvath,et al.  Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus , 2007, Journal of bacteriology.

[45]  J. Doudna,et al.  Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA , 2012, The EMBO journal.

[46]  B. Graveley,et al.  RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex , 2009, Cell.

[47]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[48]  Konstantin Severinov,et al.  Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system , 2012, Nature Communications.

[49]  Eugene V Koonin,et al.  Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems , 2011, Biology Direct.

[50]  Andrew Emili,et al.  A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair , 2011, Molecular microbiology.

[51]  J. Bever,et al.  A cooperative virulence plasmid imposes a high fitness cost under conditions that induce pathogenesis , 2012, Proceedings of the Royal Society B: Biological Sciences.

[52]  Daniel H. Haft,et al.  A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes , 2005, PLoS Comput. Biol..

[53]  R. Garrett,et al.  A putative viral defence mechanism in archaeal cells. , 2006, Archaea.

[54]  B. Tjaden,et al.  Characterization of the CRISPR/Cas Subtype I-A System of the Hyperthermophilic Crenarchaeon Thermoproteus tenax , 2012, Journal of bacteriology.