A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease.
BACKGROUND
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR.


METHODS
The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR.


RESULTS
Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii.


CONCLUSIONS
Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.
Department of Veterinary and Biomedical Sciences, Center for Infectious Disease Dynamics, Center for Molecular Immunology and Infectious Diseases, Pennsylvania State University, University Park, W213 Millennium Science Complex, University Park, PA, 16802, USA. yvi1@psu.edu.
Yury V
YV
Ivanov
Department of Food Science, Center for Infectious Disease Dynamics, Center for Molecular Immunology and Infectious Diseases, Pennsylvania State University, University Park, PA, 16802, USA. nshariat@gettysburg.edu.|Present address: Department of Biology, Gettysburg College, Gettysburg, PA, 17325, USA. nshariat@gettysburg.edu.
Nikki
N
Shariat
USDA, Agricultural Research Service, National Animal Disease Center, Ames, IA, 50010, USA. Karen.Register@ARS.USDA.GOV.
Karen B
KB
Register
Department of Veterinary and Biomedical Sciences, Center for Infectious Disease Dynamics, Center for Molecular Immunology and Infectious Diseases, Pennsylvania State University, University Park, W213 Millennium Science Complex, University Park, PA, 16802, USA. bxl29@psu.edu.
Bodo
B
Linz
Department of Veterinary and Biomedical Sciences, Center for Infectious Disease Dynamics, Center for Molecular Immunology and Infectious Diseases, Pennsylvania State University, University Park, W213 Millennium Science Complex, University Park, PA, 16802, USA. iur107@psu.edu.
Israel
I
Rivera
Department of Veterinary and Biomedical Sciences, Center for Infectious Disease Dynamics, Center for Molecular Immunology and Infectious Diseases, Pennsylvania State University, University Park, W213 Millennium Science Complex, University Park, PA, 16802, USA. kxh365@psu.edu.
Kai
K
Hu
Department of Food Science, Center for Infectious Disease Dynamics, Center for Molecular Immunology and Infectious Diseases, Pennsylvania State University, University Park, PA, 16802, USA. egd100@psu.edu.
Edward G
EG
Dudley
Department of Veterinary and Biomedical Sciences, Center for Infectious Disease Dynamics, Center for Molecular Immunology and Infectious Diseases, Pennsylvania State University, University Park, W213 Millennium Science Complex, University Park, PA, 16802, USA. harvill@psu.edu.|Lee Kong Chian School of Medicine and Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, 637551, Singapore. harvill@psu.edu.
Eric T
ET
Harvill
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001482:Base Composition / Q000235:genetics; D001884:Bordetella / Q000201:enzymology* / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001426:Bacterial Proteins; D004720:Endonucleases