Rimantas Sapranauskas

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.

BfiI recognizes the non-palindromic nucleotide sequence 5-ACTGGG-3 and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg 2 ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10I(E204Q). Detailed characterization of plasmids from 64 colonies screened from the library of ∼200,000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis.

To investigate the domain structure of proteins and the function of individual domains, proteins are usually subjected to limited proteolysis, followed by isolation of protein fragments and determination of their functions. We have developed an approach we call random gene dissection (RGD) for the identification of functional protein domains and their interdomain regions as well as their in vivo complementing fragments. The approach was tested on a two-domain protein, the type IIS restriction endonuclease BfiI. The collection of BfiI insertional mutants was screened for those that are endonucleolytically active and thus induce the SOS DNA repair response. Sixteen isolated mutants of the wild-type specificity contained insertions that were dispersed in a relatively large region of the target recognition domain. They split the gene into two complementing parts that separately were unable to induce the SOS DNA repair response. In contrast, all 19 mutants of relaxed specificity contained the cassette inserted into a very narrow interdomain region that connects BfiI domains responsible for DNA recognition and for cleavage. As expected, only the N-terminal fragment of BfiI was required to induce SOS response. Our results demonstrate that RGD can be used as a general method to identify complementing fragments and functional domains in enzymes.