Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.National Institutes of Health (U.S.) (Pioneer Award DP1MH100706)National Institutes of Health (U.S.) (Transformative Research Award)W. M. Keck FoundationMcKnight FoundationBill & Melinda Gates FoundationDamon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramEsther A. & Joseph Klingenstein Fund, Inc.Simons Foundatio

The CRISPR immunity pathway.

<p>CRISPR loci contain clusters of repeats (white boxes) and spacers (colored boxes) that are flanked CRISPR-associated (<i>cas</i>) genes. (<b>A</b>) During adaptation new spacers derived from the genome of the invading virus are incorporated into the CRISPR array by an unknown mechanism. Repeat duplication is also required. (<b>B</b>) During crRNA biogenesis a CRISPR precursor transcript is processed by Cas endoribonucleases within repeat sequences to generate small crRNAs. (<b>C</b>) During targeting the match between the crRNA spacer and target sequences specifies the nucleolytic cleavage of invading mobile genetic elements such as viruses and plasmids. (<b>D</b>) In the CRISPR-Cas system of <i>F. novicida</i>, the tracrRNA (a small RNA mediated in crRNA biogenesis in this system) contains homology to the BLP (bacterial lipoprotein) transcript. The base-pair interaction between the tracrRNA and the BLP mRNA (mediated also by another small RNA, the scaRNA, and the nuclease Cas9) regulates the expression of this immunomodulatory lipoprotein.</p

Sortase C-Mediated Anchoring of BasI to the Cell Wall Envelope of Bacillus anthracis▿

Vegetative forms of Bacillus anthracis replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of B. anthracis peptidoglycan

Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis