Biology subjects

Mougiakos, I.

Publications and source records attributed to Mougiakos, I..

4 recordsLinked to original sources

bioRxiv2022

Cas12a2 elicits abortive infection via RNA-triggered destruction of double-stranded DNA

Bacterial abortive infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1, 2. Several RNA-targeting CRISPR-Cas systems (e.g., types III and VI) cause Abi phenotypes by activating indiscriminate RNases3-5. However, a CRISPR-mediated abortive mechanism that relies on indiscriminate DNase activity has yet to be observed. Here we report that RNA targeting by the type V Cas12a2 nuclease drives abortive infection through non-specific cleavage of double-stranded (ds)DNA. Upon recognition of an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded (ss)RNA, ssDNA, and dsDNA. Within cells, the dsDNase activity induces an SOS response and impairs growth, stemming the infection. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided, RNA-targeting tool. These findings expand the known defensive capabilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.

microbiology↗

bioRxiv2021

Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons

CRISPR-Cas biology and technologies have been largely shaped to-date by the characterization and use of single-effector nucleases. In contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons. HIGHLIGHTS[bullet] PAM-DETECT for rapid determination of PAMs for Type I CRISPR-Cas systems in TXTL [bullet]Mining of Type I orthologs and characterization of extensively self-targeting systems [bullet]TXTL-based assessment of DNA target recognition and transposition by CRISPR transposons [bullet]Identification of a distinct branch of Type I-B CRISPR transposons

molecular biology↗

bioRxiv2021

A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-Lmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM- dependent arsenite methylation with formation of monomethylarsenite (MMAs) and dimethylarsenite (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9- based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus {Delta}TtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. ImportanceWe here describe the discovery of an unknown protein by using a proteomic approach with a functionally related protein as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcription regulator, controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65{degrees}C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

biochemistry↗

bioRxiv2017

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20{degrees}C and 70{degrees}C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55{degrees}C in Bacillus smithii and for gene deletion at 37{degrees}C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

microbiology↗