Project description:CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host’s genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding an astonishing 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show, based on transcriptomics analyses, that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium’s genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit Cas3 but not Cascade of the respective system. These findings reveal how a bacterium tolerates extensive self-targeting through two CRISPR-Cas systems and expand the suite of Cas3-inhibiting Acrs.
Project description:X. albilineans is one of the most important phytobacteria species which affect sugarcane production. Volatile organic compounds (VOCs) produced by microorganisms may have a noteworthy role in the control of plant diseases. Thus, this study investigated VOC-producing soil bacteria with an antagonistic effect against X. albilineans and evaluated the molecular mechanisms of growth inhibition trigged by the volatile dimethyl disulfide (DMDS). The comparative transcriptomic data of X. albilineans treated with DMDS showed that several metabolic pathways are up-regulated, such as two-component system, flagellar assembly, chemotaxis and bacterial secretion system. Interesting, although the ethanol used as DMDS solvent did not inhibit X. albilineans growth, it triggers a similar gene up-regulation and somehow, the phytopathogen can deal with this harmful compound better than DMDS.
