Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli , Yersinia pestis , and Mycobacterium smegmatis . Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria. IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC) enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE), encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP) and two DGCs (HmsT and Y3730) control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

The Rcs phosphorelay system, a non-orthodox two-component regulatory system, integrates environmental signals, regulates gene expression and alters the physiological behavior of members of the Enterobacteriaceae family of Gram-negative bacteria. Recent studies of Rcs system focused on protein interactions, functions, and the evolution of Rcs system components and its auxiliary regulatory proteins. Herein we review the latest advances on the Rcs system proteins, and discuss the roles that the Rcs system plays in the environmental adaptation of various Enterobacteriaceae species.

The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis . More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

Mycobacterium tuberculosis (Mtb) encodes an exceptionally large number of toxin-antitoxin (TA) systems, supporting the hypothesis that TA systems are involved in pathogenesis. We characterized the putative Mtb Rv1044-Rv1045 TA locus structurally and functionally, demonstrating that it constitutes a bona fide TA system but adopts a previously unobserved antitoxicity mechanism involving phosphorylation of the toxin. While Rv1045 encodes the guanylyltransferase TglT functioning as a toxin, Rv1044 encodes the novel atypical serine protein kinase TakA, which specifically phosphorylates the cognate toxin at residue S78, thereby neutralizing its toxicity. In contrast to previous predictions, we found that Rv1044-Rv1045 does not belong to the type IV TA family because TglT and TakA interact with each other as substrate and kinase, suggesting an unusual type of TA system. Protein homology analysis suggests that other COG5340-DUF1814 protein pairs, two highly associated but uncharacterized protein families widespread in prokaryotes, might share this unusual antitoxicity mechanism.

The untranslated regions (UTRs) of mRNA contain important features that are relevant to the post-transcriptional and translational regulation of gene expression. Most studies of bacterial UTRs have focused on the 5regions; however, 3UTRs have recently emerged as a new class of post-transcriptional regulatory elements. 3UTRs were found to regulate the decay and translation initiation in their own mRNAs. In addition, 3UTRs constitute a rich reservoir of small regulatory RNAs, regulating target gene expression. In the current review, we describe several recently discovered examples of bacterial regulatory 3UTRs, discuss their modes of action, and illustrate how they facilitate gene regulation in various environments.