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.

c Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT. Yersinia pestis, the agent of bubonic plague, infects fleas, its vectors, by producing bacterial biofilms that can colonize the insect foregut (19). Growth of the biofilm interferes with blood feeding by the infected fleas and potentiates regurgitative transmission. Complete blockage of the foregut by the bacterial biofilm can occur, and blocked fleas bite mammals repeatedly in futile feeding attempts, further enhancing transmission. Y. pestis biofilms, in which bacteria are surrounded by a self-synthesized polysaccharide-rich matrix, appear to be made only when the bacteria colonize fleas. Several proteins required for Y. pestis biofilms are proteolytically degraded at mammalian body temperatures (25), in vitro biofilms are greatly diminished at 37°C, and a Y. pestis strain defective for biofilm genes was nevertheless highly virulent in a mouse infection (3,22,32).Y. pestis biofilms are positively regulated by cyclic-di-GMP (cdi-GMP), which is synthesized by diguanylate cyclase (DGC) enzymes. The Y. pestis genome encodes several putative DGCs, but only two of them, HmsT and Y3730, are related to biofilm formation (3,8,22,32). Yersinia pseudotuberculosis, a bacterium closely related to Y. pestis, also makes biofilms that are regulated by hmsT (10). The biofilm-promoting activity of c-di-GMP has not been determined, but in other systems, c-di-GMP has been shown to be an allosteric activator of glycosyltransferases (27,28).Y. pestis and Y. pseudotuberculosis biofilms are negatively regulated by the Rcs phosphorelay system (33). Rcs consists of two membrane-bound proteins, RcsC and RcsD, a DNA-binding response regulator, RcsB, and an accessory protein, RcsA. In phosphorelays of this type, outputs can be dependent on RcsB alone, acting in homodimers, or on heterodimers of RcsB and RcsA (23). Genetic investigation showed that in Y. pestis, rcsA is a nonfunctional pseudogene, while in Y. pseudotuberculosis, rcsA is functional and represses biofilms (33). Although rcsD has a frameshift in Y.pestis, it is still functional and may dephosphorylate RcsB to derepress biofilms (33).The factors determining hmsT transcription have not been examined. In the present study, we show that in Y. pestis and Y. pseudotuberculosis, Rcs represses hmsT transcription. MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used are shown in Table 1. CDY497 was made by a method to insert PCR products into the chromosome using the Red recombination system (11, 33). For strains containing ⌬lacZ::Cm, the same method was used, after which reporter constr...

New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE 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.

Yersinia pestis, the cause of plague, forms a biofilm in the foregut of its flea vector to enhance transmission. Biofilm formation in Y. pestis is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP). HmsT and Y3730, the two diguanylate cyclases (DGC) in Y. pestis, are responsible for the synthesis of c-di-GMP. Y3730, which we name here as HmsD, has little effect on in vitro biofilms, but has a major effect on biofilm formation in the flea. The mechanism by which HmsD plays differential roles in vivo and in vitro is not understood. In this study, we show that hmsD is part of a three-gene operon (y3729-31), which we designate as hmsCDE. Deletion of hmsC resulted in increased, hmsD-dependent biofilm formation, while deletion or overexpression of hmsE did not affect biofilm formation. Localization experiments suggest that HmsC resides in the periplasmic space. In addition, we provide evidence that HmsC might interact directly with the periplasmic domain of HmsD and cause the proteolysis of HmsD. We propose that HmsC senses the environmental signals, which in turn regulates HmsD, and controls the c-di-GMP synthesis and biofilm formation 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.