RESUMO
Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of â¼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
Assuntos
Proteínas de Bactérias/genética , Exonucleases/genética , Edição de Genes/métodos , Genoma Bacteriano , Transformação Bacteriana , Acinetobacter/genética , Acinetobacter/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Reparo de Erro de Pareamento de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exonucleases/antagonistas & inibidores , Exonucleases/deficiência , Recombinação Homóloga , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
IMPORTANCE: To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.
Assuntos
Bacteriófagos , Vibrio cholerae , Vibrio cholerae/metabolismo , Percepção de Quorum/fisiologia , Bacteriófagos/genética , Transdução de SinaisRESUMO
Cyclic dinucleotide (cdN) second messengers are essential for bacteria to sense and adapt to their environment. These signals were first discovered with the identification of 3'-5', 3'-5' cyclic di-GMP (c-di-GMP) in 1987, a second messenger that is now known to be the linchpin signaling pathway modulating bacterial motility and biofilm formation. In the past 15 years, three more cdNs were uncovered: 3'-5', 3'-5' cyclic di-AMP (c-di-AMP) and 3'-5', 3'-5' cyclic GMP-AMP (3',3' cGAMP) in bacteria and 2'-5', 3'-5' cyclic GMP-AMP (2',3' cGAMP) in eukaryotes. We now appreciate that bacteria can synthesize many varieties of cdNs from every ribonucleotide, and even cyclic trinucleotide (ctN) second messengers have been discovered. Here we highlight our current understanding of c-di-GMP and c-di-AMP in bacterial physiology and focus on recent advances in 3',3' cGAMP signaling effectors, its role in bacterial phage response, and the diversity of its synthase family.