Editing bacterial genomes is an essential tool in research and synthetic biology applications. Here, we describe multiplex genome editing by natural transformation (MuGENT), a method for accelerated evolution based on the cotransformation of unlinked genetic markers in naturally competent microorganisms. We found that natural cotransformation allows scarless genome editing at unprecedented frequencies of ∼50%. Using DNA substrates with randomized nucleotides, we found no evidence for bias during natural cotransformation, indicating that this method can be used for directed evolution studies. Furthermore, we found that natural cotransformation is an effective method for multiplex genome editing. Because MuGENT does not require selection at edited loci in cis, output mutant pools are highly complex, and strains may have any number and combination of the multiplexed genome edits. We demonstrate the utility of this technique in metabolic and phenotypic engineering by optimizing natural transformation in Vibrio cholerae. This was accomplished by combinatorially editing the genome via gene deletions and promoter replacements and by tuning translation initiation of five genes involved in the process of natural competence and transformation. MuGENT allowed for the generation of a complex mutant pool in 1 wk and resulted in the selection of a genetically edited strain with a 30-fold improvement in natural transformation. We also demonstrate the efficacy of this technique in Streptococcus pneumoniae and highlight the potential for MuGENT to be used in multiplex genetic interaction analysis. Thus, MuGENT is a broadly applicable platform for accelerated evolution and genetic interaction studies in diverse naturally competent species.

Signaling through the second messenger cyclic di-GMP (c-di-GMP) is central to the life cycle of Vibrio cholerae. However, relatively little is known about the signaling mechanism, including the specific external stimuli that regulate c-di-GMP concentration. Here, we show that the phosphate responsive regulator PhoB regulates an operon, acgAB, which encodes c-di-GMP metabolic enzymes. We show that induction of acgAB by PhoB positively regulates V. cholerae motility in vitro and that PhoB regulates expression of acgAB at late stages during V. cholerae infection in the infant mouse small intestine. These data support a model whereby PhoB becomes activated at a late stage of infection in preparation for dissemination of V. cholerae to the aquatic environment and suggest that the concentration of exogenous phosphate may become limited at late stages of infection.Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, is a natural inhabitant of temperate aquatic ecosystems throughout the world, including salt, brackish, and some fresh waters (53). The bacterium is known to associate with various aquatic organisms, including cyanobacteria and copepods; additionally, there is evidence that V. cholerae forms biofilms on chitinous surfaces, such as the exoskeletons of zooplankton and phytoplankton (8,19,49,56).Upon entry into a human host via ingestion of contaminated food or water, the bacteria pass through the gastric acid barrier of the stomach and colonize the small intestine. As the bacterium transitions from its natural environment to that of the small intestine, it undergoes a shift from environmental to virulence gene expression (16,25,27,29,50). This transition includes induction of virulence factors, including the toxin coregulated pilus (TCP), required for colonization of the small intestine, and cholera toxin (CT), the A-B toxin responsible for the profuse secretory diarrhea that is characteristic of cholera (20,50). Additionally, the transition from environmental biofilms to the small intestine is accompanied by a decrease in transcription of Vibrio exopolysaccharide synthesis (vps) genes (42).While the overall mechanism controlling this response to changing environments remains unknown, there is evidence to suggest that the bacterial second messenger bis-(3Ј,5Ј)-cyclic di-GMP (c-di-GMP) plays a major role in mediating this adaptive response in V. cholerae (51, 52). The intracellular concentration of c-di-GMP is regulated by the opposing activities of diguanylate cyclase (DGC) and c-di-GMP phosphodiesterase (PDE) enzymes (3, 46). DGCs, containing a GGDEF domain (named for conserved amino acids), catalyze the formation of c-di-GMP from two GTP molecules, while c-di-GMP molecules are hydrolyzed by PDEs, containing an EAL or HD-GYP domain (6,9,33,38,39,41,48). c-di-GMP metabolic enzymes are well conserved throughout the bacterial kingdom. The V. cholerae genome encodes a total of 61 putative c-di-GMP metabolic enzymes: 30 GGDEF, 12 EAL, 9 HD-GYP, and 10 hybrid GGDEF-EAL domain proteins (13,...

Summary Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo-specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non-inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn-seq to identify potential mechanisms of xds regulation. Our selection identified the two-component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate-limited environment.

SummaryPhosphate is essential for life, being used in many core processes such as signal transduction and synthesis of nucleic acids. The waterborne agent of cholera, V ibrio cholerae, encounters phosphate limitation in both the aquatic environment and human intestinal tract. This bacterium can utilize extracellular DNA (eDNA) as a phosphate source, a phenotype dependent on secreted endo‐ and exonucleases. However, no transporter of nucleotides has been identified in V . cholerae, suggesting that in order for the organism to utilize the DNA as a phosphate source, it must first separate the phosphate and nucleoside groups before transporting phosphate into the cell. In this study, we investigated the factors required for assimilation of phosphate from eDNA. We identified PhoX, and the previously unknown proteins UshA and CpdB as the major phosphatases that allow phosphate acquisition from eDNA and nucleotides. We demonstrated separable but partially overlapping roles for the three phosphatases and showed that the activity of PhoX and CpdB is induced by phosphate limitation. Thus, this study provides mechanistic insight into how V . cholerae can acquire phosphate from extracellular DNA, which is likely to be an important phosphate source in the environment and during infection.