Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis.
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ABSTRACT:
SUBMITTER: Deng A
PROVIDER: S-EPMC8416114 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC4066482 | biostudies-literature
Project description:The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
| S-EPMC4590770 | biostudies-literature
Project description: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 ∼50% 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.
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Project description:Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (?recA) mutation suppresses the plasmid transformation defect of competent ?recU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ?recX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA-DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth.
| S-EPMC6050356 | biostudies-literature
Project description:Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA-SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA-SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA-SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA-SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA-SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.
| S-EPMC5587729 | biostudies-literature
Project description:Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.
| S-EPMC4381049 | biostudies-literature
Project description:We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double-mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in 2 days by using a strain containing a cre isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-flanked cassette containing both the cre IPTG-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.
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Project description:Horizontal gene transfer (HGT) is a driving force for bacterial evolution that occurs via conjugation, transduction, and transformation. Whereas conjugation and transduction depend on nonbacterial vehicles, transformation is considered a naturally occurring process in which naked DNA molecules are taken up by a competent recipient cell. Here, we report that HGT occurred between two Bacillus subtilis strains cocultured on a minimum medium agar plate for 10 h. This process was almost completely resistant to DNase treatment and appeared to require close proximity between cells. The deletion of comK in the recipient completely abolished gene transfer, indicating that the process involved transformation. This process was also highly efficient, reaching 1.75 × 106 transformants/μg DNA compared to 5.3 × 103 and 1.86 × 105 transformants/μg DNA for DNA-to-cell transformation by the same agar method and the standard two-step procedure, respectively. Interestingly, when three distantly localized chromosomal markers were selected simultaneously, the efficiency of cell-to-cell transformation still reached 6.26 × 104 transformants/μg DNA, whereas no transformants were obtained when free DNA was used as the donor. Stresses, such as starvation and exposure to antibiotics, further enhanced transformation efficiency by affecting the donor cells, suggesting that stress served as an important signal for promoting this type of HGT. Taken together, our results defined a bona fide process of cell-to-cell natural transformation (CTCNT) in B. subtilis and related species. This finding reveals the previously unrecognized role of donor cells in bacterial natural transformation and improves our understanding of how HGT drives bacterial evolution at a mechanistic level.IMPORTANCE Because DNA is easily prepared, studies of bacterial natural genetic transformation traditionally focus on recipient cells. However, such laboratory artifacts cannot explain how this process occurs in nature. In most cases, competence is only transient and involves approximately 20 to 50 genes, and it is unreasonable for bacteria to spend so many genetic resources on unpredictable and uncertain environmental DNA. Here, we characterized a donor cell-dependent CTCNT process in B. subtilis and related species that was almost completely resistant to DNase treatment and was more efficient than classical natural transformation using naked DNA as a donor, i.e., DNA-to-cell transformation, suggesting that DNA donor cells were also important in the transformation process in natural environments.
| S-EPMC6088163 | biostudies-literature
Project description:Bacillus subtilis strain B7-S screened from18 strains is an aerobic, endospore-forming, model organism of Gram-positive bacteria which is capable to form vanillin during ferulic acid bioconversion. The bioconversion of ferulic acid to vanillin by Bacillus subtilis B7-S (B. subtilis B7-S) was investigated. Based on our results, the optimum bioconversion conditions for the production of vanillin by B. subtilis B7-S can be summarized as follows: temperature 35 °C; initial pH 9.0; inoculum volume 5%; ferulic acid concentration 0.6 g/L; volume of culture medium 20%; and shaking speed 200 r/min. Under these conditions, several repeated small-scale batch experiments showed that the maximum conversion efficiency was 63.30% after 3 h of bioconversion. The vanillin products were confirmed by spectral data achieved from UV-vis, inductively coupled plasma atomic emission spectroscope (ICP-AES) and Fourier transform infrared spectrometer (FT-IR) spectra. Scanning electron microscopy (SEM) and transmission electron spectroscopy (TEM) results confirmed that the cell surface of B. subtilis plays a role in the induction of ferulic acid tolerance. These results demonstrate that B. subtilis B7-S has the potential for use in vanillin production through bioconversion of ferulic acid.
| S-EPMC4740768 | biostudies-literature
Project description:Microarray-based comparative genomic hybridization (M-CGH) is a powerful method for rapidly identifying regions of genome diversity among closely related organisms. We used M-CGH to examine the genome diversity of 17 strains belonging to the nonpathogenic species Bacillus subtilis. Our M-CGH results indicate that there is considerable genetic heterogeneity among members of this species; nearly one-third of Bsu168-specific genes exhibited variability, as measured by the microarray hybridization intensities. The variable loci include those encoding proteins involved in antibiotic production, cell wall synthesis, sporulation, and germination. The diversity in these genes may reflect this organism's ability to survive in diverse natural settings.
| S-EPMC1797320 | biostudies-literature