Optimization of RK2-based gene introduction system for <i>Bacillus subtilis</i>

Abstract

The Gram-positive bacterium Bacillus subtilis plays important roles in both industrial applications and basic research. However, transformation of competent B. subtilis cells is more difficult to achieve compared with that of Escherichia coli. It has been reported that the conjugative broad host range plasmid RK2 can be transferred to various organisms, including B. subtilis. Nevertheless, the protocol for conjugation from E. coli to B. subtilis has not been properly established. Thus, we optimized interspecies conjugation from E. coli to B. subtilis using the RK2 system. We constructed mobilizable shuttle and integrative vectors pEB1 and pEB2, respectively. pEB1 was used to evaluate the effect of mating media, time, temperature, and genetic background of the recipient and donor strains. We found that conjugation was not significantly affected by the conjugation time or genetic background of the recipient and donor strains. Conjugation on agar was more efficient than that in a liquid medium. A low temperature (16°C and lower) drastically decreased conjugation efficiency. When using the optimized protocol for homologous recombination after conjugation, we could not obtain double crossover mutants, as only single crossover mutants were observed in the initial selection. We then established a two-step homologous recombination method whereby positive colonies were cultivated further, which finally allowed efficient yield of double

Propagation of Recombinant Genes through Complex Microbiomes with Synthetic Mini-RP4 Plasmid Vectors

“…Diagnostic PCR with primers L2-F1 and Tra1-R2 (Supplementary Table S2 ) were done on 5 isolates to verify the acquisition pSEVA2a2d1-MATING (Supplementary Figure S1 ). While this level of HGT is comparatively modest, it falls within the transfer efficiencies reported for the delivery of promiscuous plasmids from E. coli to Gram-positive species such as Bacillus , Streptococcus, Lysteria , Lactobacillus , Staphylococcus , and others [ 15 , 43 – 45 ]. Finally, conjugation mixes were cast between E. coli donors bearing pSEVA222S β -MATING (Figure 1 (c)) and yeast recipients under conditions comparable to the rest of the cases.…”

“…In S2) were done on 5 isolates to verify the acquisition pSEVA2a2d1-MATING (Supplementary Figure S1). While this level of HGT is comparatively modest, it falls within the transfer efficiencies reported for the delivery of promiscuous plasmids from E. coli to Grampositive species such as Bacillus, Streptococcus, Lysteria, Lactobacillus, Staphylococcus, and others [15,[43][44][45].…”

Integrated conjugal plasmid pLS20 in the Bacillus subtilis genome produced 850‐kbp circular subgenomes transmissible to another B. subtilis

“…However, the recipient cells at present are limited to Bacillus species (Itaya et al, 2006; Itaya, Sato, et al, 2018), including B. natto (Itaya et al, 2022), because of the narrow‐host‐range characteristic of pLS20. Therefore, a broad‐host‐range conjugal transfer plasmid, such as pUB307 (Itaya, Kusakabe, et al, 2018; Yokoi et al, 2019), is suitable for widening recipients for future applications. Whether pUB307 in B. subtilis is functional with regard to the conjugation donor by constructing a binary plasmid with pUB307 and pLS20 was examined (Figure S1).…”

Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli

“…Traditionally, the yeast artificial chromosome (YAC) vector has been used for assembling megabase-sized synthetic chromosomes. − A hybrid of the bacterial artificial chromosome (BAC) vector and YAC, or BAC-YAC, enabled the shuttling of up to 0.5 Mb chromosomes from yeast to Escherichia coli . Larger synthetic bacterial chromosomes built in yeast are yet to be transplanted into bacterial cells. , Another approach is to directly use E. coli and BAC for constructing sub-megabase-sized chromosomes. , The E. coli platform would hopefully become a good alternative to yeast, because E. coli is able to transfer anywhere from over 100 kbp up to a few megabase-sized DNA to many kinds of cells via conjugation machinery. − …”