Unmarked genetic manipulation in Bacillus subtilis by natural co-transformation

Bacillus subtilis is well known as both a model organism and as a microbial cell factory. Simple and scarless gene modification is a desirable tool for basic research and industrial applications of B. subtilis. It has been demonstrated that naturally competent strains of B. subtilis can uptake multiple different DNA molecules, a phenomenon called co-transformation. Here, we describe a co-transformation-based method for generating unmarked mutants of B. subtilis. The PCR product containing the desired mutant allele is introduced into B. subtilis through co-transformation of the plasmid pUS20, which harbours a spectinomycin-resistant marker (Spcr). The target mutation is acquired by screening transformants for integration of pUS20 by resistance to spectinomycin. Due to its unstable replication in B. subtilis, pUS20 is easily cured from transformants in the absence of spectinomycin. This method allows for point mutation delivery at frequencies of approximately 30%. Deletions and insertions of long DNA fragments can also be carried out efficiently using this method. Moreover, this method is also successful in Bacillus velezensis, indicating that it may be extended to other Bacillus species that can form natural competence.