Conjugation-Mediated Plasmid Transfer Enables Genetic Modification of Diverse Bacillus Species

Abstract

Bacillus species are widely used to produce proteins and to understand microbial differentiation. Unfortunately, outside a few lab strains, genetic manipulation is difficult and can prevent thorough dissection of useful phenotypes. We developed a protocol that utilizes conjugation (plasmids that initiate their own transfer) to introduce plasmids into a diverse range of Bacillus spp. This will facilitate a deeper study of wild isolates for both industrial and pure research uses. Performing genetic manipulations in Bacillus strains is often hindered by difficulty in identifying conditions appropriate for DNA uptake. This shortcoming limits our understanding of the functional diversity within this genus and the practical application of new strains. We have developed a simple method for increasing the genetic tractability of Bacillus spp. through conjugation-mediated plasmid transfer via a diaminopimelic acid (DAP) auxotrophic Escherichia coli donor strain. We observe transfer into representatives of the Bacillus clades subtilis, cereus, galactosidilyticus , and Priestia megaterium and successfully applied this protocol to 9 out of 12 strains attempted. We utilized the BioBrick 2.0 plasmids pECE743 and pECE750, as well as the CRISPR plasmid pJOE9734.1, to generate a xylose-inducible green-fluorescent protein (GFP)-expressing conjugal vector, pEP011. The use of xylose-inducible GFP ensures ease of confirming transconjugants, which enables users to quickly rule out false positives. Additionally, our plasmid backbone offers the flexibility to be used in other contexts, including transcriptional fusions and overexpression, with only a few modifications. IMPORTANCE Bacillus species are widely used to produce proteins and to understand microbial differentiation. Unfortunately, outside a few lab strains, genetic manipulation is difficult and can prevent thorough dissection of useful phenotypes. We developed a protocol that utilizes conjugation (plasmids that initiate their own transfer) to introduce plasmids into a diverse range of Bacillus spp. This will facilitate a deeper study of wild isolates for both industrial and pure research uses.