An improved transconjugation protocol for Bacillus megaterium facilitating a direct genetic knockout

Abstract

We provide a simple but very efficient transconjugation protocol for Bacillus megaterium. By combining utile attributes of known transconjugation methods (small size of the transferred DNA, close physical contact between donor and recipient cells, and heat treatment of the latter) and by determining the appropriate donor/recipient ratio, mating approaches yielded 5 x 10(-5) transconjugants/recipient. Counter-selection for eliminating Escherichia coli donor cells from the mating mixture was possible by pasteurization in case a wild type sporulation proficient B. megaterium served as the mating partner. For nonsporulating mutants, the sacB gene from Bacillus subtilis coding for levansucrase was successfully employed to select against the E. coli donor. The transfer efficiency, up to 15,000 transconjugants acquirable in a single experiment, sufficed--for the first time in this species--to directly select a gene (uvrA) knockout in a one-step procedure. By making use of a mobilizable B. megaterium suicide vector, ten out of the 40 sampled putative transconjugants displayed the expected UV sensitivity and were found to harbor the suicide vector at the anticipated position. Along with the soon available information arising from current B. megaterium sequencing projects, the possibility to quickly inactivate genetic loci will considerably speed up genetic work with this biotechnologically relevant species.

What renders Bacilli genetically competent? A gaze beyond the model organism

“…The latter, for instance, possesses 33 orthologs of genes involved in competence development in B. subtilis (Eppinger et al 2011), and functionality of the DNA receptor protein ComEA has been demonstrated in B. subtilis (Lammers et al 2004). However, B. megaterium was never reported to develop natural genetic competency, and that is why rather time-consuming, less efficient genetic methods such as the polyethylene-glycol-mediated protoplast procedure or transconjugation has to be applied to obtain transformants Meinhardt et al 1989;Richhardt et al 2010;Vary 1994;Vary et al 1982).…”

Utilization of thermo-sensitive plasmid for chromosomal integration of Bacillus halodurans CM1 thermo-alkalophilic xylanase gene with its native promoter in Bacillus subtilis DB104

“…Thermo-alkaliphilic endo-1, 4-β-xylanases from Bacillus halodurans has been cloned and expressed in E. coli using pET21d with T7 promoter and the gene product showed the high potential in deinking of waste paper 8 To improve the gene expression stability and cut plasmid-maintenance, we firstly, cloned this gene with its original promoter then subcloned into pSKE194 vector, and integrated the gene into B. subtilis DB104 chromosome. In this study, we use pSKE194 plasmid constructed by Nahrstedt et al 9,10 which is a derivative plasmid from a combination between pE194 (plasmid for Bacillus) and pBluescript SK+ plasmid (for E. coli) 11 . Having combined features from its parental, this plasmid is a shuttle vector for both E. coli and B. subtilis which carries erythromycin (Erm) and ampicillin (Amp R ) resistance genes for recombinant selection.…”

“…It is also able to conduct spontaneous integration because of its thermo-sensitive origin of [DOI:10.24214/jcbps.B.9.3.131931.] replication which makes it fail to replicate at 42 o C and above 9,12 , allowing the target gene to integrate into chromosome without recE gene complex 7,13 .…”

“…However, as the demand for a simple downstream process increases, host like B. subtilis DB104 which can produce the target protein extracellularly has met our interests. This strain was genetically modified by the removal of both alkaline (∆aprA3) and neutral protease (∆nprE18) genes 9,15 so that it secretes low extracellular protease that can damage our target recombinant xylanase protein. Moreover, it is a non-pathogenic strain despite it is also endospores-producing strain 16 .…”

Current development in genetic engineering strategies of Bacillus species

“…However, genetic manipulation of B. megaterium is difficult, primarily because of low transformation efficiency. Richhardt et al [72] developed a simple and efficient transconjugation method for B. megaterium , combining a known transconjugation method [73] and B. subtilis sacB , which encodes levansucrase. The activity of this enzyme in the presence of sucrose is lethal to E. coli [74], so it is used as a counter-selection marker to eliminate the E. coli donor cells after mating.…”