Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol

Abstract

BackgroundThe genus Bacillus includes a great variety of species with potential applications in biotechnology. While species such as B. subtilis or B. licheniformis are well-known and used to provide various products at industrial scale, other Bacillus species are less characterized and are not yet used in commercial processes. One reason for this is the fact that genetic manipulation of new isolates is usually complicated with conventional techniques which have to be adapted to each new strain. Even in well-established strains, the available transformation protocols often suffer from low efficiencies.ResultsIn this paper, we provide a new broad host range E. coli/Bacillus shuttle vector, named pBACOV (Bacillus conjugation vector), that can be efficiently transferred to various Bacillus species using a single protocol. A variant of pBACOV carrying the sfGFP gene was successfully transferred to eight different species from the genus Bacillus and to one Paenibacillus species using triparental conjugation (“transmating”). This was achieved using a single protocol and worked for nine out of eleven tested acceptor species. The transmating procedure was used to test expression of the heterologous reporter gene sfGFP under control of the PaprE-promoter from B. subtilis in several Bacillus species in parallel. Expression of sfGFP was found in eight out of nine transmates. For several of the tested species, this is the first report of a method for genetic modification and heterologous gene expression. The expression level, analyzed by measuring the relative sfGFP-fluorescence normalized to the cell density of the cultures, was highest in B. mojavensis.ConclusionsThe new shuttle vector pBACOV can be transferred to many different Bacillus and Paenibacillus species using a simple and efficient transmating protocol. It is a versatile tool facilitating the application of recombinant DNA technology in new as well as established strains, or selection of an ideal host for heterologous gene expression from a multitude of strains. This paves the way for the genetic modification and biotechnological exploitation of the broad diversity of species of Bacillus and related genera as well as different strains from these species.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1198-4) contains supplementary material, which is available to authorized users.

Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae

“…The development of engineered conjugative plasmids such as pTA-Mob [35], pLS20 [39], pRK2013 [19], pRH210 [40], and RP4 [41] provides a simple alternative for DNA transfer to prokaryotic and eukaryotic recipient cells. As recently demonstrated, the development of conjugation methods for species such as the eukaryotic algae P. tricornutum and others allows for rapid advances in genetic engineering of these organisms [10,12,13].…”

“…Conventional transformation techniques, such as electroporation and chemical transformation [18], have been developed for many species, yet suffer from some drawbacks [19]. For one, these methods require pure, intact DNA molecules, which can be challenging to isolate, especially large molecules (>100 kbp) which are prone to damage from shear forces during purification and handling [20].…”

Trans-Kingdom Conjugation Within Solid Media from <em>Escherichia coli</em> to <em>Saccharomyces cerevisiae</em>

“…The development of engineered conjugative plasmids such as pTA-Mob [31], pLS20 [36], pRK2013 [18], pRH210 [37], and RP4 [38] provide a simple alternative for DNA transfer to prokaryotic and eukaryotic recipient cells. As recently demonstrated, the development of conjugation methods for species such as the eukaryotic algae P. tricornutum and other species allow for rapid advances in genetic engineering of these organisms [9,11,12].…”

“…Conventional transformation techniques, such as electroporation and chemical transformation [17], have been developed for many species, yet suffer from some drawbacks [18]. For one, these methods require pure, intact DNA molecules, which can be challenging since large molecules (>100 2 of 15 kbp) are prone to damage from shear forces during purification and handling [19].…”

Draft Genome Sequence of Paenibacillus polymyxa DSM 292, a Gram-Positive, Spore-Forming Soil Bacterium with High Biotechnological Potential

“…In comparison to other strains of the same species, Paenibacillus polymyxa DSM 292 shows some distinguishing traits. P. polymyxa is readily amenable to genetic modification using the transmating procedure described by Heinze et al (4). Most notably, strain DSM 292 did not form halos on milk agar plates, indicating a significantly lower exoprotease activity than strain DSM 365 or the type strain P. polymyxa DSM 36.…”