Transformation of Environmental Bacillus subtilis Isolates by Transiently Inducing Genetic Competence

Abstract

Domesticated laboratory strains of Bacillus subtilis readily take up and integrate exogenous DNA. In contrast, “wild” ancestors or Bacillus strains recently isolated from the environment can only be genetically modified by phage transduction, electroporation or protoplast transformation. Such methods are laborious, have a variable yield or cannot efficiently be used to alter chromosomal DNA. A major disadvantage of using laboratory strains is that they have often lost, or do not display ecologically relevant physiologies such as the ability to form biofilms. Here we present a method that allows genetic transformation by natural competence in several environmental isolates of B. subtilis. Competence in these strains was established by expressing the B. subtilis competence transcription factor ComK from an IPTG-inducible promoter construct present on an unstable plasmid. This transiently activates expression of the genes required for DNA uptake and recombination in the host strain. After transformation, the comK encoding plasmid is lost easily because of its intrinsic instability and the transformed strain returns to its wild state. Using this method, we have successfully generated mutants and introduced foreign DNA into a number of environmental isolates and also B. subtilis strain NCIB3610, which is widely used to study biofilm formation. Application of the same method to strains of B. licheniformis was unsuccessful. The efficient and rapid approach described here may facilitate genetic studies in a wider array of environmental B. subtilis strains.

Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis

“…Consistent with previous observations, ancestral strain 3610 was 1,000-fold less transformable than laboratory strain PY79 (Fig. 1B) (10). In comparison, the cured strain that lacked pBS32 was 100-fold more transformable than the ancestral strain (Fig.…”

“…The ancestral B. subtilis strain NCIB3610 (also known as 3610), however, retains many biological properties that were genetically bred out of the laboratory derivatives, including but not limited to floating pellicle biofilms, colonies of complex architecture, synthesis of an extracellular polysaccharide capsule, synthesis of a poly-␥-glutamate slime layer, synthesis of polyketide antimicrobials, synthesis of a nonribosomally synthesized lipopeptide surfactant, swarming and sliding surface motilities, and a large extrachromosomally maintained plasmid (3)(4)(5)(6)(7)(8)(9). Unfortunately, studies of the 3610 strain are hampered due to the fact that it is poorly competent, thus making genetic manipulation inconvenient (10).…”

A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

“…S2 in the supplemental material). It is worth noting that in comparison to the lab strain, the undomesticated strain is poorly naturally competent (42). We conclude that RapP inhibits srfA operon expression, which would, in turn, affect biofilm formation and the development of genetic competence.…”

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

“…In B. subtilis, overexpression of comK allows for genetic access of non-competent wild isolates (Duitman et al 2007;Nijland et al 2010). Artificially induced competence was successfully applied for close relatives of B. subtilis as well: in B. licheniformis MW3.1, overexpression of the homologous comK yielded up to 2.97×10 5 transformants per microgram of chromosomal DNA-an efficiency that allows for direct gene knock outs and even conditional knock outs of essential genes (Hoffmann et al 2010).…”

“…Wild isolates of B. subtilis routinely display poor genetic competence (Duitman et al 2007;Nijland et al 2010). The term "model organism" refers almost exclusively to the commonly employed laboratory strain B. subtilis 168 and its derivatives which are highly transformable.…”