Tracing the domestication of a biofilm-forming bacterium

Abstract

Over the course of more than a century of laboratory experimentation, Bacillus subtilis has become "domesticated," losing its ability to carry out many behaviors characteristic of its wild ancestors. One such characteristic is the ability to form architecturally complex communities, referred to as biofilms. Previous work has shown that the laboratory strain 168 forms markedly attenuated biofilms compared with the wild strain NCIB3610 (3610), even after repair of a mutation in sfp (a gene involved in surfactin production) previously known to impair biofilm formation. Here, we show that in addition to the sfp mutation, mutations in epsC, swrA, and degQ are necessary and sufficient to explain the inability of the laboratory strain to produce robust biofilms. Finally, we show that the architecture of the biofilm is markedly influenced by a large plasmid present in 3610 but not 168 and that the effect of the plasmid can be attributed to a gene we designate rapP. When rapP is introduced into 168 together with wild-type alleles of sfp, epsC, swrA, and degQ, the resulting repaired laboratory strain forms biofilms that are as robust as and essentially indistinguishable in architecture from those of the wild strain, 3610. Thus, domestication of B. subtilis involved the accumulation of four mutations and the loss of a plasmid-borne gene.

Fig. 1.

Colony and pellicle phenotypes of strains harboring mutations that impair biofilm formation. Colonies were grown on MSgg agar medium for 3 days at 30°C. The scale bar represents 2 mm. Rough spots often appear in the 168 sfp⁺ and 168 sfp⁺ epsC⁺ colonies, which are thought to represent spontaneous mutations in the gene for the SinR repressor for matrix operons. The strains are 168, 168 sfp⁺ (RL3090), 168 sfp⁺ epsC⁺ (AM271), 168 sfp⁺ epsC⁺ swrA⁺ (AM311), 168 sfp⁺ epsC⁺ swrA⁺ degQ⁺ (AM312), 3610 without plasmid (DS2569), and NCIB3610. Pellicles were grown in 6-well microtiter plates for 3 days at 30°C. The scale bar represents 1 cm.

Fig. 2.

Identification of congressants that were partially repaired for biofilm formation. Competent cells of a derivative of the laboratory strain 168 sfp⁺ (RL3090) were transformed with genomic DNA from LacZ⁺ Kan^r-containing derivatives of 3610 (AM52) (A) or 168 (AM172) (B) DNA under conditions (DNA excess) favoring congression. Whereas transformation with either DNA led to similar frequencies of lacZ⁺ congressants (blue colonies, some of which are labeled with black arrowheads), only transformation with 3610 DNA yielded congressants producing rougher colonies than the recipient (A; pink arrows). Colonies were grown on solid MSgg containing kanamycin and X-Gal at 37°C for 2 days.

Fig. 3.

Domesticated strains contain a mutation in epsC that impairs biofilm formation. (A) DNA-mediated transformation was carried out using the indicated transposon insertions (triangles) and drug resistance markers (rectangles), revealing linkage between the mutation responsible for the rough biofilm phenotype in strain AM128 and the epsA-O operon. The operon was sequenced, and a C · G-to-T · A missense mutation (epsC¹⁶⁸) was found in epsC at bp 827. (B) Alignment of the amino acid sequence of a region of EpsC from B. subtilis containing the predicted A276V substitution and corresponding regions of orthologs from the following related species (with GenBank accession no. in parentheses): Bacillus amyloliquefaciens (ABS75496), Bacillus licheniformis (AAU25142.1), Bacillus pumilus (ABV63735.1), Bacillus cereus (NP_981687.1), Bacillus halodurans (BAB07437.1), Staphylococcus aureus (ZP_04016170), Streptococcus pneumoniae (ZP_02713626.1), Clostridium botulinum (YP_001392387.1), and Pseudomonas aeruginosa (ABJ12373.1). WT, wild type; dom., domesticated.

Fig. 4.

The epsC¹⁶⁸ mutation impairs exopolysaccharide production. The top row contains images of the chambers of a 12-well microtiter dish containing ethanol-precipitated supernatant from the indicated strains. The bottom row contains ethanol-precipitated supernatant from the indicated strains resolved in the stacking gel of an SDS-PAGE gel stained with Stains-All. All strains contain sinR::kan and tasA::Tn10 spec mutations to increase expression of the eps operon and to liberate the EPS from the cell surface, respectively. The indicated wild-type and mutant strains are as follows: 3610 epsC³⁶¹⁰ (DS991), 3610 epsH (DS5187), 3610 epsC¹⁶⁸ (DS5733), 168 epsC¹⁶⁸ (DS5188), 168 epsC³⁶¹⁰ (DS5189), PY79 epsC¹⁶⁸ (DS5190), and PY79 epsC³⁶¹⁰ (DS5191).

Fig. 5.

Domesticated strains contain mutations in swrA and degQ that impair biofilm formation. DNA-mediated transformation was carried out using the indicated transposon insertions (triangles) and drug resistance markers (rectangles), revealing linkage to the mutation responsible for the rough biofilm phenotype in strains AM311 (A) and AM312 (B). (A) Laboratory strains are known to contain an A · T insertion mutation in the swrA gene, as indicated, and AM271 (168 sfp⁺ epsC⁺) also contains this insertion, whereas the more robust biofilm-forming AM311 strain does not. Numbered base pairs refer to 3610 sequence. (B) Illumina sequencing reveals a mutation in the degQ promoter.