Tracing the Domestication of a Biofilm-Forming Bacterium

A mutation that contributes to impaired biofilm formation.Laboratory strains of B. subtilis, such as 168, are often markedly attenuated in their ability to form robust biofilms compared to wild strains, such as 3610 (5). A known mutation that contributes to impaired biofilm formation by 168 is in the sfp gene. sfp encodes a broad-substrate-specificity phosphopantetheinyl transferase involved in the production of numerous B. subtilis secondary metabolites (18). Among these is surfactin, a lipopeptide signaling molecule known to help trigger the expression of genes involved in biofilm formation (17, 20). Accordingly, the starting point for the present investigation was a derivative of 168 (RL3090) that was corrected for the sfp mutation. As shown in Fig. 1, strain 168 itself formed flat, featureless colonies and a flat, thin pellicle, and correction of the sfp mutation (RL3090) yielded minimal changes in morphology (Fig. 1; sfp⁺). In comparison, 3610 formed an architecturally complex colony on solid biofilm-inducing medium (MSgg) and a thick and structurally complex pellicle at the air-liquid interface of a standing culture grown in MSgg.

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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.

To identify additional mutations in RL3090 responsible for defective biofilm formation, we used congression to move random segments of DNA from a strain of 3610 resistant to kanamycin and harboring a lacZ fusion (AM52) into competent cells of RL3090. Colonies from congressants showing an enhanced wrinkly morphology were visible after 2 days of incubation at 37°C (pink arrows in Fig. 2A) and at a frequency of about 3 to 5%. As a control, no such colonies were observed among congressants generated with donor DNA from a domesticated strain harboring the kanamycin resistance gene and a lacZ fusion (Fig. 2B). The more wrinkly colonies appeared at roughly the same frequency as blue colonies arising from uptake of the lacZ reporter, which appeared at a frequency of 1 to 5%. This observation is consistent with the idea that the enhanced wrinkly phenotype of each congressant resulted from correction of a single mutation or several closely linked mutations. Congressants that produced wrinkly colonies retained other characteristics of the recipient RL3090 strain; they were sensitive to chloramphenicol, LacZ⁻, and auxotrophic for tryptophan. The colonies and pellicles produced by the congressants exhibited a morphology that was intermediate between that of the recipient strain and that of the donor 3610 strain. That is, the congressants were only partially repaired in their ability to form biofilms, an observation that suggested that mutations at different loci were responsible for the defective biofilm phenotype of the laboratory strain.

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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.

A mutation impairing biofilm formation is located in epsC.To identify the mutation that had been corrected in generating the wrinkly congressants, one such congressant (AM128) was colony purified and used to create competent cells. The competent cells were transformed with genomic DNA from a library of transposon Tn10 insertions into the genome of strain PY79 (a derivative of 168). Next, we screened the transformants for those exhibiting the impaired biofilm formation phenotype of the donor strain. We then prepared chromosomal DNA from 56 such transformants and in each case verified linkage to the mutation causing impaired biofilm formation to the transposon by transformation. In 8 of the 56 cases, we observed measurable linkage, with cotransformation frequencies ranging from 6 to 30%. Finally, the location of each of the linked transposons was determined by sequencing DNA flanking chromosomal DNA. As can be seen in Fig. 3A, the transposons were clustered on either side of the epsA to -O (epsA-O) operon (triangles in Fig. 3A), suggesting that the mutation(s) was located within the operon.

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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.

That the mutation was in or near the operon was verified in transformation experiments using chromosomal DNA from strains of 3610 harboring antibiotic resistance cassettes that had been inserted into genes pnbA and sigL located near the eps operon (black bars in Fig. 3A). Both markers showed approximately 35% linkage to the mutation responsible for the rough phenotype. Since the markers flank the epsA-O operon, this again suggested that the mutation was located within the epsA-O operon.

Sequence analysis of the epsA-O operon from RL3090 and from the AM128 congressant that had been partially repaired for biofilm formation revealed a single base pair difference. This was a C · G-to-T · A transition at bp 827 of epsC, which changed valine codon 276 (GCG) present in RL3090 to an alanine codon (GTG). Further sequence analysis confirmed that the original donor strain, 3610, had the alanine codon and that two other laboratory strains, JH642 and PY79 (which are derivatives of 168), had the valine codon. Thus, some laboratory strains harbor a mutation in epsC [epsC(A276V)], which we will refer to as epsC¹⁶⁸.

We were surprised to discover that the published genome sequence for 168 has the wild-type T · A base pair, which we confirmed by resequencing this region of the chromosome from the strain that had been used in generating the Subtilist database, which we will designate as strain 168 BFA (Fig. 3A). However, when grown under biofilm conditions, 168 BFA produced more robust pellicles and more wrinkly colonies than did the other laboratory strains used in this study (data not shown). These findings confirm that the presence of a T · A at bp 827 is closely correlated with an increase in biofilm robustness. We acquired several other 168 strains, and only this 168 BFA strain contained the T · A at bp 827. Thus, we suggest that 168 strain variants that contain a repaired epsC allele should be so designated, similar to standard discussion of strains in which the tryptophan auxotrophy has been repaired. To rule out the possibility that the effect on biofilm robustness was actually due to an unknown, unlinked mutation and to produce a marker-free version of this strain, we switched the T · A base pair to a C · G base pair to repair epsC in RL3090, our original donor strain (see Materials and Methods). This newly built strain, AM271 (Fig. 1), had the same biofilm phenotype as that observed in our congressant strain.

The epsC¹⁶⁸ mutation causes decreased exopolysaccharide production.The epsC gene is thought to play a role in exopolysaccharide synthesis, but its precise function is unknown. Sequence analysis using the SMART protein domain database suggests that EpsC contains two transmembrane segments and a large catalytic domain that resembles an epimerase or dehydrogenase. The alanine-to-valine substitution identified in certain laboratory strains lies near the N-terminal end of the catalytic domain (Fig. 3B). The region containing the substitution is highly conserved, with an alanine or a glycine being found at the homologous position in orthologs from other species, such as Pseudomonas aeruginosa and Staphylococcus aureus (Fig. 3B). It is likely that the bulky side chain of valine is responsible for impairing or altering the function of EpsC, and we hypothesized that the loss of this catalytic activity prevented proper exopolysaccharide synthesis.

To investigate this possibility, we created otherwise isogenic strains with the wild-type or mutant alleles of epsC in two different domesticated backgrounds (those of 168 and PY79) and in the wild 3610 background. We introduced mutations in sinR and tasA to increase overall exopolysaccharide production and to allow the release of that exopolysaccharide into the medium (4, 8). Exopolysaccharide was first detected by the formation of aggregates following ethanol precipitation of cell supernatants. For each strain with the epsC¹⁶⁸ mutant allele, no exopolysaccharide aggregates were detected, whereas strains with the wild-type allele of epsC (epsC³⁶¹⁰) were able to produce such aggregates (Fig. 4). Additionally, when the precipitate was resolved by SDS-PAGE, only strains with the wild-type allele of epsC showed an exopolysaccharide band (Fig. 4). For comparison, an epsH mutant was also found to be defective in exopolysaccharide production (Fig. 4) (5, 13).

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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).

An additional mutation contributing to impaired biofilm formation.Although the derivative of RL3090 harboring the wild-type allele of epsC formed more robust biofilms than did the original recipient strain, the biofilms formed by this 168 sfp⁺ epsC⁺ strain were still attenuated in comparison with those of 3610. To identify other mutations responsible for the loss of biofilm robustness in domesticated strains, we used AM271 (168 sfp⁺ epsC⁺) as a recipient strain for a second round of congression with donor DNA from 3610. Once again, we were successful in identifying congressants that formed more robust biofilms than did the AM271 recipient. We introduced random insertions of Tn10 into one such congressant and obtained a Tn10 insertion that showed 60% cotransformation with a locus that caused a significantly rougher colony phenotype.

The third mutation contributing to impaired biofilm formation is located in swrA.Sequence analysis revealed the transposon was inserted in yvjA (Fig. 5A). Independent confirmation that the mutation was located in the yvjA region came from transformation experiments using a marker (spec) that had been placed between the nearby genes yvyD and yvzG. The marker showed∼50% linkage to the additional biofilm-impairing mutation, and transformation of AM271 with genomic DNA from a 3610 strain containing this marker resulted in many colonies forming more robust biofilms. One such strain, AM311, is shown in Fig. 1. This region of the genome (Fig. 5A) contains swrA, which is known to be mutated in many laboratory strains (14). The wild-type coding sequence contains an 8-bp track of A · T base pairs; laboratory strains typically contain an insertion of 1 bp, resulting in a frameshift mutation and a truncated, inactive protein. The wild-type SwrA protein stimulates transcription of the fla/che operon and is known to be needed for swarming motility and for poly-γ-polyglutamic acid synthesis (14, 23). It therefore seemed attractive to suppose that the swrA mutation in laboratory strains contributes to impaired biofilm formation and that AM311 had been corrected for the swrA mutation. Sequence analysis confirmed that transformants that formed more robust biofilms contained the wild-type (8 A · T base pairs) allele of swrA and the less robust biofilm formers contained the mutant allele of swrA (Fig. 5A). Sequence analysis of 7 kb of DNA in the region revealed no other mutations. Moreover, introduction of an insertion/deletion allele of swrA impaired the biofilm phenotype of AM311. We conclude that a strain carrying wild-type alleles of sfp, epsC, and swrA is more robust in biofilm formation than a corresponding strain that is mutant for swrA and hence that swrA also contributes to robust biofilm formation.

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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.

Despite its known role in swarming and poly-γ-polyglutamic acid production and colony mucoidy (14, 23), why was swrA not implicated in biofilm formation previously? We found that the introduction of a swrA mutation into 3610 caused only a subtle, easily overlooked effect on biofilm architecture. We therefore regard AM271 (sfp⁺ epsC⁺) as a sensitized strain in which the contribution of swrA was more readily apparent than in a fully robust, biofilm-forming strain.

A fourth mutation impairing biofilm formation is located in the promoter for degQ.Since our triply corrected laboratory strain AM311 (sfp⁺ epsC⁺ swrA⁺) was still not as robust in biofilm formation as is the wild strain 3610, we sought to identify further biofilm-attenuating mutations in the domesticated background. Another strain isolated during the same congression and transformation experiment as AM311 demonstrated more robust biofilm formation (Fig. 1), and we anticipated that this strain, AM312, contained the wild, repaired allele of an additional biofilm-impairing mutation in addition to being swrA⁺. Colonies with this phenotype appeared at a frequency of ∼5% during the transformation reactions from which AM311 were isolated. Efforts to map the mutation by linkage to a Tn10 insertion were unsuccessful. We therefore turned to whole-genome sequencing to identify differences between AM311 and AM312 (see Materials and Methods). We identified seven point mutations that differed between the strains (Table 3). We then directly sequenced the regions containing these changes in the parental 3610 and 168 strains and also from 5 independently generated congressant strains showing the same biofilm morphologies as AM311 and 5 independently generated hybrid strains showing the same morphologies as AM312. Only one of the mutations—the one near the regulatory gene degQ—met the criteria of being present in 168, AM311, and other congressants with impaired biofilm formation but absent in 3610, AM312, and independently isolated congressant strains with the more robust biofilm phenotype. Consistent with the assignment of the degQ mutation as being responsible for impaired biofilm formation in the tested strains, DNA-mediated transformation using a marker inserted in the chromosome in a gene (yueE) located near degQ revealed high linkage (55% cotransformation) between the marker and the mutation impairing biofilm formation (Fig. 5B). Gratifyingly, Kobayashi (16) had previously and independently found that mutations in degQ impair biofilm formation.

Sequence analysis revealed that the mutation was a T · A-to-C · G transition at the upstream (5′) end of the −10 region of the promoter for degQ. The wild-type T · A base pair at this position is highly conserved in promoters recognized by RNA polymerase containing σ^A (19). Indeed, during the initial characterization of degQ, Yang et al. observed that a mutant bearing this T · A base pair produced more DegQ (then called SacQ) activity, as judged by increased levels of secreted amylases, proteases, and levansucrase (1, 24). Finally, we note that a degQ null mutation has been shown to impair poly-γ-polyglutamic acid synthesis in certain wild strains, which causes a mucoid colony phenotype (16, 23). Poly-γ-polyglutamic acid, however, is not a component of the matrix, and its production is not required for robust biofilm formation (4).

A plasmid-borne gene influences biofilm morphology.It is known that 3610 contains an 80-kb plasmid. In other work, one of us (D.B.K.) generated a derivative of 3610 that was cured of the plasmid (unpublished results). Interestingly, the cured strain exhibited a colony morphology phenotype that was highly similar to that of strain AM312, the strain that had been corrected for sfp, epsC, swrA, and degQ. Thus, it seemed possible that the only remaining, relevant difference between AM312 and 3610 was the absence of the plasmid in the corrected laboratory strain.

In an earlier screen for mutations altering matrix gene expression in 3610 by transposon-mediated insertional mutagenesis, we had in fact identified an insertion in a plasmid-borne gene. We designate this gene rapP because of the similarity of its inferred product to other members of the family of response regulator aspartate phosphatases from B. subtilis (21). To investigate whether the absence of rapP in the corrected laboratory strain could account for the difference in morphology from 3610, we introduced DNA containing rapP (and the adjacent downstream gene phrP) at amyE in AM312. Strikingly, the resulting strain (AM373) formed colonies on biofilm-inducing medium that were essentially indistinguishable in architecture from those of 3610 (Fig. 1).

In toto, the domestication of B. subtilis with respect to biofilm formation can be largely, if not entirely, attributed to five genetic alterations. These are point mutations in the coding sequences of sfp, epsC, and swrA; a promoter mutation in degQ; and the absence of the plasmid-borne gene rapP.

This has been in part an historical investigation in which we have sought to identify mutations that have accumulated over time in the domestication of a laboratory bacterium. It is difficult to know precisely when, and in which order, the mutations arose. For instance, did the epsC mutation arise in the laboratory of Burkholder and Giles, or did subsequent investigators inadvertently (or conceivably purposefully) select for decreased biofilm robustness by choosing smoother, more-reproducible-looking colonies? In any event, the lesson here is that generations of investigators have robbed laboratory strains of some of the important biological properties of the ancestral strains first studied by Meyer and Gottheil over a century ago and first described by Ehrenberg and Cohn a century and a half ago (10).