Extended culture produces coexisting cell-types with varied biofilm-forming abilities
Ten independent populations of B. subtilis were founded from single colonies as either shaking or static cultures of the robust biofilm-forming strain 3610 and were serially transferred daily or every second day following vigorous vortexing. The cultures were maintained for 60 days, with a calculated average of 5.645 generations per dilution, representing over 338 generations for the shaking culture, and over 169 generations for the static culture. The asynchronicity between the shaking and static cultures was chosen to compensate for the steep oxygen gradient and lower nutrient mixing of static cultures, which disadvantages cell growth. Each of the five shaking and five static populations was sampled regularly and the types and distributions of colony morphologies were examined on LB plates (Figures 1 and 2). Within the first week of culturing, cells forming non-ancestral colony types appeared in all ten cultures, and many of these diverse colony morphologies were maintained at varying proportions for the duration of the experiment (Figure 2).
We classified the colony morphologies of our evolved strains as follows, in order of most to least robust biofilm: irregular wrinkled, wrinkled, wrinkled and fuzzy (FW), fuzzy, ancestral, smooth, or mucoid (Additional file 1: Figure S1). Cells forming these diverse colony morphologies were all visible over the course of the experiment and can be seen in plated samples of the 60-day cultures (Figure 2). Cells producing wrinkled, irregular wrinkled or FW colonies rapidly appeared in the shaking cultures, nearly taking over the culture by day 6 or 8, and then decreasing in abundance over time but never completely disappearing (Figure 2A-E, purple, dark blue and turquoise). Smooth colony-forming cells, on the other hand, appeared in the shaking cultures at later days, and their frequency generally increased over time (Figure 2A, orange). Fuzzy and mucoid colony-forming cells followed less clear patterns in the shaking cultures, becoming prevalent in some cultures but not in others.
Cells forming wrinkled or irregular wrinkled colonies were generally less common in the static cultures than in shaking cultures, whereas cells forming fuzzy or FW colonies were more common (Figure 2F-J). As was the case for the shaking cultures, the prevalence of smooth colony-forming cells in the static cultures increased at later time points. Mucoid colonies were absent from the static cultures apart from static culture 5, where this morphotype never comprised more than 2% of the total population.
Following two-month extended culture, we colony-purified 15 colonies from either static or shaking populations. We determined that colony morphology (as observed on LB plates) was a stable trait for the selected isolates. For this study, we then chose to limit further work to a representative sample of five isolates from the day 60 populations that represented the three most distinctive phenotypes: smooth, fuzzy, and wrinkled (Figure 3A, left images). Some strains, including SH1 (shaker 5, day 60, smooth) and SH2 (shaker 3, day 60, smooth), produced smooth, featureless colonies resembling those made by cells lacking the exopolysaccharide synthesis gene epsH (Figure 3B) [12],[18]. Other strains, such as ST1 (static 1, day 60, fuzzy), SH3 (shaker 3, day 60, wrinkled), and SH4 (shaker 3, day 60, wrinkled), produced colonies with more surface structure than did the ancestral strain (Figure 3A). We hypothesized that the enhanced structure resulted from overproduction of extracellular matrix. Surprisingly, despite their morphological differences on LB, all but one of the isolates characterized here exhibited an ancestral-like, wrinkled colony morphology on the standard nutrient-poor, biofilm-inducing medium MSgg (Figure 3A, right images). This observation suggests that the mutations we recovered during extended culture regulate matrix production in a nutrient-dependent manner, such that matrix gene expression is attenuated or enhanced in nutrient-rich conditions (i.e. LB), but remains the same in nutrient-poor conditions (i.e. MSgg).
Morphology robustness correlates with expression levels of matrix genes
To test our hypothesis that colony morphology on LB plates corresponds to matrix gene expression in this medium, we constructed luciferase reporters for the operons responsible for the production of matrix exopolysaccharide (epsA-O) and for the production of matrix proteins (tapA-sipW-tasA). We measured luminescence for the robust biofilm-forming strains ST1, SH3, and SH4, as well as the morphologically featureless strain SH2. Luciferase expression levels for these four evolved strains were compared to those of the ancestral strain and a mutant of the ancestral strain lacking the biofilm repressor gene sinR, which leads to constitutive overexpression of matrix genes [14].
Matrix gene expression – as measured by luminescence - correlated with colony morphology (Figure 4). SH2 did not appear to express matrix genes over basal levels in LB, while ST1, SH3, and SH4 exhibited significantly higher matrix gene expression than that of the ancestral strain (Figure 4). Although sinR null mutant cells resemble the irregular wrinkled colonies observed early on in several shaking cultures (Figure 3B), none of the fuzzy or wrinkled strains obtained at the experimental endpoint of our extended culture experiment matched the extreme colony morphology or matrix gene expression levels of the sinR null mutant. These results further support our hypothesis that the mutations we recovered following extended culture resulted in fine-tuning of matrix expression, but caused neither constitutive expression nor, when we additionally consider the MSgg colony phenotypes, a complete absence of biofilm matrix.
Wrinkled colony-forming evolved strains have mutations in the regulatory gene sinR
Due to the significant role of sinR in biofilm development, we focused our genetic analysis of our evolved strains on the sinR locus. SinR tetramers act as transcriptional repressors of matrix genes during vegetative growth, whereas during stationary phase sinR monomers form a complex with either SinI or SlrR. SinI is an antirepressor and can sequester SinR, while SlrR-SinR complexes release repression of the matrix operons and instead repress genes needed for planktonic growth [14],[19],[20]. Sequencing the sinR locus revealed that strains ST1 and SH3 each carry a mutation in the protein-interaction domain of SinR (Figure 5A), which is the domain responsible for homodimerization and binding with SinI and SlrR. These mutations may modulate SinR binding, by either decreasing SinR dimerization or tetramerization, or possibly by increasing SinR-SlrR or SinI-SinR binding [21],[22]. A T-to-C point mutation in ST1 changes amino acid 107 from a serine to a proline. In SH3, a G-to-A point mutation changes amino acid 89 from a glycine to an arginine. SH4 harbors a silent mutation in sinR at serine 57 changing the codon from TCG to TCA. Subramaniam et al. showed that serine codon bias within sinR serves to couple SinR translation to serine levels in the cell [23]. Interestingly, the mutation in SH4 does not fit the general model posed by Subramaniam et al., who observed that mutations changing TCA codons to TCG codons led to higher SinR production and more wrinkled colonies. Instead, we observed the opposite mutation, which nonetheless led to more wrinkled colonies. Regardless, the phenotype of SH4 could be recapitulated by introducing its silent sinR mutation into a wild-type background. One possible explanation is that this silent serine mutation, which is located N-terminally, could impair the stability of the sinR transcript (Y. Chai, personal communication).
We note that while the robust biofilm-forming strains ST1, SH3, and SH4 harbored mutations in the master biofilm regulator sinR, the smooth colony-forming strains SH1 and SH2 did not, nor did the smooth isolates have mutations in sinI, the anti-repressor of sinR. Given the presence of sinR mutations in some of our evolved strains and the fact that SinR is involved in regulating sporulation initiation in addition to biofilm formation, we recognized the possibility that sporulation was also affected in our extended cultures. Thus, we specifically induced and measured sporulation in the evolved isolates and the ancestral strain. Our results did not indicate a correlation between the presence of a mutation in sinR and sporulation efficiency (Additional file 2: Figure S2).
Point mutations in sinR contribute to the colony morphologies of evolved strains
Although the identified sinR mutations seemed likely to be responsible for the observed biofilm phenotypes in the evolved strains, we wanted to rule out a coincidental role for these mutations. To verify that the sinR mutations in our evolved strains were sufficient to achieve enhanced biofilm robustness, we introduced these specific point mutations into the ancestral strain. Specifically, we reconstituted the sinR point mutations from ST1, SH3, or SH4 in the ancestor to create the strains R1 (sinR
319T>C), R3 (sinR
265G>A), andR4 (sinR
171G>A), respectively. As demonstrated by colony phenotype on LB agar, each of the reconstituted strains exhibits significant matrix overexpression when compared to the ancestor (Figure 5B). R3 and R4 closely phenocopied SH3 and SH4, respectively, indicating that these sinR mutations alone may be responsible for the evolved colony morphologies. R1 partially phenocopied the fuzzy colony-producing strain ST1 (Figure 5B). R1 had more pronounced biofilm structure than did ST1, suggesting that ST1 harbors at least one other mutation that tempers the phenotype caused by the sinR mutation. Overall, we found that point mutations in sinR arose readily during extended culture of B. subtilis, that these mutations are sufficient for robust biofilm formation in wrinkled colony types, and that these mutations can contribute to fuzzy colony types as well.