Species and condition shape the mutational spectrum in experimentally evolved biofilms

Abstract

Biofilm formation is a vital factor for the survival and adaptation of bacteria in diverse environmental niches. Experimental evolution combined with the advancement of whole-population genome sequencing provides us a powerful tool to understand the genomic dynamic of evolutionary adaptation to different environments, such as during biofilm development. Previous studies described the genetic and phenotypic changes of selected clones from experimentally evolved Bacillus thuringiensis and Bacillus subtilis that were adapted under abiotic and biotic biofilm conditions. However, the full understanding of the dynamic evolutionary landscapes was lacking. Furthermore, the differences and similarities of adaptive mechanisms in B. thuringiensis and B. subtilis were not identified. To overcome these limitations, we performed longitudinal whole-population genome sequencing to study the underlying genetic dynamics at high resolution. Our study provides the first comprehensive mutational landscape of two bacterial species' biofilms that is adapted to an abiotic and biotic surface.

Keywords: Bacillus subtilis; experimental evolution; parallelism; population size.

Fig 1

Mutation spectrum, dynamics, and diversity. (a) Distribution of detected mutations of each type in four adaptation models over time. Shaded bars show the distribution of different mutation types in each time point or total. (b) Dynamic distribution of detected mutations in each lineage over time. (c) dN/dS ratio. The ratio of non-synonymous to synonymous mutations (dN/dS) in the entire pool of detected mutations of each condition, this ratio is normalized by the relative number of synonymous and non-synonymous sites of B. thuringiensis and B. subtilis, respectively. Boxes indicate Q1–Q3, lines indicate the median, black circles filling with different color indicate the ratio of each lineage. (d) Genotype diversity. Dynamic distribution of genotype alpha diversity in each population of four adaptation models over time calculated using Shannon method. Genotypes and frequencies were generated by Lolipop software package in genotype and genealogy analysis.

Fig 2

Genealogy and genotype frequencies over time. Each shade or color represents a different genotype, and vertical area corresponds to genotype frequency, inferred by Lolipop. Dominant genotypes that contain the high-frequency mutated genes, which are shared in different populations, are highlighted in certain colors within each adaptation model. The nested genotypes of the dominant genotype are highlighted with the same color in Bth_bead, Bs_pellicle, and Bs_root, except for the nested genotypes which also belong to the dominant genotypes. In Bth_root, for the complex combination of mutations among different lineages and early fix of new mutations, nested genotypes are not highlighted. Other genotypes are in gray.

Fig 3

Evolvability. The number of accumulated mutations in each genotype at the last time point in each condition. Each circle represents one genotype. Boxes indicate Q1–Q3, lines indicate the median, and circles with no filling indicate the accumulated mutation number of each genotype.

Fig 4

Parallelism. (a) Parallelism in each adaptation model and species. For all plots, the null distribution (shown in gray) was obtained by simulating random mutations to genes, considering the number of mutations in each model and species in our data and the relative length of each gene. The green dotted line indicates the number of lineages in each condition. (b) Degree of parallelism within each condition estimated by Jaccard index. Asterisks at the top indicate significant differences between biotic and abiotic evolved conditions of each species (***P < 0.001, Student’s unpaired two-tailed t-test was performed). Boxes indicate Q1–Q3, lines indicate the median, black circles filling with different color indicate the J value of each combination, and gray dots indicate the outliers.

Fig 5

Genes mutated more than one time in B. thuringiensis and B. subtilis. Each column represents one replicate lineage. Thirty-five genes and 48 genes mutated more than one time in B. thuringiensis (left) and B. subtilis (right), respectively, were distributed non-randomly. Color indicates the highest frequency of mutations in that gene in that lineage. Detailed information of these genes can be found in Data Set S2.