The Genetic Basis of Laboratory Adaptation in Caulobacter crescentus

The dimorphic bacterium Caulobacter crescentus has evolved marked phenotypic changes during its 50-year history of culture in the laboratory environment, providing an excellent system for the study of natural selection and phenotypic microevolution in prokaryotes. Combining whole-genome sequencing with classical molecular genetic tools, we have comprehensively mapped a set of polymorphisms underlying multiple derived phenotypes, several of which arose independently in separate strain lineages. The genetic basis of phenotypic differences in growth rate, mucoidy, adhesion, sedimentation, phage susceptibility, and stationary-phase survival between C. crescentus strain CB15 and its derivative NA1000 is determined by coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. This study evidences multiple genetic mechanisms of bacterial evolution as driven by selection for growth and survival in a new selective environment and identifies a common polymorphic locus, zwf, between lab-adapted C. crescentus and clinical isolates of Pseudomonas aeruginosa that have adapted to a human host during chronic infection.Colonization of new environments or changes in resource availability, predatory regime, or climate can drive adaptive evolution. Determining the genetic basis of these changes informs our understanding of the evolution of diversity and the nature of selection. Domestication of crop plants, adaptive radiations, and in-host evolution during chronic microbial infection are characterized by the evolution of a suite of phenotypes that are advantageous in the new environment. Recent work has successfully identified several of the polymorphisms responsible for this type of adaptive evolution in a variety of species (3, 7, 11, 12, 15, 22, 25, 35-37). With comparative genome sequencing emerging as a powerful tool for identifying genetic polymorphism (5, 14, 23), these studies are becoming faster and easier. Still, large genome sizes and countless sequence differences between individuals, isolates, strains, and species have made comprehensive analyses intractable.Upon isolation and introduction into the laboratory, model research organisms experience extreme environmental changes, with associated selection pressures. Indeed, adaptation to life in captivity has been observed in a wide range of domesticated and model research organisms (2) and in zoo populations of endangered species (31). Many phenotypes that evolve in these nonnative environments do so repeatedly and become common features of human-cultured, -raised, or -cultivated organisms (2), providing evidence of positive selection. Likewise, the aquatic bacterium Caulobacter crescentus has evolved marked phenotypic changes during the 50 years it has been cultured in the laboratory environment. At least six phenotypic differences (Fig. ​(Fig.1)1) between two closely related strains (NA1000 and CB15) derived from the same common ancestor have evolved over decades of laboratory cultivation. It is presumed that these phenotypes evolved in response to the dynamic culture conditions and associated selection pressures experienced by bacteria in the laboratory environment. However, the extent of genetic divergence between these strains was uncharacterized, and it was not known whether the phenotypes could be explained by a few single nucleotide polymorphisms (SNPs), insertions/deletions, or genome rearrangements or by the accumulation of many mutations, each with a small contribution to particular phenotypes. In an effort to comprehensively characterize their divergence, we identified the genetic basis of all known phenotypic differences between two laboratory strains (NA1000 and CB15) of C. crescentus.Open in a separate windowFIG. 1.Evolved phenotypic differences between CB15 (Crosson₂) and NA1000 (Crosson₁). (A) Caulobacter cells divide asymmetrically to yield a swarmer and a stalked cell, which are mixed in culture. NA1000 stalked and predivisional cells (light gray) pellet less efficiently than swarmer cells (dark gray), allowing them to be physically separated. Synchrony capacity is quantified by calculating the proportion of cultured cells remaining in suspension. Error bars are ±standard errors of the mean (SEM). (B) When patched and grown on high-sugar media, NA1000 colonies develop a mucoid morphology, while CB15 colonies do not. (C) The transducing phage φCR30 efficiently infects and lyses CB15 cells, resulting in clear plaques, while infection of NA1000 with the same phage lysate results in fewer plaques that are visually turbid. (D) Holdfast-mediated attachment to a surface can be measured using a crystal violet assay. CB15 cells attach, resulting in robust staining, while NA1000 exhibits negligible adherence. (E) Upon continued aeration and incubation of stationary-phase Caulobacter cultures, NA1000 (▪) loses viability more rapidly than CB15 (○). Error bars are ±SEM. (F) In glucose minimal medium, NA1000 generation time is 20% shorter than that of CB15. Error bars are ±SEM.Our study revealed 11 coding, noncoding, and insertion/deletion polymorphisms between these two strains, five of which completely account for the evolved differences between the strains. The results presented herein provide insight into prokaryotic evolution driven by selection for growth and survival in a research laboratory and demonstrate the utility of combining whole-genome sequencing and alignment with molecular genetic tools to reveal the genetic basis of multiple derived phenotypes. Our work demonstrates that rapid adaptation of C. crescentus to the laboratory environment occurred in both strain lineages and is characterized by relatively few genetic changes, including nonsynonymous mutation, noncoding regulatory changes, acquisition of new genes, and inactivation of existing genes, each with a large phenotypic effect.