The Genetic Basis of Laboratory Adaptation in Caulobacter crescentus

Abstract

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. 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.

FIG. 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.

MATERIALS AND METHODS

Strains, media, and culture conditions.

Strains and plasmids are listed in Table 1. Rich medium was PYE (0.2% Bacto peptone, 0.1% yeast extract, 1 mM MgSO₄, 0.5 mM CaCl₂). Solid medium was PYE with 1.5% Bacto agar (16). Top agar was PYE with 0.3% Bacto agar and 0.3% glucose. High-sugar PYE medium was PYE with 1.5% glucose or 3% sucrose. Minimal medium was M2G (20 mM Na₂HPO₄, 20 mM KH₂PO₄, 9.3 mM NH₄Cl, 0.5 mM MgSO₄, 0.5 mM CaCl₂, 1 μM FeSO₄, 1 μM EDTA, 0.2% glucose). All cultures were grown at 30°C, unless otherwise noted.

TABLE 1.

Strains and plasmids

Strain or plasmid	Description, genotype, relevant characteristics, and/or date frozen (mo/day/yr)	Source; reference
Caulobacter strains
FC19	CB15; wild-type, sequenced strain; Crosson2	Lucy Shapiro; 30
FC20	NA1000; synchronizable derivative of CB15; Crosson1	Lucy Shapiro; 18
FC746	CB15 with NA1000 allele at SNP1	This study
FC747	CB15 with NA1000 allele at SNP2	This study
FC748	CB15 with NA1000 allele at SNP3	This study
FC749	CB15 with NA1000 allele at SNP4	This study
FC750	CB15 with NA1000 allele at SNP5	This study
FC751	CB15 with NA1000 allele at SNP6	This study
FC752	CB15 with NA1000 allele at SNP9	This study
FC753	CB15 with NA1000 allele at SNP10	This study
FC754	CB15 with NA1000 allele at SNP7	This study
FC755	CB15 with NA1000 allele at SNP8	This study
FC756	NA1000 with CB15 allele at SNP1	This study
FC757	NA1000 with CB15 allele at SNP2	This study
FC758	NA1000 with CB15 allele at SNP3	This study
FC759	NA1000 with CB15 allele at SNP4	This study
FC760	NA1000 with CB15 allele at SNP5	This study
FC761	NA1000 with CB15 allele at SNP6	This study
FC762	NA1000 with CB15 allele at SNP9	This study
FC763	NA1000 with CB15 allele at SNP10	This study
FC764	NA1000 with CB15 allele at SNP7	This study
FC765	NA1000 with CB15 allele at SNP8	This study
FC766	NA1000 with a deletion of the large indel	This study
FC1049	CB15 with NA1000 alleles at SNP8 and SNP10	This study
FC1050	NA1000 with CB15 alleles at SNP8 and SNP10	This study
FC846	CB15 (ATCC 19089, ML28); frozen in 2001	Mike Laub
FC858	NA1000 (Laub); frozen in 2001	Mike Laub
FC859	NA1000 (Smit); frozen in 1983	John Smit
FC860	NA1000 (Ely); frozen in 1979	Bert Ely
FC847	CB15 (Brun4); frozen 1/11/94	Yves Brun
FC855	NA1000 (Brun1); frozen 6/11/90	Yves Brun
FC856	NA1000 (Brun2); frozen 2/2/97	Yves Brun
FC857	NA1000 (Brun3); frozen 11/8/01	Yves Brun
FC854	CB15 (Poindexter, YB1358); frozen 10/15/97	Yves Brun (originally from Jeanne Poindexter)
FC1054	CB15/pRKlac290-PzwfCB15	This study
FC1055	CB15/pRKlac290-PzwfNA1000	This study
FC1056	CB15/pRKlac290-PzwfNA1000JS	This study
Escherichia coli strains
FC728	Top10/pNPTS138-SNP1CB15	This study
FC730	Top10/pNPTS138-SNP2CB15	This study
FC732	Top10/pNPTS138-SNP3CB15	This study
FC734	Top10/pNPTS138-SNP4CB15	This study
FC736	Top10/pNPTS138-SNP5CB15	This study
FC738	Top10/pNPTS138-SNP6CB15	This study
FC740	Top10/pNPTS138-SNP9CB15	This study
FC742	Top10/pNPTS138-SNP10CB15	This study
FC727	Top10/pNPTS138-SNP7CB15	This study
FC744	Top10/pNPTS138-SNP8CB15	This study
FC729	Top10/pNPTS138-SNP1NA1000	This study
FC731	Top10/pNPTS138-SNP2NA1000	This study
FC733	Top10/pNPTS138-SNP3NA1000	This study
FC735	Top10/pNPTS138-SNP4NA1000	This study
FC737	Top10/pNPTS138-SNP5NA1000	This study
FC739	Top10/pNPTS138-SNP6NA1000	This study
FC741	Top10/pNPTS138-SNP9NA1000	This study
FC743	Top10/pNPTS138-SNP10NA1000	This study
FC725	Top10/pNPTS138-SNP7NA1000	This study
FC745	Top10/pNPTS138-SNP8NA1000	This study
FC1051	Top10/pRKlac290-PzwfCB15	This study
FC1052	Top10/pRKlac290-PzwfNA1000(Crosson1)	This study
FC1053	Top10/pRKlac290-PzwfNA1000(Smit)	This study
Plasmids
pNPTS138	Allele replacement vector	M. R. K. Alley
pRKlac290	β-Galactosidase vector	M. R. K. Alley and James Gober

DNA sequencing.

C. crescentus NA1000 cells (10 ml) were grown in PYE and pelleted by centrifugation. DNA was extracted and purified using standard guanidium thiocyanate extraction and isopropanol/ethanol precipitation and sheared for library preparation, and quality was assayed using an Agilent 2100 bioanalyzer (Santa Clara, CA). The genome was sequenced using a GS FLX (Roche, Branford, CT) sequencer to a depth of 20-fold and assembled de novo with the Roche-454 Life Sciences Newbler assembler (see Table S1 in the supplemental material).

Polymorphism confirmation and genome finishing.

The 151 contigs resulting from assembly were aligned against the CB15 genome sequence (GenBank accession number AE005673) using Mauve 2.0.0 (10) and Sequencher 4.7 (Gene Codes, Ann Arbor, MI). Gaps between contigs were generally small (850 bp, average size) and occurred mostly at sites of low sequence complexity or repeated elements. PCR primers flanking the gap were used to amplify the region from NA1000 and directly sequenced (primer sequences are available upon request). We designed primers flanking each position at which the fully assembled genomes disagreed (Table 2 and see Tables S2 and S3 in the supplemental material), amplified them from NA1000 by PCR, and directly sequenced the PCR product using an ABI3730xl sequencer (Applied Biosystems, Foster City, CA). Polymorphisms that were confirmed in strain NA1000 were resequenced in CB15 to determine whether the differences were real polymorphisms (Table 2) or errors in the published CB15 genome sequence (see Table S3 in the supplemental material).

TABLE 2.

Confirmed differences between Caulobacter crescentus CB15 and NA1000 genomes^a

SNP name	CB15 position	NA1000 position	Gene annotation	CB15 gene	NA1000 gene	Erroneous allele (changed aa or base)	Corrected allele (changed aa or base)	Type of mutation
Mobile element	473068.5	473069-499098	Several genes involved in polysaccharide biosynthesis and metabolism	NA	NA	Not present	Present	I
SNP1	525889	551919	Intergenic	CC_0502-0503	CCNA_00536-00537	NA	NA	NC
SNP2	1499722	1525749	Transcriptional regulator, TetR family	CC_1345	CCNA_01407	GCC (Ala)	GTC (Val)	NS
SNP3	1548826	1548820	Cytochrome cbb3 oxidase subunit I, ccoN	CC_1401	CCNA_02097-02098	CTC (Leu)	TTC (Phe)	NS
SNP4	2221557	2247533	Intergenic	CC_2018-2019	CCNA_02070	NA	NA	NC
SNP5	2268285	2294261	Intergenic	CC_2057-2058	CCNA_02136-02137	NA	NA	NC
SNP6	2604988	2630969	4-Coumarate—CoA ligase	CC_2400	CCNA_02483	CGG (Ile)	CGA (Ile)	S
SNP7	2638118.5	2664099	O-antigen chain length regulator, hfsA	CC_2431	CCNA_02513	Intact ORF (ΔG)	Frameshifted ORF (G)	I
SNP8	3042436.5	3068416	TonB-dependent receptor	CC_2820	CCNA_02910	Frameshifted ORF (ΔT)	Intact ORF (T)	I
SNP9	3197735	3223720	Hypothetical protein	CC_2978	CCNA_03073	GAG (Ala)	ACG (Thr)	NS
SNP10	3234417	3260402	TonB-dependent receptor	CC_3013	CCNA_03108	GAC (Asp)	GGC (Glc)	NS

^aPositions and gene numbers for CB15 are consistent with the published genome sequence, GenBank accession number AE005673. Positions and gene numbers for NA1000 are consistent with the newly assembled and annotated genome, GenBank accession number CP001340. Gene annotations were determined using the Integrated Genomics gene annotation algorithm and comparison with protein sequences in GenBank. Erroneous and corrected alleles are represented in the CB15 (AE005673) and NA1000 (CP001340) genome sequences, respectively. aa, amino acid; NA, not applicable; I, insertion/deletion; NC, noncoding; S, synonymous; NS, nonsynonymous; CoA, coenzyme A.

PCR conditions.

PCR for direct sequencing or diagnostic purposes was carried out using GoTaq green (Promega, Madison, WI) 2× reaction mix with 0.5 to 1.0 μM primer, 5% dimethyl sulfoxide (DMSO), and a template (0.5 to 1.0 μl fresh Caulobacter culture). We used the thermal cycling protocol 95°C for 3 min; 30 cycles of 94°C for 15 s, 55°C for 15 s, and 72°C for 1 min/kb; 72°C for 5 min; and maintenance at 4°C. When used as a template for Sanger sequencing, 2.5 μl of the fresh PCR product was mixed with 2.0 μl water and 0.5 μl ExoSAP-IT (USB Corporation, Cleveland, OH), incubated at 37°C for 30 min and at 80°C for 15 min, and stored at −20°C. PCR for cloning was carried out using KOD Hot Start polymerase (Novagen, Madison, WI) with 0.5 to 1.0 μM primer in 1× KOD buffer, 5% DMSO, 0.16 mM deoxynucleoside triphosphates (dNTPs), 2.5 mM MgSO₄, and the template (0.5 to 1.0 μl fresh Caulobacter culture). We used the following thermal cycling protocol: 95°C for 3 min; 30 cycles of 94°C for 15 s, 55°C for 15 s, and 68°C for 1 min/kb; 68°C for 5 min; and maintenance at 4°C. Primer sequences are available upon request.

Caulobacter strain history.

The history of these Caulobacter strains was pieced together from information gathered from personal communications with longstanding members in the Caulobacter field (John Smit, Bert Ely, and Ellen Quardokus) and through careful reading of the Caulobacter literature (Fig. 2) (13, 17, 18, 32, 33).

FIG. 2.

History of laboratory-cultivated Caulobacter crescentus strains CB15 and NA1000. (A) The synchronizable CB15/NA1000 ancestor was originally isolated in 1960 (▵) and deposited with the ATCC (○). Following spontaneous loss of synchronizability, a new synchronizable derivative was isolated (NA1000). The source and freeze date for each CB15 (○) and NA1000 (▪) isolate, as well as the approximate timing of phenotypic evolution in both lineages, are indicated. The Brun, Laub, and Crosson strains are all endpoints originally derived from stock strains in the laboratory of Lucy Shapiro (Stanford University). (B) A DNA parsimony tree based on the 11 polymorphic sites forms two clades representing the NA1000 and CB15 lineages, and strain positions within the tree are consistent with their freeze dates (Table 1). The branches where SNPs evolved are indicated. Branch lengths are not drawn to scale. (C) Genotype data for the archived strains and the inferred CB15/NA1000 ancestral genotype. SNPs are numbered according to genomic position (Fig. 3).

Phylogenetic analysis.

We obtained CB15 and NA1000 strains that were archived at various points during the 50 years that Caulobacter has been in culture in the lab (Fig. 2). We genotyped each of the 11 polymorphisms between CB15 (Crosson₂) and NA1000 (Crosson₁) that we identified in all of these strains using PCR amplification and direct sequencing. From these data, we constructed an unrooted phylogenetic tree using DNA parsimony with 100 bootstrap replicates with PHYLIP (19) and Drawtree (28).

Genome annotation.

Open reading frames (ORFs) for the complete NA1000 genome sequence were called and annotated using ERGO (Integrated Genomics, Chicago, IL). We manually curated the annotation to verify that experimentally characterized genes were annotated correctly. Furthermore, as there is a tendency of automated gene-calling algorithms to misannotate start sites in GC-rich genomes, we made manual adjustments to the translation start positions for approximately 10% of ERGO-predicted genes. The adjustments were based on alignments to homologous sequences in GenBank.

Strain construction.

For each polymorphism that was confirmed by resequencing in both CB15 and NA1000, we built a pair of allele substitution strains using a standard double-crossover allele replacement strategy (34). One-kilobase fragments surrounding each polymorphism were PCR amplified (reaction conditions are described above), cloned using the Zero Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad, CA), subcloned into the suicide knockout plasmid pNTPS138 (Table 1), and electroporated into either CB15 or NA1000. Counterselection for the second chromosomal crossover event resulting in allele replacement was selected for by plating cells that had been outgrown in nonselective liquid medium for 12 h on PYE-sucrose plates. Allele substitutions were verified by PCR amplification and direct sequencing (reaction conditions are described above).

Synchrony assay.

C. crescentus cells divide asymmetrically to yield a flagellated swarmer cell and a stalked cell; exponentially growing C. crescentus cultures contain a mixture of these cell types. In strain NA1000, the stalked and predivisional cells can be physically separated from newly born swarmer cells by centrifugation in Ludox or Percoll or by repeated centrifugation and washing. Cell types from strain CB15, on the other hand, cannot be physically separated by these methods. To quantify the capacity of C. crescentus strains to be synchronized, liquid cultures were grown to an optical density at 660 nm (OD₆₆₀) of 0.1 to 0.3. Percoll (50 μl; Sigma Chemical, St. Louis, MO) was added to 500 μl of culture and spun at 14,000 rpm in a 260D microcentrifuge (Denville Scientific, Metuchen, NJ) for 1 min. Under these conditions, all cells of strain CB15 collect in the pellet, while only swarmer cells pellet in strain NA1000, leaving the stalked and predivisional cells in a suspension as part of the supernatant (Fig. 1). The supernatant and cell suspension were collected, and the pellet was resuspended in 500 μl fresh M2G. We measured the OD₆₆₀ of the supernatant (OD_s) containing the cell suspension and the OD₆₆₀ of the resuspended cell pellet (OD_p) separately using a GeneSys 20 spectrophotometer (Thermo Fisher Scientific, Asheville, NC). To control for differences in total culture density, we defined synchrony capacity as OD_s/(OD_s + OD_p).

Mucoidy assay.

We assessed colony mucoidy by patching each strain on high-sugar medium, incubating at 30°C for 48 h, and storing at 4°C for 24 h before scoring. CB15 colonies and streaks appear dry and dull, while NA1000 colonies and streaks appear moist and shiny when grown under these conditions (Fig. 1).

Phage susceptibility assay.

Phage susceptibility was determined by examining φCR30 plaque morphology. CB15 plaques have sharp edges and clear centers, while NA1000 plaques have indistinct edges and turbid centers (Fig. 1). Phage susceptibility was assessed by plating 100 μl of culture in 2 ml of PYE-glucose top agar on 60-mm PYE-glucose plates. Dilutions (10 μl) of φCR30 (17) lysate were spotted on the cooled top agar lawns and incubated overnight at 30°C. Plaques were photographed with illumination from below and appear lighter than the surrounding lawn of C. crescentus cells.

Surface attachment assay.

Measuring cellular attachment to a polystyrene culture dish assessed the development of a functional holdfast. Liquid PYE (1 ml) was inoculated with 2 μl fresh culture in a 96-well polystyrene culture dish (Corning, Corning, NY). Cultures were incubated at 30°C overnight, with shaking. The OD₆₆₀ for an aliquot from each culture was measured using a GeneSys 20 spectrophotometer (Thermo Fisher Scientific, Asheville, NC) before the wells were washed thoroughly with deionized water, stained for 15 min with 0.1% crystal violet, washed thoroughly with deionized water, and solubilized in 1 ml 100% ethanol, and the OD₅₄₀ was measured using a GeneSys 20 spectrophotometer (Thermo Fisher Scientific, Asheville, NC). Quantitative attachment is reported relative to CB15 levels.

Stationary-phase survival assay.

Liquid PYE (5 ml) was inoculated with freshly grown overnight culture and incubated at 30°C, with shaking, for 96 h. Dilutions were plated on PYE plates and incubated at 30°C for 48 h, and colonies were counted.

Measurement of population doubling time.

Doubling times were measured in 2-ml cultures in M2G (16) at 30°C with shaking in an Infors Multitron shaker (Infors, Bottmingen, Switzerland). Optical density measurements were taken every 20 to 40 min between an OD₆₆₀ of 0.05 and an OD₆₆₀ of 0.250 using a GeneSys 20 spectrophotometer (Thermo Fisher Scientific, Asheville, NC) and fit to a single exponential-growth function using Prism 4.0c (GraphPad Software, Inc., La Jolla, CA). To account for slight variations in growth rate due to different batches of media, all doubling times were normalized to the average CB15 doubling time for a given experiment and measured independently at least five times.

Expression analysis.

The zwf promoter region (285 bp) from each strain (Crosson₁, Crosson₂, Smit) was PCR amplified using KOD polymerase, cloned using the Zero Blunt TOPO PCR cloning kit (Invitrogen, Carlsbad, CA), subcloned into the lacZ transcriptional fusion plasmid pRKLac290, and mated into CB15 and NA1000 using Escherichia coli strain FC03, carrying the RK600 helper plasmid (20). Reporter strains were grown in M2G plus 1 μg/ml tetracycline. All β-galactosidase assays were conducted with 100 μl exponential-phase culture (with an OD₆₆₀ between 0.01 and 0.20), solubilized in chloroform, mixed with Z buffer (700 μl; 60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7), and incubated in the presence of an o-nitrophenyl-β-d-galactopyranoside (ONPG) substrate (4 mg/ml ONPG in 0.1 M KPO₄, pH 7). Reactions were stopped by adding 1 M Na₂CO₃ (1 ml). OD₄₂₀ was measured using a GeneSys 20 spectrophotometer (Thermo Fisher Scientific, Asheville, NC), and Miller units (U) were calculated as U = (OD₄₂₀·1,000)/(OD₆₆₀·tv), where t is the reaction time (minutes) and v is the volume of culture used in the assay (ml) (27).

Genomic GC content analysis.

The C. crescentus NA1000 genome (GenBank accession number NC_011916) was downloaded into MATLAB R2008a (the MathWorks, Natick, MA) using the getgenbank command. The GC-AT composition of the entire chromosome was calculated using a 1,000-bp sliding window with the ntdensity command. This analysis requires the MATLAB Bioinformatics toolbox.

Statistical analyses.

Quantitative phenotypes were compared between an allele replacement strain and its parental-background strain using pairwise two-tailed t tests calculated in Prism 4.0c (GraphPad Software, Inc., La Jolla, CA). Strains with a phenotype significantly different from the CB15 or NA1000 phenotype of the parental background are indicated with an asterisk or double asterisk, respectively, in Fig. 5D to F. Sample sizes, phenotype means, and P values are listed in Table 4.

FIG. 5.

Simultaneous mapping of laboratory-evolved Caulobacter phenotypes by comparison of allele replacement strains with their parental backgrounds (Fig. 1 and Table 1). Strains are organized by background (CB15, left; NA1000, right), CB15 (white) and NA1000 (black) phenotypes are shown for reference, and strains that differ significantly from their parent are indicated (*, CB15; **, NA1000) (Table 4). The SNP numbers at the top and bottom apply to all graphs and tables. The capacity of cultures to be physically synchronized (A), their development of mucoid colony morphology on high-sugar media (M, mucoid; R, rough nonmucoid) (B), and their φCR30 susceptibility (C, clear plaques; T, turbid plaques) (C) map to the presence of the large indel (φ and the yellow bar in panel A and the yellow letters in panels B and C). (D) The difference in adhesion between NA1000 and CB15, as measured by a crystal violet assay, maps to SNP7, a frameshift mutation in the holdfast synthesis gene hfsA. (E) Increased survival in stationary phase is conferred by the presence of CB15 alleles at SNP8 and SNP10, a frameshift mutation and a single amino acid change, respectively, in two different TBDRs. Single-allele-replacement strains have intermediate survival rates (red and pink bars), while the double-allele-replacement strains show parental levels of survival (striped red and pink bars). (F) The faster generation time in NA1000 maps to SNP5 (green bars). All error bars are ±SEM.

TABLE 4.

Quantitative phenotypic analysis of Caulobacter crescentus CB15 and NA1000 allele substitution strains^a

Strain	SNP(s)	Strain background	Synchrony capacity					Relative attachment					Mid-stationary-phase survival					Relative doubling time
			n	Mean	SEM	P value (versus CB15)	P value (versus NA1000)	n	Mean	SEM	P value (versus CB15)	P value (versus NA1000)	n	Mean	SEM	P value (versus CB15)	P value (versus NA1000)	n	Mean	SEM	P value (versus CB15)	P value (versus NA1000)
FC019		CB15	10	0.07	0.01		<0.0001	6	1	0		<0.0001	19	9.63 × 108	2.4 × 108		0.001	19	1.0	0.01		<0.0001
FC020		NA1000	11	0.71	0.04	<0.0001		6	2.65 × 10−3	2.8 × 10−4	<0.0001	ND	16	1.65 × 106	6.8 × 105	0.001		16	0.8	0.01	<0.0001
FC746	1	CB15	10	0.09	0.01	0.341	ND	6	0.813	0.143	0.25	ND	10	1.30 × 109	3.5 × 108	0.424	ND	6	1.0	0.02	0.411	ND
FC747	2	CB15	11	0.11	0.03	0.264	ND	6	0.878	0.061	0.1	ND	10	7.27 × 108	1.5 × 108	0.512	ND	6	1.0	0.02	0.255	ND
FC748	3	CB15	11	0.11	0.02	0.104	ND	6	0.929	0.167	0.69	ND	10	7.92 × 108	1.6 × 108	0.635	ND	6	1.0	0.01	0.107	ND
FC749	4	CB15	10	0.08	0.01	0.593	ND	6	0.745	0.105	0.06	ND	10	7.87 × 108	1.4 × 108	0.622	ND	5	1.0	0.01	0.126	ND
FC750	5	CB15	10	0.04	0.01	0.04	ND	6	0.869	0.133	0.37	ND	8	9.56 × 108	2.2 × 108	0.985	ND	11	0.8	0.02	<0.0001	0.676
FC751	6	CB15	11	0.11	0.02	0.169	ND	6	0.863	0.11	0.27	ND	10	2.06 × 109	5.5 × 108	0.045	ND	9	1.0	0.01	0.051	ND
FC752	7	CB15	11	0.11	0.03	0.222	ND	6	0.871	0.108	0.28	ND	8	2.02 × 109	8.3 × 108	0.114	ND	6	1.0	0.01	1	ND
FC753	8	CB15	11	0.07	0.01	0.6	ND	6	0.989	0.113	0.93	ND	17	3.10 × 107	8.3 × 106	0.001	0.002	9	1.0	0.01	0.554	ND
FC754	9	CB15	11	0.07	0.01	0.966	ND	6	2.39 × 10−3	2.4 × 10−4	<0.0001	0.503	8	1.93 × 109	6.7 × 108	0.102	ND	6	1.0	0.02	0.025	ND
FC755	10	CB15	11	0.08	0.01	0.52	ND	6	1.14	0.175	0.46	ND	17	6.58 × 107	1.3 × 107	0.001	<0.0001	7	1.0	0.01	0.065	ND
FC756	1	NA1000	11	0.72	0.03	ND	0.77	6	2.63 × 10−3	1.6 × 10−4	ND	0.97	9	1.48 × 106	3.0 × 105	ND	0.854	15	0.8	0.01	ND	0.271
FC757	2	NA1000	11	0.81	0.02	ND	0.04	6	2.72 × 10−3	2.7 × 10−4	ND	0.86	11	5.62 × 106	2.3 × 106	ND	0.064	13	0.8	0.01	ND	0.240
FC758	3	NA1000	11	0.79	0.02	ND	0.09	6	2.77 × 10−3	2.1 × 10−4	ND	0.727	8	4.13 × 106	2.0 × 106	ND	0.160	6	0.8	0.01	ND	0.438
FC759	4	NA1000	11	0.74	0.02	ND	0.53	6	2.66 × 10−3	2.0 × 10−4	ND	0.964	8	2.43 × 106	8.4 × 105	ND	0.500	6	0.8	0.01	ND	0.674
FC760	5	NA1000	10	0.75	0.03	ND	0.48	6	2.69 × 10−3	2.6 × 10−4	ND	0.902	8	3.1 × 106	1.1 × 106	ND	0.257	12	1.0	0.01	0.065	<0.0001
FC761	6	NA1000	11	0.83	0.03	ND	0.02	6	3.37 × 10−3	3.6 × 10−4	ND	0.144	8	3.27 × 106	1.2 × 106	ND	0.210	6	0.9	0.01	ND	0.024
FC762	7	NA1000	11	0.75	0.02	ND	0.38	6	2.87 × 10−3	2.8 × 10−4	ND	0.589	8	2.78 × 106	1.3 × 106	ND	0.413	6	0.8	0.01	ND	0.337
FC763	8	NA1000	11	0.74	0.02	ND	0.49	6	2.93 × 10−3	2.8 × 10−4	ND	0.492	17	1.39 × 107	4.6 × 106	0.001	0.016	7	0.8	0.02	ND	0.593
FC764	9	NA1000	11	0.78	0.02	ND	0.16	6	0.901	0.125	0.46	<0.0001	8	3.96 × 106	1.1 × 106	ND	0.079	6	0.8	0.01	ND	0.165
FC765	10	NA1000	10	0.81	0.03	ND	0.06	6	2.92 × 10−3	3.1 × 10−4	ND	0.53	17	2.09 × 107	4.3 × 106	0.001	0	7	0.8	0.03	ND	0.952
FC766	NA (large indel)	NA1000	10	0.07	0.02	0.793	<0.0001	6	2.77 × 10−3	2.2 × 10−4	ND	0.731	8	2.65 × 106	8.3 × 105	ND	0.390	6	0.8	0.01	ND	0.243
FC1049	8 and 10	CB15	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	8	4.93 × 106	2.2 × 106	0.018	0.081	ND	ND	ND	ND	ND
FC1050	8 and 10	NA1000	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	8	1.58 × 109	6.3 × 108	0.273	0.002	ND	ND	ND	ND	ND

^aFor graphical representation of these data, see Fig. 5. Synchrony capacity is defined as OD_s/(OD_s + OD_p). Attachment is measured relative to that of CB15. Mid-stationary-phase survival is measured as the number of CFU/ml after 96 h of growth in liquid culture. Doubling times are measured relative to that of CB15. P values were calculated using unpaired two-tailed t tests to compare the phenotype of a strain with that of its parent and, only if significantly different, with that of the other parent. n, number of replicates; NA, not applicable; ND, not done.

Nucleotide sequence accession numbers.

The complete, annotated NA1000 genome was submitted to GenBank under accession numbers CP001340 and NC_011916.

RESULTS

The dimorphic aquatic bacterium Caulobacter crescentus has evolved marked phenotypic changes during the 50 years that it has been in culture in the laboratory environment. The history of the experimental strains has been documented in the literature and scientists' memories, making C. crescentus an excellent system for studying the nature of selection and phenotypic microevolution in prokaryotes (Fig. 2 and Table 1). Upon isolation, the CB15/NA1000 ancestor, called CB15 in the literature, was adherent and slow growing, and the two cell types (the motile swarmer cell and the nonmotile stalked cell) could be physically separated (i.e., synchronized) by centrifugation (32). It was maintained in slant culture before deposition as strain ATCC 19089, and after additional years of culture, CB15 lost the capacity to be synchronized in at least one laboratory. A synchronizable derivative was reisolated from this stock, maintained in serial liquid and slant culture for several years, and subsequently named NA1000. As both strains were serially passaged throughout their history, the opportunity for evolution and divergence existed in both lineages. An increased survival rate during stationary phase is likely strongly selected under this type of strain maintenance regime. During the years after the NA1000 derivative was isolated, it lost adherence, acquired a faster generation time, and was disseminated to numerous labs across North America (18). Spontaneous mutants for both of these phenotypes are positively selected in a serially propagated culture, as faster-growing cells rapidly increase in frequency, while cells that adhere to the side of a flask or culture tube are culled from the population each time the culture is diluted. CB15 (Crosson₂) exhibits higher survival in stationary phase, has greater susceptibility to phage φCR30, and is less mucoid than NA1000 (Crosson₁) (Fig. 1) (13). Due to its inherent capacity to be physically synchronized by centrifugation, strain NA1000 has become the predominant experimental C. crescentus strain throughout the world.

The sequencing and annotation of C. crescentus NA1000.

To define the genetic basis of phenotypic divergence in C. crescentus, we used a combination of whole-genome sequencing and classical molecular genetic tools to simultaneously map the mutations responsible for all known phenotypic differences between strain CB15 (Crosson₂) and its derivative NA1000 (Crosson₁). Chromosomal DNA of C. crescentus NA1000 (Crosson₁) was sequenced using a model 454-GS FLX instrument and assembled de novo into 151 contigs (see Table S1 in the supplemental material). Direct sequencing of PCR products spanning gap regions completed the assembly to produce the 4.04-Mb NA1000 genome (GenBank accession number CP001340). Enhanced sequence quality, improved prokaryotic gene prediction algorithms, and more extensive protein sequence representation in GenBank resulted in an improved C. crescentus CB15 genome annotation relative to that of the previously published C. crescentus genome (30).

Comparison of the completed NA1000 genome (GenBank accession number CP001340) with the published CB15 sequence (accession number AE005673) (30) revealed 76 polymorphisms and no genomic rearrangements. Upon resequencing the polymorphic sites in both strains, we identified 20 errors in the 454 sequence data and 45 of the differences as errors in the published CB15 sequence (Fig. 3A; see also Tables S2 and S3 in the supplemental material) (30). The remaining 11 differences represent the entire set of real polymorphisms between these strains and include eight SNPs and two single-base and one large (26-kb) insertion-deletion site (indel) (Fig. 3B and Table 2).

FIG. 3.

Schematic representation of errors in the CB15 genome sequence and polymorphism between CB15 and NA1000. Caulobacter crescentus CB15 genome sequence (GenBank accession number AE005673) was aligned with the NA1000 sequence (accession number CP001340), and all differences identified were resequenced in both CB15 and NA1000. (A) Of the 76 differences identified, 20 were errors in the 454 sequence data (see Table S2 in the supplemental material) and 45 were errors in the published CB15 genome sequence, as both NA1000 and CB15 carry alleles identical to the newly generated NA1000 sequence (see Table S3 in the supplemental material). (B) The remaining 11 polymorphisms represent the true genotypic differences between CB15 and NA1000. They are numbered sequentially starting at the origin of replication (ori) and proceeding clockwise through the terminus (ter) and back to the origin. Figures were drawn with assistance from the Genome Tools Project software package (24). Sequencing statistics and details about sequence errors and polymorphisms are listed in Table 2 and Tables S1, S2, and S3 in the supplemental material.

The chromosomal location and predicted ORFs within the large indel suggest that it is a mobile element. We measured the GC composition of the NA1000 genome and identified this indel as the region most different from average, indicating that it was likely acquired from a foreign source (Fig. 4). Within the indel are 22 predicted ORFs, including a P4 family integrase, an excisionase, conjugation and DNA transfer proteins, and several predicted membrane polysaccharide biosynthesis genes (Table 3). Sequence analysis of the ORFs in the indel reveals homology across a diverse group of bacterial taxa, precluding identification of its original source. This indel is inserted into a serine tRNA gene (KEGG database accession number CCNA_R0007), causing a small duplication that leaves the tRNA intact and functional (Fig. 4). Using PCR, we detected spontaneous deletion of the large indel from the NA1000 genome at a low frequency in exponentially growing culture (Fig. 4E), demonstrating that this mobile element is a cause of instability in the CB15/NA1000 ancestral and NA1000 genetic backgrounds.

FIG. 4.

The large indel in the NA1000 genome is a mobile element. (A) Analysis of the base composition of the NA1000 genome using a 1,000-bp sliding window reveals a substantially reduced GC content in the large indel, suggesting that it was acquired by horizontal transfer. Two other regions with known differences from average NA1000 GC content correspond to rRNA and tRNA loci. (B) The large indel is inserted into the 3′ end of serine tRNA gene CCNA_R0007 (long white arrow), causing a short duplication (short white arrows) but preserving the integrity of the gene. Four primers (A, B, C, and D; small black arrows) were used to determine if a strain carries the mobile element. Primer sets AB and CD amplify fragments spanning the left and right junctions, respectively. (C) Primer set AD fails to amplify a product in strains carrying the insertion but readily amplifies a product across the repaired junction. (D) Many mobile elements excise through a circular intermediate (8); the presence of a circular intermediate in NA1000 can be assayed with primer set BC. (E) Spontaneous deletion of this element occurs at low levels in exponentially growing NA1000 cultures. The only product amplified from a CB15 culture is the repaired junction (AD), whereas both the left and right junctions (AB and CD, respectively) are amplified from NA1000. A faint repaired junction (AD) product is detected in NA1000 cultures, suggesting that a small fraction of the cells in the population carry spontaneous deletions of the large indel. The AD products are identical in size and sequence in CB15, NA1000, and NA1000Δφ, indicating that deletion of this region is highly reproducible. We were unable to identify conditions that induced or enhanced excision and did not detect a circular intermediate with primers CB, suggesting that spontaneous excision events are rare, the intermediates are unstable, or excision of this element occurs via an alternate mechanism.

TABLE 3.

Annotated genes within the large indel (mobile) element present in the Caulobacter crescentus NA1000 genome

Gene	Annotationa	Strand
CCNA_00460	Hypothetical protein	+
CCNA_00461	UDP-glucose 6-dehydrogenase (pseudogene)	+
CCNA_00463	Hypothetical protein	−
CCNA_00464	Hypothetical protein	+
CCNA_00465	UDP-galactopyranose mutase	+
CCNA_00466	Glycosyltransferase	+
CCNA_00467	Oligosaccharide translocase/flippase	+
CCNA_00468	Hypothetical protein	−
CCNA_00469	Glycosyltransferase	−
CCNA_00470	O-antigen polymerase	−
CCNA_00471	GDP-l-fucose synthase	−
CCNA_00472	GDP-mannose 4,6 dehydratase	−
CCNA_00473	Hypothetical protein	−
CCNA_00474	DNA relaxase/conjugal transfer nickase-helicase, trwC	−
CCNA_00475	Bacterial conjugation protein, ATP binding domain, trwB	−
CCNA_00476	Hypothetical protein	−
CCNA_00477	Excisionase	−
CCNA_00478	Hypothetical protein	−
CCNA_00479	Transcriptional regulatory protein	+
CCNA_00480	Bacteriophage P4 integrase	−
CCNA_00481	Transcriptional regulatory protein	+
CCNA_00482	Protein HipA	+

^aGene annotations were determined using the Integrated Genomics gene annotation algorithm and by comparison with protein sequences in GenBank.

Evidence of parallel adaptive evolution in C. crescentus laboratory strains.

During adaptation to the laboratory environment, C. crescentus diverged phenotypically into the contemporary CB15 and NA1000 strains. To better understand their evolutionary trajectory, we genotyped the 11 polymorphic sites in strains that had been frozen and stored at different times since their original isolation (Fig. 2A and Table 1) and constructed an unrooted parsimony tree. Comparing the tree architecture with historical information, we demonstrate that both the CB15 and NA1000 lineages have undergone evolution in the lab since they diverged; the genotyped strains cluster into two distinct monophyletic clades (Fig. 2B). Over time, three independent mutations arose in the same TonB-dependent receptor (CCNA_03108, SNP10) and two independent regulatory alleles arose upstream of the gene encoding glucose-6-phosphate dehydrogenase (G6PD) (zwf; SNP5a and SNP5b) (Fig. 2B and C). The repeated, parallel evolution of these genomic loci provides evidence that strains of C. crescentus cultivated in labs across North America are subject to similar selective pressures and that these mutations confer an adaptive advantage on C. crescentus cultured in the laboratory (22).

Simultaneous mapping of positively selected phenotypes.

New phenotypes can evolve through positive selection or genetic drift acting on newly acquired genes, regulatory or coding changes of major effect, or accumulation of many mutations, each with a small phenotypic contribution (5). To assess the genetic mechanisms in the evolution of C. crescentus CB15 and NA1000, we simultaneously mapped the causative mutation(s) for each phenotypic difference between the strains (Crosson₂ and Crosson₁, respectively). This was accomplished by constructing a series of reciprocal allele replacement strains for each of the 8 SNPs and 2 single-base indels, isolating an NA1000 strain with a spontaneous deletion of the large indel (NA1000Δ26kbφ), and measuring all phenotypes in each of these and the two parental strains (Fig. 5 and Tables 1 and 4).

Synchrony, mucoidy, and phage susceptibility phenotypes are determined by a putative mobile element.

Using our simultaneous mapping strategy, we determined that the ability to physically separate swarmer and stalked cells (i.e., synchronizability), mucoid colony morphology, and reduced φCR30 susceptibility require the presence of the large indel sequence. NA1000 exhibits all of these phenotypes, but CB15 and NA1000Δ26kbφ do not (Fig. 1A to C and 5A to C and Table 4). Many of the genes contained within the large indel have predicted functions in carbohydrate metabolism and biosynthesis that may alter the C. crescentus capsular phenotype (Table 3) (41). Indeed, the capsule likely plays significant roles in all three of the phenotypes that map to this region. Alteration in the abundance or complement of polysaccharides in the capsule can affect cellular sedimentation, and thus synchronizability, by differentially altering the density and hydrodynamic properties of the cell (6). Furthermore, φCR30 infection is dependent on attachment to the bacterial S-layer (13), and the presence of a mucoid capsule likely interferes with binding of the phage. As bacteriophage present a significant source of bacterial mortality in oligotrophic environments (4), the presence of a gene or genes that contribute even a modest protective effect can provide a significant selective advantage in the natural environment. As discussed above, we have shown that this sequence is inherently unstable and spontaneously excises in a subpopulation of cells in culture (Fig. 4), providing a mechanism for the host population to toggle between two disparate capsular states, depending on selective conditions.

Loss of holdfast-mediated adhesion in NA1000 is due to a frameshift mutation in the holdfast synthesis gene, hfsA.

Caulobacter species are well known for their ability to attach to inorganic and organic substrates through the holdfast organelle at the end of the stalk (26, 32, 40). Across the years of laboratory cultivation, CB15 (Crosson₂) retained the ability to attach to surfaces and to other cells, while NA1000 (Crosson₁) did not. Cell attachment is easily assayed by quantifying adhesion to a polystyrene surface using crystal violet staining (Fig. 1D). Analysis of the allele substitution strains reveals that the attachment defect in NA1000 is due to a single base insertion (SNP7) causing a frameshift in the middle of the known holdfast synthesis gene, hfsA (Table 2) (39). CB15 cells with the NA1000 SNP7 allele lose their ability to attach to surfaces, while the CB15 allele completely restores attachment in an NA1000 background (Fig. 5D and Table 4).

Altered survival in stationary phase is determined by mutations in two related TonB-dependent outer membrane receptors.

In liquid culture, C. crescentus reaches stationary phase within 36 h. After 96 h of culture time, 6% of CB15 (Crosson₂) and 0.01% of NA1000 (Crosson₁) cells remain culturable on solid agar (Fig. 1E). This difference in survival during stationary phase involves two TonB-dependent outer membrane receptor (TBDR) genes (encoding SNP8 [CCNA_02910] and SNP10 [CCNA_03108]; see Table S5 in the supplemental material). All single-allele-replacement strains involving these TBDRs have survival rates that are intermediate between those of their parental strains, and no single mutation can fully explain the dramatic difference in stationary-phase survival between CB15 (Crosson₂) and NA1000 (Crosson₁) (Fig. 5E and Table 4). Replacement of both TBDR alleles, however, results in recapitulation of the parental phenotypes (Fig. 5E and Table 4). Thus, cell survival during stationary phase is the result of an additive effect of distinct mutations in two genes encoding related TBDRs. SNP10 has mutated independently in at least three different branches of the C. crescentus strain phylogeny (Fig. 2B), suggesting that it is under strong positive selection in the laboratory environment.

A single base pair regulatory mutation in the zwf promoter increases growth rate.

Like increased survival under stress, increased growth rate is a strongly selected phenotype that often evolves during laboratory cultivation (1, 2). Cell growth and division are complex and highly regulated processes, and there are theoretically many ways to elicit a fast-growth phenotype. We find that NA1000 (Crosson₁) carries a single nucleotide polymorphism (SNP5) in the promoter of zwf, which encodes the metabolic enzyme glucose-6-phosphate dehydrogenase (Fig. 6 and Table 1). We observe an inverse relationship between zwf expression level and growth rate: the NA1000 allele results in a 70% reduction in zwf expression (Fig. 6) and is entirely responsible for the 20%-faster generation time in NA1000 (Fig. 1F and 5F and Table 4).

FIG. 6.

Regulatory mutations that reduce zwf expression increase the growth rate of Caulobacter. (A) SNP5 is located 7 bp upstream of the zwf transcriptional start site in isolate NA1000 (Crosson₁). An independently evolved allele (SNP5b) in NA1000 (Smit) is located 3 bp downstream of the zwf transcriptional start. (B) Both NA1000 promoters show reduced activity relative to those of CB15, as measured using transcriptional fusions to lacZ (one-way analysis of variance; P < 0.0001). (C) Model of carbon flux in CB15 cells (white arrows) and NA1000 cells (black arrows). Glucose (G) is phosphorylated to glucose-6-phosphate (G6P), which serves as the primary substrate for three metabolic pathways in Caulobacter. G6P can enter either the pentose phosphate (PP) or the Entner-Doudoroff (ED) pathway following dehydrogenation by the gene product of zwf, glucose-6-phosphate 1-dehydrogenase. Alternatively, G6P can be isomerized to fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. As dehydrogenation of G6P is the rate-limiting step in both the PP and ED pathways, reduced zwf expression is known to increase the amount of G6P available for isomerization into F6P and, consequently, increase the concentration of substrates used for cell membrane and cell wall biogenesis (29).

G6PD catalyzes the first step in both the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways (Fig. 6C). Together, these pathways generate ATP, NADH, NADPH, and pyruvate, as well as five carbon sugars important for nucleic acid biosynthesis (9). We propose a model in which reduced G6PD expression limits carbon flux through both the ED and PP pathways, resulting in a concomitant increase of G6P isomerization to fructose-6-phosphate (F6P) (Fig. 6C). F6P is a precursor for a range of biomolecules, including peptidoglycan and lipid A, and cannot be used as a substrate for glycolysis because the Caulobacter genome does not encode a phosphofructokinase (9). It has been shown experimentally that decreased zwf expression in Escherichia coli modulates carbon flux through central metabolic pathways, resulting in increased concentrations of F6P (29). Similarly, synthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa requires F6P as a precursor (38). Our data provide evidence that C. crescentus growth rate is not limited by energy production (e.g., ATP, NADH, NADPH) when cultivated under laboratory conditions but rather by the availability of the cellular building block F6P.

Clearly, regulatory changes in the expression of key metabolic genes can lead to the rapid evolution of adaptive traits (11, 12). We have identified an independent mutation in the NA1000 (Smit) strain that also results in fast growth and reduced zwf expression (Fig. 6). The parallel evolution of fast growth in these NA1000 strains via two independent zwf promoter mutations suggests that this simple regulatory change is the most facile means of producing a faster-growing cell. Notably, the evolved deregulation of zwf expression in clonal isolates of Pseudomonas aeruginosa from chronically infected cystic fibrosis patients (38) suggests that changes in carbon metabolism via zwf are advantageous during the establishment of chronic infection and may be more broadly involved in the adaptive evolution of bacteria to new environments.

DISCUSSION

Distinguishing between positive selection and neutral evolution is challenging and typically requires large data sets and thorough statistical analyses (3, 21, 35, 36). While the small number of differences between C. crescentus CB15 and NA1000 makes it impossible to rigorously test these hypotheses, it is notable that repeated, parallel evolution of the same phenotype via similar genetic changes is compelling evidence that the phenotypes that we characterized are under positive selection (22). The documented parallel evolution of a TonB-dependent receptor gene and the regulatory region of zwf are linked to increased survival during stationary phase and a faster generation time, respectively. We note that both rapid growth and increased stress resistance characterize adaptation to life in captivity across a broad taxonomic section of domesticated research organisms (1, 2) and endangered zoo populations derived from long-term breeding programs (31). In short, human cultivation of prokaryotic and eukaryotic species selects for fast growth and increased stress resistance. We have deciphered the genetic bases of these, and other, adaptive phenotypes in lab-adapted C. crescentus.

All together, our data provide evidence that positive selection is responsible for the evolution of C. crescentus during its domestication as a laboratory organism. Moreover, adaptive evolution in bacteria proceeds through multiple genetic mechanisms, including nonsynonymous mutation, noncoding regulatory changes, acquisition of new sequences, and inactivation of existing genes. Future work will provide further insight into the prevalence of each of these mechanisms in the evolution of bacterial populations during domestication, infection, or colonization of other new environments.