umuDC found in various plant-associated Pseudomonas species [13]. This system is both ecologically relevant and experimentally tractable, making it ideally suited to investigate the influence of MDR on adaptation. In our previous study, parallel lineages of initially isogenic P. cichorii 302959 were maintained in a serial transfer regime for 500 generations during which half received daily MDR activation in the form of UVR exposure [12]. During that evolution experiment, mutations that conferred any compet- itive advantage under the lineage conditions were favored by natural selection. The results of our initial study suggested that regular activation of inducible mutability in the form of MDR was not detrimental to fitness, but rather could contribute to adaptation and genetic diversity [12]. Another recent study has also suggested that when fitness improvements are available, adaptation may be facilitated by moderately enhanced mutagenesis [14]. In our previous study, relative fitness measurements of UVR-exposed lineages indicated a specificity of adaptation observable as higher fitness gains under UVR conditions than under non-UVR conditions that could not be attributed to increases in UVR tolerance alone. Conversely, lineages not exposed to daily UVR displayed comparable impro- vements in relative fitness under both UVR and non-UVR conditions. Such specialization is a general feature of adaptive evolution [15,16]. However, given the common growth medium in both the UVR and non-UVR lineage environments, we expected that beneficial mutations would contribute to enhanced growth under both conditions. In the present study, we investigated adaptive specificity in experimental lineages of P. cichorii 302959 by examining physio- logical components of relative fitness improvements. Relative fitness measurements indicate an overall growth advantage that we hypothesized could be subdivided into discrete improvements in bacterial physiology to reveal a more detailed understanding of the adaptation of P. cichorii 302959 to specific laboratory environ- ments. Our results suggest that improvements in doubling and lag times contributed to specific relative fitness gains by both UVR and non-UVR lineages in their respective environments. Surpris- ingly, specific fitness improvements appeared relatively early in all experimental lineages, independent of regular MDR activation. Materials and Methods Bacterial strains, growth conditions, and general molecular biology techniques The ancestral ‘round’ strain of P. cichorii 302959 was originally isolated in Japan [13]. In a previous study, sixteen populations of P. cichorii 302959 were derived from a single ancestral colony and propagated by serial transfer for 500 generations in minimal liquid media [12]. Eight UVR lineages (numbered 25–32) received a single, daily dose of UVC (254 nm) radiation to activate muta- genic DNA repair. Cultures were individually mixed 1:1 with saline (0.85% NaCl) in a glass Petri dish and exposed to ,40 J m22 of UVC (254 nm) radiation from an XX-15 UV lamp (UVP Products, San Gabriel, CA) that resulted in ,10% survival by each lineage population. The energy output of the lamp was monitored with a UV-X radiometer fitted with a UV-25 sensor (UVP Products, San Gabriel, CA) and determined to be 1.5 J m22 s21. Following UVR irradiation, cultures were diluted 1:100 into fresh medium and incubated under dark conditions to minimize photoreactivation. These lineages diversified into a fluctuating coexistence of ‘round’ (R) and ‘fuzzy’ (F) colony morphotypes. Eight non-UVR lineages (numbered 33–40) were diluted daily into fresh medium without MDR activation by UVR exposure and contained only the R morphotype. The culture Inducible Mutability and Components of Adaptation transfer strategies for both UVR and non-UVR lineages resulted in a 1000-fold daily increase in the growth of each population, representing ,10 generations of binary fission. Samples from each lineage were periodically preserved in a nonevolving state in 10% glycerol at 280uC. All evolved isolates in this study were derived from the ancestral genotype following either 250 or 500 generations of selection in the lineage experiment (Figure 1). We define ‘‘lineage isolates’’ as single- colony isolates from any of the replicate lineages in this experiment. Evolved isolates are denoted by lineage replicate and morphotype (e.g., 25R refers to an isolate from lineage 25 that exhibits the round colony morphotype). Each lineage contained ,1010 total P. cichorii 302959 cells at stationary phase, and the relative fitness of these communities has been shown to rely on the ecological interactions of sympatric genotypes [12]. Therefore, lineage communities were also analyzed in this study as population samples; these samples contained ,108 cells thawed from preserved UVR lineages to maintain the assemblage of diverse genotypes. Isolates and population samples from generation 500 characterized here are identical to those reported previously [12]. Strains of P. cichorii were cultured at 28uC in Luria–Burtani (LB) broth (Difco, Detroit, MI), in Davis Minimal broth supplemented with 25 mg l21 glucose (DM25) (Difco), or on King’s medium B (KB) agar [17]. Antibiotics were used where appropriate at the following concentrations: carbenicillin 50 mg ml21, gentamicin 10 mg ml21. Competent cell preparation was performed according to Choi et al. [18]. Plasmid DNA was isolated using the QIAprep Miniprep kit (QIAGEN, Valencia, CA). Transformation by electroporation, standard agarose gel electrophoresis, and other recombinant DNA techniques were performed according to Sambrook et al. [19]. Competition experiments and relative fitness calculations The relative fitness of all isolates and population samples was determined by direct competition with the ancestor under both UVR and non-UVR conditions to identify patterns of specific or general adaptation as described previously [12]. Briefly, competi- tions were performed under the same UVR and non-UVR conditions as the lineage experiment described above. To ensure that competitors were comparably acclimated to the competition environment, isolates were simultaneously removed from glycerol stocks maintained at 280uC, individually grown in LB broth overnight, and then individually grown for 24 hr in the competition environment. Competitors were mixed at a 1:1 volumetric ratio, and appropriate dilutions were spread on KB agar in triplicate at 0 and 24 hr to estimate the initial and final density of each. Strain differentiation was accomplished by plasmid-encoded catechol 2,3- dioxygenase (xylE) which causes expressing colonies on agar media to turn yellow when sprayed with 0.1 M catechol. The relative fitness (W) of the two competitors was calculated as the ratio of their realized growth rate as described previously [12]. When the two competitors are equally fit in the competition environment, W = 1. The six replicates of each relative fitness measurement were analyzed by two-tailed, independent t-tests against the hypothesis of mean equal fitness (W = 1). Fitness measurements were compared using two-tailed paired t-tests to determine any significant differences in adaptation between growth intervals (generations 250 vs. 500) or growth environments (UVR vs. non- UVR conditions). Growth curves and calculations Before optical density (OD) measurement, lineage isolates and population samples were grown overnight in LB broth and subcultured into 10 ml DM25 at 28uC for 24 hr acclimation. Cultures were diluted 1000-fold into fresh DM25, and 100 ml PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e15975