The isolation of aerobic citrate-utilizing Escherichia coli (Cit ؉ ) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of E. coli Cit ؉ mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type E. coli strain B and with K-12 and three K-12 derivatives: an E. coli ⌬rpoS::kan mutant (impaired for stationaryphase survival), an E. coli ⌬citT::kan mutant (deleted for the anaerobic citrate/succinate antiporter), and an E. coli ⌬dctA::kan mutant (deleted for the aerobic succinate transporter). E. coli underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent E. coli Cit ؉ mutants were isolated from all E. coli derivatives except the E. coli ⌬citT:: kan mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry. E. coli Cit ؉ mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four E. coli Cit ؉ mutants revealed the required sequence of mutational events leading to a refined Cit ؉ mutant. These events showed amplified citT and dctA loci followed by DNA rearrangements consistent with promoter capture events for citT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE. H ow genetic information evolves to generate new phenotypes/ species is a central issue in biology. Long-term evolution experiments (LTEE) using microorganisms have been initiated by several groups, in part to empirically observe this phenomenon (1). LTEE using bacteria, bacteriophage, or yeast have distinct advantages that include high population numbers, rapid generation times, and the opportunity to freeze intermittent populations (frozen fossils) to track mutations over time. Coupled with wholegenome sequencing, evolutionary changes can be genetically characterized to identify a mutation(s) required for a specific phenotypic change and frozen intermediates can be revived to replay and confirm the events. The most famous and meticulously documented LTEE are those, initiated in 1988, in Richard Lenski's laboratory (2). Twelve parallel cultures of Escherichia coli REL606 (an E. coli B strain) have been growing aerobically in minimal salts medium with low glucose concentrations (0.0025%) for 27 years. Cultures are transferred daily into fresh medium. Frozen samples are preserved for each culture every 500 generations, providing a tremendous resource to study long-term bacterial adaptation under controlled conditio...

10Pathogenic bacteria recognize environmental cues to vary gene expression for host adaptation. 11Moving from ambient to host temperature, Yersinia enterocolitica responds by immediately 12repressing flagella synthesis and inducing the virulence plasmid (pYV)-encoded type III secretion 13 system. In contrast, shifting from host to ambient temperature requires 2.5 generations to restore 14 motility suggesting a link to the cell cycle. We hypothesized that differential DNA methylation 15 contributes to temperature-regulated gene expression. We tested this hypothesis by comparing 16 single-molecule real-time (SMRT) sequencing of Y. enterocolitica DNA from cells growing 17 exponentially at 22°C and 37°C. The inter-pulse duration ratio rather than the traditional QV scoring 18 was the kinetic metric to compare DNA from cells grown at each temperature. All 565 YenI 19 restriction sites were fully methylated at both temperatures. Among the 27,118 DNA adenine 20 methylase (Dam) sites, 42 had differential methylation patterns while 17 remained unmethylated 21 regardless of temperature. A subset of the differentially methylated Dam sites localized to promoter 22 regions of predicted regulatory genes including LysR-type and PadR-like transcriptional regulators, 23 and a cyclic-di-GMP phosphodiesterase. The unmethylated Dam sites localized with a bias to the 24 replication terminus, suggesting they were protected from Dam methylase. No cytosine methylation 25 was detected at Dcm sites. 26 27 28 DATA SUMMARY 29 30 All methylation/base modification data are available at figshare at 31 https://dx.doi.org/10.6084/m9.figshare.3493247 and 32 https://dx.doi.org/10.6084/m9.figshare.3493310. 33 34 35 IMPACT STATEMENT 36 37 Organisms sense and respond to their environment, in part, by epigenetic variation mediated by 38 DNA methylation. Pathogenic bacteria vary gene expression to allow survival and activate virulence 39 systems in response to host temperature. Yersinia enterocolitica, a facultative intracellular 40 pathogen, respond by immediately repressing flagella synthesis and inducing the virulence plasmid-41 encoded type III secretion system. In this work, we examined the locations of DNA methylation 42 throughout the Y. enterocolitica genome. While most methylation target sites were fully methylated, 43 we identified sites with disparate temperature-dependent methylation. Several of these sites were 44 within promoter regions of predicted regulatory genes. Differences in DNA methylation in promoter 45 sequences are often responsible for variations in transcription. Identification of these differences in 46 methylation provide likely candidates for regulators responsible for temperature-dependent 47 phenotypes. 48 49 50