Tracing the Evolution of Competence in Haemophilus influenzae Heather Maughan¤*, Rosemary J. Redfield Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada Abstract Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake. Citation: Maughan H, Redfield RJ (2009) Tracing the Evolution of Competence in Haemophilus influenzae. PLoS ONE 4(6): e5854. doi:10.1371/ journal.pone.0005854 Editor: Igor Mokrousov, St. Petersburg Pasteur Institute, Russian Federation Received December 11, 2008; Accepted May 11, 2009; Published June 10, 2009 Copyright: 2009 Maughan, Redfield. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by NIH Kirschstein (5F32GM078861-03) and Killam postdoctoral fellowships to H.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: heather.maughan@utoronto.ca ¤ Current address: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada Introduction Many bacteria develop a physiological state called natural competence, enabling cells to take up DNA from the environment and occasionally recombine it with the chromosome; a cell is said to be transformed if recombination changes its genotype. This genetically programmed process differs from artificially induced competence, where divalent cations or electric fields are used to permeabilize cell membranes. Competence has important impli- cations for the ecology and evolution of bacteria: the nucleotides salvaged from the degradation of incoming DNA provide nutrients, and the alleles introduced by transformation can provide genetic novelty and templates for DNA repair. Because the ability to be take up DNA and become transformed is sporadically distributed at all phylogenetic levels in bacteria, its evolutionary origin(s) are unclear. Many well-studied major clades have both transformable and non-transformable members, a state that is consistent with either a single origin and many losses or multiple independent origins (reviewed in [1,2]. Comparison of the cellular machineries used for DNA uptake does not resolve the issue. Although almost all bacteria that have been studied use a modified type IV pilus system for DNA uptake, which would seem to support a single evolutionary origin [3,4], the type IV pilus machinery also functions to generate forces for adhesion and motility [5–7] and may have been independently co-opted for DNA uptake in different lineages. On a smaller scale but parallel to what is seen in the major clades, all species that have been examined include both transformable and non-transformable isolates [8–14]. This variability between strains may also account for the observed differences in competence at higher phylogenetic levels, as decisions about the competence of bacterial species are custom- arily based on the phenotype of a single strain. Differences between strains of the same species indicate that changes in competence are recent and provide a framework for identifying the microevolutionary changes responsible for the macroevolu- tionary patterns of competence. Haemophilus influenzae is the model organism for studies of competence in the gamma-proteobacteria (e.g. [15]). Although H. influenzae is an obligate commensal of the human respiratory tract, it also causes opportunistic infections in both the respiratory tract and normally sterile sites, especially in infants, the elderly, and individuals with weak immune systems. In previous work we have characterized the variation in competence in H. influenzae by measuring the amount of DNA taken up and recombined by 35 genetically diverse strains [16]. Although most strains took up DNA and were transformed, the amounts of DNA taken up and the frequencies of transformation varied over several orders of PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5854