Pneumococcal Transformation: Mutations in the Primary Sigma Factor Bypass the Critical ComW Requirement
thesisposted on 2016-07-01, 00:00 authored by Yanina Tovpeko
Streptococcus pneumoniae is an opportunistic pathogen that resides in the human nasopharynx and is naturally transformable, or able to take up and integrate exogenous DNA into its genome. Competence for genetic transformation is tightly regulated, transient, and occurs in two phases, early and late. The early genes encode a quorum-sensing system and peptide pheromone that signal an entire population to become competent in synchrony. One early gene encodes σX, the only known streptococcal alternative σ factor, which is responsible for coordinated synthesis of the late genes, those which are necessary for DNA uptake and recombination. In S. pneumoniae, elevated σX is insufficient for development of full competence without co-expression of a second competence-specific protein, ComW, which is regulated by the same pheromone circuit that controls σX. comW mutants display several phenotypes, a 104-fold reduction in the amount of transformants, a 10-fold reduction in σX activity, and a 10-fold reduction in the amount of σX protein. To identify proteins that may be interacting with ComW during competence, a suppressor screen was performed seeking mutants that were partially restored for transformation in the ΔcomW mutant background. Whole genome sequencing of suppressor strains revealed ten different single-base substitutions in rpoD, the gene encoding the primary σ factor, σA, that each bypass the ComW requirement for transformation. Late gene expression was also restored in ΔcomW, σA mutants. Eight of the ten single-base substitutions mapped to residues previously implicated in σA binding affinity to core RNA polymerase. Together, these data suggest that ComW increases σX access to core RNA polymerase, pointing to a role for ComW in σ factor exchange during genetic transformation in S. pneumoniae.