Adaptive mutation and evolution of the RNA polymerase subunit delta during Streptococcus pneumoniae biofilm development.

Abstract

Streptococcus pneumoniae typically resides asymptomatically within the respiratory tract, sinuses and nasopharynx of healthy carriers. However, for children, the elderly, or immunocompromised individuals, S. pneumoniae is the cause of considerable morbidity and mortality globally. Many of the pathologies caused by this organism are associated with the surface adhered microbial communities known as biofilms. Biofilms of the S. pneumoniae clinical isolate 22F ST433 have been observed to rapidly exhibit parallel evolution with mutations to the RNA polymerase (RNAP) subunit δ gene rpoE repeatedly occurring. All rpoE mutants displayed a Small Colony Variant (SCV) morphology; this phenotype is associated with carriage and disease states in S. pneumoniae, and therefore improved understanding of biofilm formation may be key to reducing the burden of disease of this pathogen. The rpoE mutations within the SCV population are heterogenous, ranging from single nucleotide polymorphisms to large-scale deletions of the C-terminal domain of RpoE. As a result of these mutation SCVs display significant altered phenotypes relative to the 22F wild-type including metabolic changes, reduced virulence and increased biofilm formation. However, these changes are likely to be due to altered RpoE function rather than a complete loss as we also observed that SCVs were phenotypically distinct from a complete rpoE knock-out. Alteration to, rather than loss of RpoE function within SCVs is supported by our observations that for several important characteristics, including biofilm formation, the magnitude of the phenotypic change correlates to the length of the C-terminal domain of RpoE. Phenotypic differences between the 22F wild-type and SCVs are therefore likely to be due to the C-terminal domain’s roles in RNAP-δ-DNA interactions, the positioning of RNAP within gene promoter regions and open complex formation where C-terminal domain length is likely to be an important factor for RpoE function. Differences observed between SCVs and rpoE KO are likely due to the conserved N-terminal domain of the SCVs, which we predict to have DNA binding and enzymatic activity, and the complete loss of which causes dysregulation. These findings further highlight the diversity that can be generated within even single species biofilms and the mechanisms through which S. pneumoniae RpoE functions.

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ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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