title: Parallel Evolution in Streptococcus pneumoniae Biofilms.
abstract: Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.
authors.affiliation: Centre for Biological Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom Institute for Life Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom.
authors.forename: Nicholas W V
authors.initials: NW
authors.lastname: Churton
authors.affiliation: Genomics Research Unit, Microbiology Services, Public Health England, Colindale, United Kingdom.
authors.forename: Raju V
authors.initials: RV
authors.lastname: Misra
authors.affiliation: Centre for Biological Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom Institute for Life Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom NIHR Southampton Respiratory Biomedical Research Unit, Southampton, United Kingdom.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Howlin
authors.affiliation: Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom Institute for Life Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom NIHR Southampton Respiratory Biomedical Research Unit, Southampton, United Kingdom Southampton NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton NHS Foundation Trust, United Kingdom.
authors.forename: Raymond N
authors.initials: RN
authors.lastname: Allan
authors.affiliation: Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom Institute for Life Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom NIHR Southampton Respiratory Biomedical Research Unit, Southampton, United Kingdom.
authors.forename: Johanna
authors.initials: J
authors.lastname: Jefferies
authors.affiliation: Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom NIHR Southampton Respiratory Biomedical Research Unit, Southampton, United Kingdom Southampton NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton NHS Foundation Trust, United Kingdom.
authors.forename: Saul N
authors.initials: SN
authors.lastname: Faust
authors.affiliation: Genomics Research Unit, Microbiology Services, Public Health England, Colindale, United Kingdom.
authors.forename: Saheer E
authors.initials: SE
authors.lastname: Gharbia
authors.affiliation: Centre for Biological Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom Institute for Life Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Edwards
authors.affiliation: Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom Institute for Life Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom NIHR Southampton Respiratory Biomedical Research Unit, Southampton, United Kingdom Public Health England, Southampton, United Kingdom.
authors.forename: Stuart C
authors.initials: SC
authors.lastname: Clarke
authors.affiliation: Centre for Biological Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom Institute for Life Sciences, Faculty of Natural and Environmental Sciences, University of Southampton, United Kingdom NIHR Southampton Respiratory Biomedical Research Unit, Southampton, United Kingdom j.s.webb@soton.ac.uk.
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Webb
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D018441:Biofilms / Q000254:growth & development*; D005075:Biological Evolution; D016680:Genome, Bacterial; D006801:Humans; D009154:Mutation / Q000235:genetics; D011008:Pneumococcal Infections / Q000382:microbiology*; D013296:Streptococcus pneumoniae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins
keywords: Streptococcus pneumoniae; biofilm; genomic diversification; parallel evolution