RT Journal Article SR Electronic T1 Moonlighting proteins activate transformation in epigenetically-differentiated phase variants of multidrug-resistant Streptococcus pneumoniae JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.03.07.483185 DO 10.1101/2022.03.07.483185 A1 Min Jung Kwun A1 Alexandru V. Ion A1 Marco R. Oggioni A1 Stephen D. Bentley A1 Nicholas J. Croucher YR 2022 UL http://biorxiv.org/content/early/2022/04/04/2022.03.07.483185.abstract AB Transformation of Streptococcus pneumoniae GPSC1 has enabled vaccine evasion and the acquisition of antibiotic resistance. Epigenetic phase variants of GPSC1 isolate RMV7, differentiated by rearrangements at the tvr restriction-modification locus, differed ∼100-fold in their transformation efficiency. This variation was recapitulated in knock-in mutants of the relevant tvr alleles. RNA-seq showed the difference was due to blocking of the early competence regulatory cascade. The more transformable variant upregulated expression of manLMN, encoding a carbon source importer. This was shown to be necessary for efficient competence induction, despite being dispensable for growth in rich media. Transformation was promoted by import of N-acetylglucosamine, which activated competence through an orthologue of the gram-negative competence regulator TfoX, and an enzyme likely involved in nucleotide-mediated signalling. A mobile genetic element was more active in the less transformable variant, which limited competence induction through inhibiting CIRCE motif binding by the chaperone regulator HrcA. Correspondingly, both heat shock and decreased Ca2+ concentrations reduced competence by limiting HrcA binding regulatory sequence motifs. Hence both ManLMN and HrcA moonlighted as activators of competence. Such proteins may be important in potentiating horizontal DNA exchange, as selection on their primary function likely constrains them from mutating into alleles that selfishly downregulate transformation.Competing Interest StatementNJC has received an investigator-initiated award from GlaxoSmithKline and consulted for Pfizer.