Moonlighting proteins activate transformation in multidrug-resistant Streptococcus 1 pneumoniae epigenetic phase variants

Transformation of Streptococcus pneumoniae GPSC1 has enabled vaccine evasion and the 16 acquisition of antibiotic resistance. Epigenetic phase variants of GPSC1 isolate RMV7, 17 resulting from rearrangements at the tvr restriction-modification locus, differed ~100-fold in 18 their transformation efficiency. This variation was recapitulated in knock-in mutants of the 19 relevant tvr alleles. RNA-seq showed the difference was due to blocking of the early 20 competence regulatory cascade. The more transformable variant more highly expressed 21 manLMN , encoding a carbon source importer. This was shown to be necessary for efficient 22 competence induction, despite being dispensable for growth in rich media. Transformation 23 was promoted by import of N -acetylglucosamine, which activated competence through an 24 orthologue of the gram-negative competence regulator TfoX, and an enzyme likely involved 25 in nucleotide-mediated signalling. The less transformable variant more highly expressed 26 mobile element genes, causing a protein misfolding stress response mediated by HrcA. This 27 chaperone regulator caused the decrease in transformation efficiency associated with heat 28 shock, and the increase associated with elevated Ca 2+ concentrations. Hence both ManLMN 29 and HrcA moonlighted as activators of competence. Such proteins, conserved by selection 30 on their primary function, are five

chromosomal island (PRCI; also known as a phage-inducible chromosomal island, or PICI) 11 integrated adjacent to dnaN (PRCIdnaN; Fig. S23; Table S3). This is one of two PRCIs 12 associated with GPSC1 [7,28], the other being integrated near uvrA (Fig. 2). The regulatory 13 mechanisms of these elements are not thoroughly characterised [50], and in the absence of mutation had no significant effect in RMV7wt (Fig S25). This suggests HtrA inhibits induction 36 of competence, but is unlikely to explain much of the difference between these variants.

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 7, 2022.

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We tested whether analogues of either of these pathways existed in S. pneumoniae.

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 7, 2022. ; https://doi.org/10.1101/2022.03.07.483185 doi: bioRxiv preprint pneumoniae ATCC 700669 [68]. In RMV7, this gene (tfoXSpn) is conserved in the same 3 position, two genes upstream of the comEA competence operon (Fig. S28). The amino acid 4 sequence corresponded to only the N-terminal region of the gram-negative orthologue, and 5 was predicted to form a four-strand beta sheet flanked by alpha helices (Fig. S29). The gene 6 could be both disrupted, and restored, in RMV7rare. Transformation assays using a panel of 7 sugars demonstrated the elevated induction of competence by GlcNAc in this variant was 8 dependent on TfoXSpn (Fig. 3C).

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A gene encoding a candidate adenylate cyclase, yjbK, was also identified in RMV7. This 11 protein is predicted to have a β-barrel structure, as observed for orthologous enzymes 12 synthesising 3',5'-cAMP (Fig. S30). The yjbK gene could be both disrupted, and restored, in

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To test whether TfoX and YjbK had a similar role in mediating the larger response to GlcNAc 24 in RMV7wt, both were also knocked out in this background. However, the results showed a 25 different pattern (Fig. 3F). The disruption of tfoX had no effect on GlcNAc's ability to induce 26 competence, whereas RMV7wt yjbK::Janus failed to activate competence unless GlcNAc or 27 sialic acid were supplemented in the media. These results were consistent with ManLMN 28 affecting competence through at least two pathways, one of which is dependent upon TfoX 29 and YjbK. Disruption of these genes in the laboratory strain R6x was also found to reduce 30 transformation efficiency, although detecting these effects required culturing in a low-sugar  CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

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To test whether any other part of the PRCI might inhibit transformation, the entire element 9 was removed, either with (RMV7wt PRCIdnaN+att::Janus) or without (RMV7wt PRCIdnaN::Janus) the flanking att sites. In both cases, a ~5-fold increase in transformation rates was observed 11 (Fig. 4A). This implied the PRCI encoded an activity that inhibited the activation of the 12 competence system. To identify where this was located within the MGE, four large mutations 13 were generated within the PRCI: one removing the regulatory genes; one removing the 14 regulatory genes and IONPJBJN_00496; one removing the replication genes; and one 15 removing both the regulatory and replication genes. However, none of these mutations had 16 such a large effect on transformation rates as the elimination of the entire element (Fig. 4A).
This suggested the inhibition of competence induction was not the consequence of a single gene product, but instead the activity of the MGE itself.

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A qRT-PCR assay was employed to test whether activation of PRCIdnaN could affect 21 transformation through upregulating other stress signals within the cell. This found neither ciaR nor manL expression was altered when the PRCI was removed (Fig. 4C). However, 23 transcription of the chaperone regulator hrcA was halved in the absence of the MGE, 24 consistent with the higher expression of this gene in RMV7domi pre-CSP (Table S3) Disruption of hrcA was confirmed to relieve repression of dnaK and groL expression through had a lower transformation efficiency than either RMV7rare, or RMV7rare hrcA restored (Fig.   7   5B). This difference grew as the Ca 2+ concentration increased, consistent with HrcA-CIRCE 8 binding increasing transformation efficiency. These effects could be reproduced in S.

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pneumoniae R6x grown in a chemically-defined medium (Fig. S32). Unfortunately, the 10 essential nature of the chaperones regulated by HrcA prevented any further identification of 11 the specific mechanism by which competence was inhibited.

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Replicating previous observations, a 40 °C heat shock reduced the transformation efficiency

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To check that this regulation was independent of the ManLMN-mediated effects on 23 competence, the transformation efficiency of the RMV7rare hrcA::Janus manLMN::cat double 24 mutant was compared to that of the progenitor genotype, and the single mutants (Fig. 5D).

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This demonstrated an approximately five-fold decrease in transformation for each single 26 mutant, and a ~25-fold reduction for the double mutant. This is consistent with HrcA and 27 ManLMN both activating competence independently.

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The transformation efficiency of S. pneumoniae is highly heterogeneous across populations (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

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This protein may be considered a candidate for generating an alternative signalling 27 molecule.

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It seems likely there is at least one more mechanism by which GlcNAc and ManLMN affect   rich media (Fig. 3), and the chaperone regulator was not necessary to enable competence at 28 higher temperatures (Fig. 5).

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Their activation of competence may instead be a consequence of gene-level selection.
to be altered by transformation, even if they are affected by homologous recombination. If 1 synchronising competence across populations is important, then conserved regulators 2 (ManLMN and HrcA in S. pneumoniae; the Catabolite Repression Protein in H. influenzae) 3 may acquire secondary competence-promoting activities. This is because purifying selection 4 for their primary function will limit population-wide sequence diversity, meaning they

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 7, 2022. ; https://doi.org/10.1101/2022.03.07.483185 doi: bioRxiv preprint Cell culture and mutagenesis 3 Genotypes used in this study are described in Table S5. Unless otherwise stated, 4 encapsulated S. pneumoniae were cultured statically at 35 °C with 5% CO2 in 10 mL of a 5 mixed liquid media, consisting of a 2:3 ratio of Todd-Hewitt media with 0.5% yeast extract 6 (Sigma-Aldrich), and Brain-Heart Infusion media (Sigma-Aldrich). Transformation

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 7, 2022. significance of the comparison between variants from the same day of the passage, as 28 calculated using a Wilcoxon rank sum test. Across all panels, significance is coded as: p < 29 0.05, *; p < 0.01, **; p < 10 -3 , ***; p < 10 -4 , ****. All p values were subject to a Holm-

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Bonferroni correction within each panel.

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is     Relative abundance Genotype RMV7 wt RMV7 wt PRCI d naN +at t :: Janus C . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 7, 2022. ; https://doi.org/10.1101/2022.03.07.483185 doi: bioRxiv preprint quantified by qRT-PCR, in the genotypes assayed in panel B. IONPJBJN_00507 is a coding 1 sequence within the PRCI that is absent from RMV7wt PRCIdnaN+att::Janus. The six points for 2 each gene correspond to three technical replicate assays on each of two biological 3 replicates. The horizontal line on the violin plot shows the median relative abundance for 4 each gene in each genotype. Across all panels, significance is coded as: p < 0.05, *; p < 5 0.01, **; p < 10 -3 , ***; p < 10 -4 , ****. All p values were subject to a Holm-Bonferroni correction 6 within each panel.   (CRP), which is activated by 3',5'-cAMP, generated by the CyaA adenylate cyclase under 10 carbon source starvation conditions. Hence there are parallels with the TfoX orthologue, and 11 adenylate cyclase-like protein YjbK, responding to GlcNAc in S. pneumoniae RMV7. GlcNAc 12 also appears to promote competence through a TfoX/YjbK-independent route, based on the 13 behaviour of RMV7wt. There are no such parallels for the regulation of competence by HrcA, 14 the binding of which to CIRCE sequences is promoted by Ca 2+ , thereby activating