Extended Data Fig. 8: Signaling mediated by c-di-GMP in E. coli biofilm.

Signaling mediated by c-di-GMP in E. coli biofilm. (a) Differential expression of genes encoding c-di-GMP processing enzymes in biofilm relative to planktonic bacteria; Shown is log₂(fold change) between the maximum in biofilm (n = 24) and the maximum in planktonic culture (n = 14). (b) Bulk profiling cannot identify the local activation of c-di-GMP processing genes in biofilm. Shown is the log₂(fold change) of genes encoding c-di-GMP processing enzymes in biofilm relative to planktonic lifestyle; a computationally synthetic biofilm transcriptome by averaging all 24 spatially resolved samples was generated to mimic a bulk biofilm sample, which was compared with the maximum from planktonic culture (n = 14). (c) Regulatory logic of PdeR/DgcM cascade via local c-di-GMP signaling to modulate MlrA activity, which drives the transcription of csgD; note that PdeR inhibits MlrA, either directly or indirectly via DgcM; and DgcM activates MlrA, either directly or indirectly via PdeR. (d) Gene expression levels of csgD at the interior of biofilm (n = 5) and the stationary phase of planktonic culture (n = 2). Data are presented as mean ± std (P = 0.0009, two tailed Mann-Whitley U test). (e) Different responses of DGCs and PDEs between biofilm and planktonic culture when stepping into famine from feast; x axis presents the log₂(fold change) between famine (interior) and feast (periphery) in biofilm; y axis presents the log₂(fold change) between famine (stationary stage) and feast (exponential stage) in planktonic culture; blue dots are genes encoding DGCs and red for PDEs; diagonal line highlights the equivalent feast-to-famine expression change between biofilm and planktonic culture; the region between y = x + 1 and y = x − 1 is shaded to highlight those outlier genes; these genes exhibit different expression change from feast to famine between biofilm and planktonic culture. (f) Above are the spatial expression profiles (log₂(TPM + 1)) of dgcM and pdeR in biofilm; below are the temporal profiles of these two genes in planktonic culture. See ‘Signaling mediated by AI-2 QS and c-di-GMP in biofilms uncovered by RAINBOW-seq’ subsection in Methods for more discussions about c-di-GMP signaling in biofilm learned from RAINBOW-seq data.

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