Crl activates the transcription of many RpoS-dependent promoters including the rssB promoter.

A) Plasmid maps for construction of transcriptional fusions. The empty vector pQE80L was used as the backbone to construct the pSB19 plasmid by Gibson assembly, replacing the Lac promoter/operator and the RBS by the coding sequence of mCherry following a consensus RBS sequence (see dotted lines for both pQE80L and pSB19). The promoter regions of yodD, osmY, gadB and osmE or the 1658 bp upstream of rssB were then introduced into pSB19, obtaining the plasmids pSB21, pSB22, pSB23, pSB38 and pSB37 respectively. B) The activity of the transcriptional mCherry fusions of the RpoS-dependent promoters of yodD (pSB21, top left panel), osmY (pSB22, top right panel), gadB (pSB23, bottom left panel) and osmE (pSB38, bottom right panel) in the WT, ΔrpoS (AB165), Δcrl (INH24), iraP::kan (AB006) and iraP::kan Δcrl (INH26) strains. Strains were grown from exponential to stationary phase in a microplate reader in MOPS minimal medium that measured mCherry fluorescence and OD600 every 20 minutes. The transcriptional fusion construct is shown above each graph (see Material and methods for details). C) Comparison of relative average expression of the transcriptional fusions of the four RpoS-dependent promoters to WT, after 16 hours of growth, from data as in S4B (n > 3). D) Relative expression of the four RpoS-dependent transcriptional fusions in the ΔrpoS (AB165), Δcrl (SB147), ΔiraP (SB151) and ΔiraP Δcrl (INH26) strains, with WT (MG1655) set to 100, during stationary phase. E) Comparison of the activity of the gadB promoter fused to mCherry, expressed either from the pQE80L-derived plasmid pSB23 or from the same fusion expressed as a single chromosomal copy, in WT (MG1655; blue symbols) and ΔrpoS (AB165; red symbols) strains. Strains were grown and the RFU measured as described in Fig 4A. F) Fluorescence over time of the translational fusion contained on pSB37, carrying the upstream region of rssB fused to mCherry, as shown in S4 Fig. The fusion extends 1597bp upstream of the rssB coding gene, including the P1 and P2 promoters, as shown in S3A and S4A Figs. The strains used in S4B Fig were transformed with the plasmid containing the rssB fusion and followed during growth.

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