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. 2014 Nov 25;3:e03587. doi: 10.7554/eLife.03587

Figure 7. Transcriptional and translational regulation of hvyA depends on PleC-CtrA, SciP, and MucR1/2.

(A) Beta-galactosidase activity of PhvyA-lacZ transcriptional fusion. Left panel: transcription of hvyA is strongly reduced in ctrA401 (T170I, temperature sensitive) and ΔpleC strains compared to WT Caulobacter. Mutation of the kinase DivJ partially restores hvyA transcription in the ΔpleC ΔdivJ strain. Transcription from PhvyA-lacZ is significantly increased in the ΔmucR1/2 mutant. Right panel: beta-galactosidase activity of PhvyA-lacZ in ΔmucR1/2 cells complemented with Caulobacter mucR1 (CC_R1, WT or mutant derivatives Y97C and R85C) or the MucR paralogs from S. fredii NGR234 (Sf_a00320 and Sf_c07580) on plasmid under control of Pvan. Values are expressed as percentages (activity in WT NA1000 or WT carrying the empty vector set at 100%). (B) Beta-galactosidase activity of PhvyA-hvyA::lacZ translational fusion in the same strains shown in panel (A). Translation of hvyA is strongly reduced in cells expressing the ctrA401 allele and in the ΔpleC strain compared to WT Caulobacter. Mutation of DivJ partially restores hvyA translation in the ΔpleC ΔdivJ strain. PhvyA-hvyA::lacZ activity is also significantly decreased in the ΔmucR1/2 and ΔpleC ΔmucR1/2 mutants, consistently with the ‘light’ buoyancy of these strains. PhvyA-hvyA::lacZ activity is restored in the ΔmucR1/2 double mutant carrying ctrA(T170A), sciP(T24I), or sciP(T65A) alleles (ctrA* and sciP*). Values are expressed as percentages (activity in WT NA1000 or WT carrying the empty vector set at 100%). (C) Translational control of hvyA by CtrA and SciP. Beta-galactosidase activity of the PhvyA-hvyA::lacZ fusion in WT Caulobacter cells over-expressing sciP(T65A) or sciP(WT) from Pvan on plasmid. Whereas sciP(T65A) does not affect the activity of the translational PhvyA-hvyA::lacZ fusion, over-expression of sciP(WT) significantly decreases the activity of the fusion, consistently with the model presented in Figure 8. The activity is expressed as percentage of the activity in WT cells carrying the empty vector. Occupancy of CtrA, MucR1, and MucR2 at the hvyA promoter region as determined by ChIP-seq analysis is shown in Figure 7—figure supplement 1. The effect of the CtrA(D51E) allele on PhvyA-lacZ is shown in Figure 7—figure supplement 2. The ability of heterologous MucR to restore PhvyA-lacZ repression or PhvyA-hvyA::lacZ activity in Caulobacter ΔmucR1/2 is reported in Figure 7—figure supplement 3.

DOI: http://dx.doi.org/10.7554/eLife.03587.019

Figure 7.

Figure 7—figure supplement 1. Occupancy at the hvyA promoter region as determined by ChIP-seq analysis.

Figure 7—figure supplement 1.

(A) Traces show occupancy of MucR1 (in blue), MucR2 (in purple), CtrA in WT cells (in green), and CtrA in ΔpleC cells (in red). In the absence of PleC, the occupancy of the hvyA promoter by CtrA is significantly reduced. (B) Same as panel A, but at a higher magnification to show the CtrA and MucR2 peaks that are otherwise masked by the MucR1 peak.
Figure 7—figure supplement 2. Transcriptional control of hvyA by PleC-CtrA.

Figure 7—figure supplement 2.

Beta-galactosidase activity of the PhvyA-lacZ transcriptional fusion in WT and ΔpleC cells harbouring ctrA(WT) or the phosphomimetic ctrA(D51E) allele under control of Pvan on plasmid. The assay shows that the phosphomimetic ctrA(D51E) allele is unable to restore hvyA transcription in ΔpleC cells. The activity is expressed as percentage of the activity in WT cells carrying the empty vector.
Figure 7—figure supplement 3. Transcriptional and translational control of hvyA by heterologous MucRs.

Figure 7—figure supplement 3.

Beta-galactosidase activity of the PhvyA-lacZ (transcriptional) and PhvyA-hvyA::lacZ (translational) fusions in ΔmucR1/2 cells harbouring heterologous mucR genes under control of Pvan on plasmid. Cc_R1, Caulobacter mucR1 (WT or mutant alleles); Cc_R1 long, original annotation of Cc_R1 (CCNA_00982); Sf_a00320, S. fredii NGR_a00320; Sf_c07580, S. fredii NGR_c07580; At_ROS, A. tumefaciens mucR paralog; Bs_mucR, B. suis mucR paralog; Bh_mucR, B. henseleae mucR paralog. The assay shows that in the presence of a heterologous MucR increased translational activity of PhvyA-hvyA::lacZ is always accompanied with a commensurate repression of the transcriptional PhvyA-lacZ. The activity is expressed as percentage of the activity in WT cells carrying the empty vector.