Effector-mediated interaction of CbbRI and CbbRII regulators with target sequences in Rhodobacter capsulatus

Abstract

In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbb(I) and cbb(II) operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbR(I) and CbbR(II)) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbR(I) and CbbR(II) were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbR(I) and CbbR(II) to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbR(I) and CbbR(II) for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH(2)PO(4) were found to enhance only CbbR(I) binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbR(II). The DNase I footprint of CbbR(I) was reduced in the presence of RuBP, while reductions in the CbbR(II) DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH(2)PO(4). The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell.

FIG. 1.

Gene organization of the R. capsulatus cbb_I and cbb_II operons. Gene designations: cbbR, positive transcriptional regulator; cbbL, large subunit, form I RubisCO; cbbS, small subunit, form I RubisCO; cbbQ and cbbO, unknown function; cbbF, fructose-1,6/sedoheptulose-1,7-bisphosphatase; cbbP, phosphoribulokinase; cbbT, transketolase; cbbG, glyceraldehyde 3-phosphate dehydrogenase; cbbA, fructose-1,6-bisphosphate aldolase; cbbM, large subunit, form II RubisCO; cbbE, ribulose-5-phosphate epimerase; cbbZ, 2-phosphoglycolate phosphatase; cbbY, unknown function. The direction of transcription and the extent of potential transcripts are indicated by arrows.

FIG. 2.

Binding of CbbR_I and CbbR_II to their cognate promoters in the presence of various metabolites. A phosphorimage of a gel mobility shift assay is shown with CbbR_I (0.08 nmol) and a cbb_I probe (A) and CbbR_II (0.71 nmol) and a cbb_II probe (B). Lane 1, probe only; lane 2, CbbR and probe with no metabolite added; lane 3, 2-phosphoglycerate; lane 4, RuBP; lane 5, 2-phosphoglycolate; lane 6, 3-phosphoglycerate; lane 7, phosphoenolpyruvate; lane 8, NADPH; lane 9, NADH; lane 10, ATP; lane 11, fructose-6-phosphate; lane 12, fructose-1,6-bisphosphate; lane 13, ribose-5- phosphate; lane 14, KH₂PO₄. All metabolites were present at a concentration of 1 mM. All reactions contained 3.7 μg of poly(dI-dC)::poly(dI-dC) and 15,000 cpm of ³²P-labeled probe. Arrows indicate unbound probe (U) and shifted protein-DNA complexes.

FIG. 3.

Concentration dependence of RuBP on the binding of CbbR_I and CbbR_II to their cognate promoters. Phosphorimage of gel mobility shift assays are shown with CbbR_I (0.08 nmol) and a cbb_I probe (A) and CbbR_II (0.71 nmol) and a cbb_II probe (B). Lane 1, CbbR with no metabolite added; lane 2, 1 μM RuBP; lane 3, 10 μM RuBP; lane 4, 100 μM RuBP; lane 5, 1.0 mM RuBP; lane 6, probe only. All reactions contained 3.7 μg of poly(dI-dC)::poly(dI-dC) and 15,000 cpm of ³²P-labeled probe. Arrows indicate unbound probe (U) and shifted protein-DNA complexes.

FIG. 4.

Effect of RuBP on the DNase I-protected region of CbbR_I binding to the cbb_I promoter. The phosphorimage of a DNase I footprint is shown. The probe fragment used spans nucleotides −156 to +58 relative to the cbbL transcription start and is labeled on the top (A) and bottom (B) strands. Brackets indicate regions of protection, and asterisks indicate DNase I-hypersensitive sites. A control lane to which no CbbR_I was added to the binding reaction is shown along with a lane containing a standard Maxim-Gilbert A+G sequencing ladder of the probe. The concentration of RuBP present in each reaction is indicated. Unless otherwise indicated, all reactions contained 88 nM CbbR_I.

FIG. 5.

Effect of fructose-1,6-bisphosphate on the DNase I-protected region caused by CbbR_II binding to the cbb_II promoter. The phosphorimage of a DNase I footprint is shown. The probe fragment used spans nucleotides −151 to +46 relative to the cbbF transcription start for the top strand (A) and −151 to +79 on the bottom strand (B) is labeled on the top and bottom strands. Brackets indicate regions of protection, and asterisks indicate DNase I-hypersensitive sites. A control lane that does not contain CbbR_II is shown along with a lane containing a standard Maxim-Gilbert A+G sequencing ladder of the probe. The concentration of fructose-1,6-bisphosphate present in each reaction is indicated. Unless otherwise indicated, all reactions contained 3.7 μM CbbR_II.

FIG. 6.

Effect of 3-phosphoglycerate and phosphoenolpyruvate on the pattern of DNase I protection caused by CbbR_II binding to the cbb_II promoter. The phosphorimage of a DNase I footprint of CbbR_I binding to a cbb_II promoter probe fragment alone (A) and in the presence of 1 mM phosphoenolpyruvate (PEP) (B) or 1 mM 3-phosphoglycerate (PGA) (C). The probe is labeled on the top strand and spans nucleotides −151 to +46 relative to the cbbF transcription start. Brackets indicate regions of protection, and asterisks indicate DNase I hypersensitive sites. The concentration of CbbR_II in each reaction is indicated above each lane. A standard lane containing a Maxim-Gilbert A+G sequencing ladder of the probe is also shown.

FIG. 7.

Summary and model of DNase I footprinting results for CbbR_I binding to the cbb_I promoter in the presence and absence of the effector RuBP (A) and CbbR_II binding to the cbb_II promoter in the presence and absence of fructose-1,6-bisphosphate (B). Bars indicate regions of protection on the top (upper) and bottom (lower) DNA strands in the presence and absence of effector. The black portion of each bar represents the region of protection in the presence of effector. Asterisks indicate DNase I-hypersensitive sites. Asterisks in boxes indicate hypersensitive sites that disappear or diminish in intensity in the presence of effector. Circled asterisks indicate hypersensitive sites that appear only in the presence of effector. Arrows indicate conserved inverted repeat (IR) sequences. A and T residues within the LysR consensus binding motif (T-N₁₁-A) sequences are in bold italics.