Abstract
In a previous study (Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000) J. Biol. Chem. 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbb(I)) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides. To determine how these regulators interact with the form II (cbb(II)) promoter, three cbbF(II)::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbb(II) operon of R. sphaeroides CAC. Expression of beta-galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA. The binding sites for both CbbR and RegA were determined by DNase I footprinting. A region of the cbb(II) promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbb(II) expression. The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbb(II) expression under all growth conditions. Unlike the cbb(I) operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were required for maximal cbb(II) expression. Two potentially novel and specific cbb(II) promoter-binding proteins that did not interact with the cbb(I) promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of the cbb(I) operon is RegA independent, indicated that the mechanisms controlling cbb(I) and cbb(II) operon expression during chemoautotrophic growth are quite different.
Highlights
Sole carbon source, and the CBB cycle is the primary source for most of the fixed carbon utilized by the cell
Regulation of cbbII Operon Gene Expression two-component regulatory system was implicated in cbb regulation by genetic studies that demonstrated that a R. sphaeroides regB insertion mutant exhibited reduced cbbI and cbbII expression during photoautotrophic growth in a 1.5% CO2/ 98.5% H2 atmosphere [17]
Previous studies [25] using transcriptional fusions to xylE determined that the cbbII promoter was located within 991 bp of the cbbFII translation start. It was with this in mind that we constructed cbbFII::lacZ translational fusion plasmids containing 1025 bp, 545 bp, and 227 bp of sequence upstream of the cbbFII transcription start (Fig. 1). These plasmids were transferred into R. sphaeroides CAC, a spontaneous gain of function mutant of R. sphaeroides HR that has the ability to grow chemoautotrophically in the presence of 10% O2 concentrations [6]
Summary
Bacterial Strains, Plasmids, and Culture Conditions—R. sphaeroides strains CAC [6], and CAC::regA⍀ [17] (Table I) were grown photoautotrophically under a gas atmosphere of 1.5% CO2/98.5% H2 and chemoautotrophically under a gas atmosphere of 5% CO2/45% H2/50% air as described previously [11, 13]. Gel mobility shift probes were generated by either filling in restriction fragment overhanging ends with DNA polymerase I Klenow fragment in the presence of [␣-32P]dCTP and dNTPs or PCR amplification using [␥-32P] end-labeled oligonucleotides as described earlier for DNaseI footprint analysis. A 606-bp EcoRI/BglII fragment of pKII⌬Xho was ligated into EcoRI/BamHI-digested pMC1403 to generate pMCCIIXho. EcoRI-digested pMCCIIXho was ligated into the EcoRI site of the conjugative plasmid pVK101 to yield pVKCIIXho. The method used for the construction of pVKCIIXcm was the same as that for pVKCIIXho except that XcmI was used in place of XhoI to generate plasmids pKII⌬Xcm and pMCCIIXcm. The method used for the construction of pVKCIIXcm was the same as that for pVKCIIXho except that XcmI was used in place of XhoI to generate plasmids pKII⌬Xcm and pMCCIIXcm This resulted in a lacZ fusion containing 227 bp upstream of the cbbII transcription start
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