Two functionally distinct regions upstream of the cbbI operon of Rhodobacter sphaeroides regulate gene expression

Abstract

A number of cbbFI::lacZ translational fusion plasmids containing various lengths of sequence 5' to the form I (cbbI) Calvin-Benson-Bassham cycle operon (cbbFIcbbPIcbbAIcbbLIcbbSI) of Rhodobacter sphaeroides were constructed. Expression of beta-galactosidase was monitored under a variety of growth conditions. It was found that 103 bp of sequence upstream of the cbbFI transcription start was sufficient to confer low levels of regulated cbbI promoter expression; this activity was dependent on the presence of an intact cbbR gene. Additionally, R. sphaeroides CbbR was shown to bind to the region between 9 and 100 bp 5' to the cbbFI transcription start. Inclusion of an additional upstream sequence, from 280 to 636 bp 5' to cbbFI, resulted in a significant increase in regulated cbbI promoter expression under all growth conditions tested. A 50-bp region responsible for the majority of this increase occurs between 280 and 330 bp 5' to cbbFI. The additional 306 bp of upstream sequence from 330 to 636 bp also appears to play a positive regulatory role. A 4-bp deletion 281 to 284 bp 5' to cbbFI significantly reduced cbbI expression while the proper regulatory pattern was retained. These studies provide evidence for the presence of two functionally distinct regions of the cbbI promoter, with the distal domain providing significant regulated promoter activity that adheres to the normal pattern of expression.

FIG. 1

Nucleotide sequence of the region used for construction of cbbF_I::lacZ translational fusion plasmid pVKC1. (A) The nucleotide sequence of the 718-bp AvaII fragment was used to construct plasmid pVKC1, spanning 571 bp of the cbbR gene, the cbbR-cbbF_I intergenic region, and 57 bp of cbbF_I. Potential cbbF_I mRNA start sites at +1 and +9 bp, as well as the +4-bp start site determined for the LacZ fusion pVKC1, are indicated by vertical arrows and are described in the text. The sequence is numbered relative to the distal mRNA start at +1 bp. Two imperfect inverted repeat sequences containing LysR-type binding motifs (T-N₁₁-A) are indicated. (B) Primer extension mapping of the R. sphaeroides cbbF_I transcript 5′ endpoint(s). Shown are results of primer extension experiments with total RNA extracted from photoautotrophically grown R. sphaeroides HR (lane 1) and a synthetic oligonucleotide of the sequence 5′-GTATGGCATCGGGGTGGGTG-3′ (cbbF1-ex). The extension products are sized against a sequencing ladder with the same oligonucleotide primer and plasmid p12EH as template. The initiation points of the extension products are indicated by arrows.

FIG. 1

Nucleotide sequence of the region used for construction of cbbF_I::lacZ translational fusion plasmid pVKC1. (A) The nucleotide sequence of the 718-bp AvaII fragment was used to construct plasmid pVKC1, spanning 571 bp of the cbbR gene, the cbbR-cbbF_I intergenic region, and 57 bp of cbbF_I. Potential cbbF_I mRNA start sites at +1 and +9 bp, as well as the +4-bp start site determined for the LacZ fusion pVKC1, are indicated by vertical arrows and are described in the text. The sequence is numbered relative to the distal mRNA start at +1 bp. Two imperfect inverted repeat sequences containing LysR-type binding motifs (T-N₁₁-A) are indicated. (B) Primer extension mapping of the R. sphaeroides cbbF_I transcript 5′ endpoint(s). Shown are results of primer extension experiments with total RNA extracted from photoautotrophically grown R. sphaeroides HR (lane 1) and a synthetic oligonucleotide of the sequence 5′-GTATGGCATCGGGGTGGGTG-3′ (cbbF1-ex). The extension products are sized against a sequencing ladder with the same oligonucleotide primer and plasmid p12EH as template. The initiation points of the extension products are indicated by arrows.

FIG. 2

β-Galactosidase and RubisCO assay results for R. sphaeroides HR::lacZ::cbbF_I translational fusion strains. Maps depicting the sequences used to construct each fusion plasmid are shown. Multiple assays of several independent growth experiments were performed, and the standard deviations for each determination are denoted by the line above each bar. The average value for these determinations is also shown above each bar. The β-galactosidase (A, C, and E) and RubisCO (B, D, and F) activities for R. sphaeroides HR containing various cbbF_I::lacZ translational fusion plasmids grown under chemoheterotrophic (A and B), photoheterotrophic (C and D), and photoautotrophic (E and F) conditions are shown. The average β-galactosidase activities (two independent experiments) for the negative control strain HR::pVK1403 were 0.0 nmol/min/mg under chemoheterotrophic and photoheterotrophic growth conditions and 0.5 nmol/min/mg under photoautotrophic growth conditions. The average RubisCO activities for HR::pVK1403 were 1.0, 38, and 295 nmol/min/mg under chemoheterotrophic, photoheterotrophic, and photoautotrophic growth conditions, respectively.

FIG. 3

Binding of CbbR to the cbb_I promoter. Shown is the phosphorimage of a gel mobility shift assay (B) with the 190-bp BssHII-BamHI fragment (A) of pKC1-5 as a probe and extracts (2 μg) of BL21(DE3) containing either pET11R-11 (11R) or pET11a (11A). Lane 1, no extract added; lane 2, 11R plus 5 min of incubation at 100°C; lane 3, 11R plus proteinase K; lane 4, 11R; lane 5, 11R plus 100-fold molar excess of unlabeled probe; lane 6, 11R plus 200-fold excess of salmon sperm DNA; lane 7, 11R plus 100-fold molar excess of the 92-bp BssHII-SacII fragment; lane 8, 11R plus 100-fold molar excess of the 98-bp SacII-BamHI fragment; lane 9, 11A. All binding reaction mixtures contained 3 μg of poly(dI-dC) · poly(dI-dC). The arrow indicates the position of the unbound DNA (B). Putative CbbR binding sites (>>---<<) and transcription start sites (->) are indicated (A). The phosphorimage was generated with a Storm 840 PhosphorImager in conjunction with ImageQuant version 4.2 software (Molecular Dynamics, Inc.).

FIG. 4

Comparison of the cbbF_I-cbbR intergenic region of R. sphaeroides with that of the X. flavus cbb operon. Vertical arrows represent mapped putative transcription start sites. Vertical lines represent nucleotides conserved between the two sequences. Putative CbbR binding sites (>>---<<) in the R. sphaeroides sequence are indicated. The underlined sequence is the site of CbbR binding to the X. flavus cbbL promoter. Hyphens indicate gaps inserted to maximize sequence identity. Bold-face italic sequences are mentioned in the text. Numbers refer to the distance upstream from the transcription start of R. sphaeroides cbbF_I.