Metabolic signals that lead to control of CBB gene expression in Rhodobacter capsulatus

Abstract

Various mutant strains were used to examine the regulation and metabolic control of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus. Previously, a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient strain (strain SBI/II) was found to show enhanced levels of cbb(I) and cbb(II) promoter activities during photoheterotrophic growth in the presence of dimethyl sulfoxide. With this strain as the starting point, additional mutations were made in genes encoding phosphoribulokinase and transketolase and in the gene encoding the LysR-type transcriptional activator, CbbR(II). These strains revealed that a product generated by phosphoribulokinase was involved in control of CbbR-mediated cbb gene expression in SBI/II. Additionally, heterologous expression experiments indicated that Rhodobacter sphaeroides CbbR responded to the same metabolic signal in R. capsulatus SBI/II and mutant strain backgrounds.

FIG. 1.

cbb_I and cbb_II gene clusters of R. capsulatus. The sites of gene disruptions in the mutant strains are indicated. Solid arrows indicate direction of transcription; dotted arrows indicate orientation of the gentamicin resistance cassette used to inactivate the respective genes. The genes encoding the enzymes of the CBB pathway in R. capsulatus are organized in two operons, each preceded by a separate cbbR gene (encoding a LysR-type transcriptional regulator). cbbL cbbS encodes form I RubisCO, while cbbM encodes form II RubisCO. cbbM is clustered with genes encoding other enzymes of the CBB pathway: cbbF (fructose 1,6/sedoheptulose 1,7-bisphosphatase), cbbP (phosphoribulokinase), cbbT (transketolase), cbbG (glyceraldehydes-3-phosphate dehydrogenase), cbbA (fructose 1,6-bisphosphate aldolase), and cbbE (epimerase). The cbb genes are oriented with respect to other familiar genes in this organism: pgm, phosphoglucomutase; qor, quinol oxidoreductase; anfA, regulator for the alternative nitrogenase genes.

FIG. 2.

Immunoblot analysis and levels of RubisCO and PRK activities in strain SBI/P during photoheterotrophic growth. Antibodies raised against form I Synechococcus 6301 RubisCO (A) and R. sphaeroides form II RubisCO (B) were utilized. Approximately 5 μg of protein was added per lane (A and B). Levels of RubisCO activity (below the lanes) and PRK activity (below the lanes, in parentheses) (both in nanomoles per minute per milligram) were determined from three independent cultures grown photoautotrophically (1.5%CO₂-98.5% H₂) (lane 1) or photoheterotrophically in the presence of DMSO (lanes 3 to 5). Lanes 1, extract from wild-type strain SB1003; lanes 2, purified Synechococcus sp. strain 6301 RubisCO (A) and R. sphaeroides form II RubisCO (B); lanes 3, extract from SB1003; lanes 4, extract from SBI; lanes 5, extract from SBI/P. ND, not determined.

FIG. 3.

cbb_I::lacZ (A) and cbb_II::lacZ (B) promoter activity during photoheterotrophic growth of R. capsulatus strains. β-Galactosidase activities were determined from three to nine independent cultures assayed in duplicate. NG, no growth during photoheterotrophic conditions with ammonia as the nitrogen source in the absence of DMSO.

FIG. 4.

Promoter activity using an R. sphaeroides cbbR-cbb_I::lacZ promoter fusion (pVKD1) (9) in R. capsulatus strains during photoheterotrophic growth. β-Galactosidase activities were determined from three independent cultures assayed in duplicate. NG, no growth in the absence of an alternative electron acceptor.