Differential Accumulation of Form I RubisCO in Rhodopseudomonas palustris CGA010 under Photoheterotrophic Growth Conditions with Reduced Carbon Sources

Nonsulfur purple (NSP) photosynthetic bacteria are metabolically versatile and can grow under a wide variety of physiological conditions. They assimilate CO2 via the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway with ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzing the actual CO2 fixation reaction (19, 27). In Rhodobacter sphaeroides and Rhodobacter capsulatus, the structural genes encoding enzymes of the CBB pathway are organized into two cbb operons, cbbI and cbbII. Each operon contains genes encoding one of two distinct forms of RubisCO. The cbbI operon contains the cbbLS genes encoding the large and small subunits of form I RubisCO, while the cbbM gene in the cbbII region encodes the large subunit of form II RubisCO (3, 8, 18). In all organisms studied so far, transcriptional regulation of the cbb operons is mediated by a positive regulator, CbbR, encoded by the divergently transcribed cbbR gene. CbbR belongs to the LysR family of transcriptional regulators (4-6, 9, 19, 24, 25, 30, 33). With CO2 as the sole source of carbon (during photoautotrophic and chemolithoautotrophic growth), NSP bacteria derive cellular carbon via CO2 fixation through the CBB pathway. The availability of fixed carbon (photoheterotrophic growth) changes the primary role of the CBB cycle, enabling CO2 to serve as an electron sink for reducing equivalents derived from the oxidation of organic carbon (6, 20-27, 31). Rhodopseudomonas palustris is an NSP photosynthetic bacterium that belongs to the alphaproteobacterial group. The extreme metabolic versatility of this organism allows photosynthetic growth by fixation of CO2 (photoautotrophy) or by assimilation of organic carbon (photoheterotrophy), as well as aerobic chemoheterotrophic and chemolithoautotrophic growth (17, 20, 21). The organization of the cbb genes in R. palustris is unique, with juxtaposition of genes encoding a novel two-component regulatory (CbbRRS) system in the cbbI region (20, 21) (Fig. ​(Fig.1).1). The CbbRRS genes encode an atypical two-component system that is comprised of a large hybrid sensor kinase (CbbSR) and two response regulator proteins (CbbRR1 and CbbRR2), and there are no apparent DNA binding domains on any of these proteins (21). The cbbRRS genes are found between the major transcription regulator gene (cbbR) and genes encoding the large (cbbL) and small (cbbS) subunits of form I RubisCO. Recently, it was shown that this two-component system modulates the expression of the cbbI operon under photoautotrophic growth conditions, possibly in response to a redox signal (20-22). The presence of genes encoding this system in the cbbI region (Fig. ​(Fig.1)1) and the role of this system in modulating the activity of form I RubisCO and expression of cbbLS in photoautotrophically grown cells (21) are unique to R. palustris, as similar systems have not been described for related NSP bacteria, such as R. sphaeroides and R. capsulatus (6, 20). Inasmuch as this two-component system specifically influences cbbLS transcription in photoautotrophically grown R. palustris cells, the role of the cbbRRS system under photoheterotrophic benzoate growth conditions was analyzed in the current study. FIG. 1. Gene arrangement in the cbbI and cbbII regions of R. palustris CGA010. R. palustris can metabolize a wide array of organic compounds, which serve as electron donors and major carbon sources. These compounds include organic acids, such as malate, succinate, and butyrate, as well as aromatic acids, such as benzoate. Among the NSP bacteria, R. palustris stands out because of its capacity for anaerobic photoheterotrophic growth on reduced aromatic compounds, such as benzoate (12, 17, 29). Photoheterotrophic growth of R. palustris on benzoate requires bicarbonate/CO2 or some other added electron acceptor. R. palustris can degrade benzoate anaerobically through a reductive benzoate pathway in which benzoyl-coenzyme A (benzoyl-CoA) is the central intermediate. The final products of benzoate degradation are 3 molecules of acetyl-CoA, 1 molecule of CO2, and 6 reducing equivalents. Acetyl-CoA is used for the synthesis of cell building blocks (12, 13). In this study, we demonstrated that photoheterotrophic growth on benzoate in the presence of low levels of bicarbonate obligately required synthesis of form I RubisCO and that the CbbR regulator protein played an essential role. Unlike its role under photoautotrophic growth conditions, the CbbRRS two-component system did not appear to play a regulatory role under benzoate growth conditions, suggesting that there is differential utilization of this regulatory system in R. palustris.