Calvin Cycle Mutants of Photoheterotrophic Purple Nonsulfur Bacteria Fail To Grow Due to an Electron Imbalance Rather than Toxic Metabolite Accumulation

Abstract

Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO 2 -fixing Calvin cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO) However, we also demonstrate that Rs. rubrum and Rp. palustris Calvin cycle phosphoribulokinase mutants that cannot produce RuBP cannot grow photoheterotrophically on succinate unless an electron acceptor is provided or H 2 production is permitted. Thus, the Calvin cycle is still needed to oxidize electron carriers even in the absence of toxic RuBP. Surprisingly, Calvin cycle mutants of Rs. rubrum, but not of Rp. palustris, grew photoheterotrophically on malate without electron acceptors or H 2 production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated. P urple nonsulfur bacteria (PNSB) are renowned for their ability to employ versatile metabolic modules to thrive under different growth conditions. PNSB can grow photoautotrophically using light for energy, inorganic compounds other than water (e.g., thiosulfate, Fe 2ϩ ) for electrons, and CO 2 for carbon. The Calvin cycle is well-known for permitting autotrophic growth by converting CO 2 into organic precursors for biosynthesis (Fig. 1). In this pathway phosphoribulokinase (PRK) expends ATP to generate ribulose 1,5-bisphosphate (RuBP). RuBP is then combined with CO 2 via ribulose 1,5-bisphosphate carboxylase (RubisCO), resulting in two molecules of 3-phosphoglycerate. CO 2 fixation generates relatively oxidized metabolites that accept electrons from NAD(P)H via glyceraldehyde-3-phosphate dehydrogenase.,PNSB can also grow photoheterotrophically using light for energy and organic compounds for carbon and electrons. Unlike the process in a respiring heterotroph, reducing power from oxidative pathways [e.g., NAD(P)H] is not used to reduce a terminal electron acceptor and generate ATP by oxidative phosphorylation. Rather, ATP generation is largely decoupled from the oxidative pathways of central metabolism as photoheterotrophs repeatedly energize electrons and shuttle them through a H ϩ -pumping electron transfer chain to generate ATP by cyclic photophosphorylation (Fig. 1). Nevertheless, photoheterotrophs generate ample reducing power that must be oxidized to replenish pools of oxidized electron carriers [e.g., NAD(P) ϩ ] and maintain metabolic flow. CO 2 fixation was first hypothesized to fulfill this essential role of maintaining oxidized electron carriers during photoheterotrophic growth in 1933 by Muller (1), prior to the elucidation of the Calvin cycle itself (2) (Fig. 1, model 1). Muller devised this hypothesis to explain why there was net CO 2 fixation when PNSB grew photoheterotrophically on compounds, like butyrate, that are more electron rich than the average carbon in biomass (1).Supporting this hypothesis, it was later shown that PNSB could be grown photoheterotrophically on butyrate without added CO 2 if an electron acceptor like dimethyl sulfoxide (DMSO) was provided (3) or i...

Carbon roadmap from syngas to polyhydroxyalkanoates in Rhodospirillum rubrum

“…). These findings are in agreement with previous observation under photoheterotrophic conditions in the presence of organic carbon, such as acetate, that suggested that the CBB cycle is involved in the maintenance of the redox balance in the cell, but not in CO 2 fixation (McKinlay and Harwood, 2010; 2011; Gordon and McKinlay, ).…”

Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

“…). Nase functions in both nitrogen acquisition and electron balance, while the primary role of CO 2 fixation during photoheterotrophic growth is as an electron sink (Hallenbeck et al ., ; Falcone and Tabita, ; Hädicke et al ., ; Farmer et al ., ; Gordon and McKinlay, ). Total electron flux through Nase and Rubisco increases with the growth rate for the wild type, Mo‐Nase and V‐Nase strains, but is always high for the Fe‐Nase strain (Fig.…”

“…1, Arrow E), such that electrons from the organic substrate are fully allocated between biomass, H 2 and CO 2 . Under these conditions, Nase is important both for nutrient acquisition and as an electron sink (Muller, 1933;Hillmer and Gest, 1977;Harwood, 2010a, 2011;Farmer et al, 2014;Gordon and McKinlay, 2014;McCully and McKinlay, 2016). To determine how simultaneous nitrogen acquisition and electron balancing constraints influence growth based on each Nase isoform, we cultured wild type R. palustris and mutant strains known to utilize only a single isoform (hereafter Mo-Nase, V-Nase and Fe-Nase strains, Oda et al, 2005) in diazotrophic photoheterotrophic media with organic substrates of varying oxidation states and assimilation pathways (Table 1) and measured growth, N 2 and CO 2 fixation, H 2 production, biomass composition and global protein abundance patterns.…”

N2gas is an effective fertilizer for bioethanol production byZymomonas mobilis

“…Cultures were grown in 10-mL volumes in 27-mL anaerobic test tubes or in 60-mL volumes in 160-mL serum vials at 30°C. Media were made anaerobic by bubbling with either N 2 or Ar and sealed as previously described (23). Test tubes were laid flat, and serum vials were left upright.…”