Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle

Abstract

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term “phosphoglycolate salvage.” Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.

Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli

“…The E. coli genome encodes enzymes of a ‘glycerate pathway’ that could serve as a means of phosphoglycolate salvage. Indeed, this pathway was recently shown to be the primary route of phosphoglycolate salvage in C. necator , a proteobacterial chemolithoautotroph that notably lacks carboxysome genes ( Claassens et al, 2020 ). The glycerate pathway proceeds by dephosphorylating 2PG to glycolate, oxidizing glycolate to glyoxylate, and converting two units of glyoxylate into tartronate semialdehyde via a decarboxylating lyase reaction.…”

“…However, the Δ gph knockout was challenging to transform by electroporation, consistent with a proposed role in DNA repair ( Teresa Pellicer et al, 2003 ). We reasoned that 2PG salvage might be required in CCMB1, as photorespiratory genes are essential in cyanobacteria ( Eisenhut et al, 2008 ) and chemolithoautotrophic bacteria ( Claassens et al, 2020 ; Desmarais et al, 2019 ) even though both groups often express carboxysome CCMs. We therefore proceeded leaving the genes of the putative glycolate pathway intact.…”

“…Plants, cyanobacteria and other autotrophs uniformly express ‘photorespiratory’ pathways to process the rubisco oxygenation product 2-phosphoglycolate, or 2PG ( Eisenhut et al, 2008 ). Although these are typically called photorespiratory pathways after their discovery in plants, where they were named based on the reproducible observation of light-induced CO 2 production in many C 3 plant species ( Zelitch, 1979 ), we refer to them as ‘phosphoglycolate salvage pathways’ following Claassens et al, 2020 because they are also found in chemolithoautotrophic organisms lacking photosynthesis. 2PG salvage appears to be essential in plants, cyanobacteria, and chemolithoautotrophic proteobacteria ( Claassens et al, 2020 ; Eisenhut et al, 2008 ; Somerville and Ogren, 1980 , Somerville and Ogren, 1979 ).…”

Wide range of metabolic adaptations to the acquisition of the Calvin cycle revealed by comparison of microbial genomes

“…These findings suggest that some CBB-positive microbes may benefit from having phosphoglycolate phosphatase isozymes like observed in Cyanobacteria. Glyoxylate carbons can be recycled or eliminated for example by the glycerate pathway in Ralstonia eutropha , the malate cycle, or the photorespiratory C2 cycle [ 68 , 69 ]. Tartronate semialdehyde reductase (EC 1.1.1.60; TARS to G in Fig 5 ) and tartrate dehydrogenase (EC 1.1.1.93; OGLYC to TAR/m-TAR in Fig 5 ) represent the glycerate pathway, but these enzymes showed no enrichment, and only the former was significant in the ACE analysis, reporting both positive (subtree 5) and negative correlation (subtree 4).…”

Ecophysiology of the cosmopolitan OM252 bacterioplankton (Gammaproteobacteria)

“…Multiple pathways for phosphoglycolate salvage have recently been investigated in the model chemolithoautotrophic organism Cupriavidas necator H16 (77). We found annotated genes supporting the presence of the C2 cycle ( glyA , hprA , and associated aminotransferases) and the malate cycle ( aceB , maeB , pyruvate dehydrogenase aceEF ) in Ca.…”