"Candidatus Galacturonibacter soehngenii" Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex.

Laura C Valk,Morten S Dueholm,Mark C M Van Loosdrecht,Gerben R Stouten,Jack T Pronk,Per H Nielsen,Martijn Diender,Jette F Petersen

doi:10.3389/fmicb.2020.00063

Abstract

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of “Candidatus Galacturonibacter soehngenii,” from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of “Ca. G. soehngenii” was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the “Ca. G. soehngenii” MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of “Ca. G. soehngenii” enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of “Ca. G. soehngenii.”

Highlights

Over the course of multiple decades, seven carbon-fixing pathways capable of supporting autotrophic growth have been identified and intensively studied; the Calvin-Benson-Bassham (CCB) reductive pentose-phosphate cycle, the reductive citricacid cycle (Arnon-Buchanan (AB) cycle), the hydroxypropionate (Fuchs-Holo) bi-cycle, the 3-hydroxypropionate/4hydroxybutyrate cycle, dicarboxylate/hydroxybutyrate cycle, the reductive acetyl-CoA (Wood-Ljungdahl) pathway and the reductive glycine pathway (Berg, 2011; Fuchs, 2011; Figueroa et al, 2018)
In a recent study on D-galacturonate-limited, anaerobic enrichment cultures, we identified the dominant bacterium as a species from a novel genus within the Lachnospiraceae, for which we proposed the name “Candidatus Galacturonibacter soehngenii.”
In these experiments, a low in situ hydrogen partial pressure limited in vivo Wood-Ljungdahl pathway (WLP) activity, as it was expected that hydrogen was used as reductant for the production of acetate from formate or CO2

Summary

Introduction

Over the course of multiple decades, seven carbon-fixing pathways capable of supporting autotrophic growth have been identified and intensively studied; the Calvin-Benson-Bassham (CCB) reductive pentose-phosphate cycle, the reductive citricacid cycle (Arnon-Buchanan (AB) cycle), the hydroxypropionate (Fuchs-Holo) bi-cycle, the 3-hydroxypropionate/4hydroxybutyrate cycle, dicarboxylate/hydroxybutyrate cycle, the reductive acetyl-CoA (Wood-Ljungdahl) pathway and the reductive glycine pathway (Berg, 2011; Fuchs, 2011; Figueroa et al, 2018). The first five pathways are primarily used for carbon fixation and the reductive glycine pathway for recycling of electron carriers. The Wood-Ljungdahl pathway (WLP) acts as a primary pathway for energy conservation in anaerobes (Fuchs, 2011; Bar-Even et al, 2012b; Schuchmann and Müller, 2014). Methanogens use methanofuran (MFR), tetrahydromethanopterin and coenzyme-F420 as cofactors while acetogens rely on NAD(P)H, tetrahydrofolate (THF) and ferredoxin (Fd) (Fuchs, 2011; Adam et al, 2018). Reduction of CO2 to acetate via the WLP requires 8 electrons (Equation 1, Ragsdale and Pierce, 2008; Schuchmann and Müller, 2014)

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