FNR-Type Regulator GoxR of the Obligatorily Aerobic Acetic Acid Bacterium Gluconobacter oxydans Affects Expression of Genes Involved in Respiration and Redox Metabolism

Abstract

Gene expression in the obligately aerobic acetic acid bacterium Gluconobacter oxydans responds to oxygen limitation, but the regulators involved are unknown. In this study, we analyzed a transcriptional regulator named GoxR (GOX0974), which is the only member of the FNR family in this species. Evidence was obtained that GoxR contains an iron-sulfur cluster, suggesting that GoxR functions as an oxygen sensor similar to FNR. The direct target genes of GoxR were determined by combining several approaches including a transcriptome comparison of a ΔgoxR mutant with the wild type and detection of in vivo GoxR binding sites by ChAP-Seq. Prominent targets were the cioAB genes encoding a cytochrome bd oxidase with low O2 affinity, which were repressed by GoxR, and the pnt operon, which was activated by GoxR. The pnt operon encodes a transhydrogenase (pntA1A2B), an NADH-dependent oxidoreductase (GOX0313), and another oxidoreductase (GOX0314). Evidence was obtained for GoxR being active despite a high dissolved oxygen concentration in the medium. We suggest a model in which the very high respiration rates of G. oxydans due to the periplasmic oxidations cause an oxygen-limited cytoplasm and insufficient reoxidation of NAD(P)H in the respiratory chain, leading to an inhibited cytoplasmic carbohydrate degradation. GoxR-triggered induction of the pnt operon enhances fast interconversion of NADPH and NADH by the transhydrogenase and NADH reoxidation by the GOX0313 oxidoreductase via reduction of acetaldehyde formed by pyruvate decarboxylase to ethanol. In fact, small amounts of ethanol were formed by G. oxydans under oxygen-restricted conditions in a GoxR-dependent manner. IMPORTANCE Gluconobacter oxydans serves as cell factory for oxidative biotransformations based on membrane-bound dehydrogenases and as model organism for elucidating the metabolism of acetic acid bacteria. Surprisingly, to our knowledge none of the more than 100 transcriptional regulators encoded in the genome of G. oxydans has been studied experimentally up to now. In this work, we analyzed the function of a regulator named GoxR, which belongs to the FNR family. Members of this family serve as oxygen sensors by means of an oxygen-sensitive [4Fe-4S] cluster and typically regulate genes important for growth under anoxic conditions by anaerobic respiration or fermentation. Because G. oxydans has an obligatory aerobic respiratory mode of energy metabolism, it was tempting to elucidate the target genes regulated by GoxR. Our results show that GoxR affects the expression of genes that support the interconversion of NADPH and NADH and NADH reoxidation by reduction of acetaldehyde to ethanol.

The l-rhamnose-dependent regulator RhaS and its target promoters from Escherichia coli expand the genetic toolkit for regulatable gene expression in the acetic acid bacterium Gluconobacter oxydans

“…Activation of P rhaSR by RhaR in such a manner is not possible in G. oxydans since CRP is absent. The protein showing the highest similarity to CRP was shown to function as an iron–sulfur cluster-containing FNR-type transcriptional regulator (GOX0974/GOX_RS06010) of genes involved in respiration and redox metabolism ( Schweikert et al, 2021 ). In G. oxydans , the presence of RhaR even decreased the RhaS-dependent P rhaBAD activity ( Figure 1 ).…”

“…Furthermore, in E. coli the l -rhamnose metabolism is under catabolite repression by glucose, which is overcome by the binding of the cAMP receptor protein (CRP) to consensus recognition sequences found in all three P rha promoters and interaction of CRP with the RNA polymerase, which depends on the bending of the promoter DNA by RhaS or RhaR. In G. oxydans CRP is absent since the predicted CRP gene (GOX0974/GOX_RS06010) was shown to encode an iron–sulfur cluster protein termed GoxR, an FNR-type transcriptional regulator of genes involved in respiration and redox metabolism ( Schweikert et al, 2021 ). Overall, it seemed very interesting to analyze how RhaS, RhaR, and the promoters P rhaBAD , P rhaT , and P rhaSR perform in G. oxydans and if they could be useful for regulatable gene expression in this AAB.…”

Regulation Mechanism of Nitrite Degradation in Lactobacillus plantarum WU14 Mediated by Fnr

“…The gene encoding Fnrwas rst discovered by John Guest et al in the mid-1970s during the identi cation of fumarate and nitrate reduction mutants (Yan et al 2008). Fnris a global regulator of the anaerobic metabolism of Escherichia coli, controlling the synthesis of multiple genes (Schweikert et al 2021). It belongs to the large family of regulators, containing 4Fe-4S clusters with oxygen instability (Mao et al 2022).…”

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5‐ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase

“…Our knowledge on many aspects of metabolism and in particular regulation in G . oxydans is very limited compared with other bacteria used as multipurpose production hosts in biotechnology, although there is continuous progress (Bringer and Bott, 2016 ; Schweikert et al ., 2021 ). These features impede the development of G .…”