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Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.
BACKGROUND: Acetogenic bacteria are able to use CO2 as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H2. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H2+CO2 and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO2 to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD+ with the export of Na+ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui.
RESULTS: The genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G+C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H2+CO2 was dependent on Na+ and acetate formation was inhibited by a protonophore, indicating that H+ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F1FO ATP synthase does not have the conserved Na+ binding motif. A search for potential H+-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech).
CONCLUSIONS: The thermophilic acetogen T. kivui does not use Na+ but H+ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H2+CO2 make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.
Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
More than 2 million tons of glycerol are produced during industrial processes each year and, therefore, glycerol is an inexpensive feedstock to produce biocommodities by bacterial fermentation. Acetogenic bacteria are interesting production platforms and there have been few reports in the literature on glycerol utilization by this ecophysiologically important group of strictly anaerobic bacteria. Here, we show that the model acetogen Acetobacterium woodii DSM1030 is able to grow on glycerol, but contrary to expectations, only for 2–3 transfers. Transcriptome analysis revealed the expression of the pdu operon encoding a propanediol dehydratase along with genes encoding bacterial microcompartments. Deletion of pduAB led to a stable growth of A. woodii on glycerol, consistent with the hypothesis that the propanediol dehydratase also acts on glycerol leading to a toxic end-product. Glycerol is oxidized to acetate and the reducing equivalents are reoxidized by reducing CO2 in the Wood–Ljungdahl pathway, leading to an additional acetate. The possible oxidation product of glycerol, dihydroxyacetone (DHA), also served as carbon and energy source for A. woodii and growth was stably maintained on that compound. DHA oxidation was also coupled to CO2 reduction. Based on transcriptome data and enzymatic analysis we present the first metabolic and bioenergetic schemes for glycerol and DHA utilization in A. woodii.
Acetogenic bacteria are already established as biocatalysts for production of high-value compounds from C1 substrates such as H2 + CO2 or CO. However, little is known about the physiology, biochemistry and bioenergetics of acetogenesis from formate, an interesting feedstock for biorefineries. Here, we analysed formate metabolism in the model acetogen Acetobacterium woodii. Cells grew optimally on 200 mM formate to an optical density of 0.6. Formate was exclusively converted to acetate (and CO2) with a ratio of 4.4:1. Transcriptome analyses revealed genes/enzymes involved in formate metabolism. Strikingly, A. woodii has two genes potentially encoding a formyl-THF synthetase, fhs1 and fhs2. fhs2 forms an operon with a gene encoding a potential formate transporter, fdhC. Deletion of fhs2/fdhC led to a reduced growth rate, formate consumption and optical densities. Acetogenesis from H2 + CO2 was accompanied by transient formate production; strikingly, formate reutilization was completely abolished in the Δfhs2/fdhC mutant. Take together, our studies gave the first detailed insights into the formatotrophic lifestyle of A. woodii.
Background: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown.
Results: Here we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs.
Conclusions; We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.
Energy-conserving dimethyl sulfoxide reduction in the acetogenic bacterium Moorella thermoacetica
(2022)
Moorella thermoacetica is one of the well-studied thermophilic acetogenic bacteria. It grows by oxidation of organic substrates, CO or H2 coupled to CO2 reduction to acetate. Here, we describe that M. thermoacetica can also use dimethyl sulfoxide as terminal electron acceptor. Growth of M. thermoacetica on glucose or H2 + CO2 was stimulated by dimethyl sulfoxide (DMSO). Membranes showed a DMSO reductase activity, that was induced by growing cells in presence of DMSO. The enzyme used reduced anthraquinone-2,6-disulfonate, benzyl- and methyl viologen as electron donor, but not NAD(P)H. Activity was highest at pH 5 and 60°C, the Km for DMSO was 2.4 mM. Potential DMSO reductase subunits were identified by peptide mass fingerprinting; they are encoded in a genomic region that contains three potential dmsA genes, three dmsB genes and one dmsC gene. Transcriptome analysis revealed that two different dmsAB gene clusters were induced in the presence of DMSO. The function of these two and their predicted biochemical features are discussed. In addition, the data are in line with the hypothesis that M. thermoacetica can use DMSO alongside CO2 as electron acceptor and DMSO reduction is catalysed by an energy-conserving, membrane-bound electron transport chain with DMSO as final electron acceptor.
Tree-related microhabitats (TreMs) describe the microhabitats that a tree can provide for a multitude of other taxonomic groups and have been proposed as an important indicator for forest biodiversity (Asbeck et al., 2021). So far, the focus of TreM studies has been on temperate forests, although many trees in the tropics harbour exceptionally high numbers of TreMs. In this study, TreMs in the lowland tropical forests of the Choco (Ecuador) and in the mountain tropical forests of Mount Kilimanjaro (Tanzania) were surveyed. Our results extend the existing typology of TreMs of Larrieu et al. (2018) to include tropical forests and enabled a comparison of the relative recordings and diversity of TreMs between tropical and temperate forests. A new TreM form, Root formations, and three new TreM groups, concavities build by fruits or leaves, dendrotelms, and root formations, were established. In total, 15 new TreM types in five different TreM groups were specified. The relative recordings of most TreMs were similar between tropical and temperate forests. However, ivy and lianas, and ferns were more common in the lowland rainforest than in temperate forests, and bark microsoil, limb breakage, and foliose and fruticose lichens in tropical montane forest than in lowland rainforest. Mountain tropical forests hosted the highest diversity for common and dominant TreM types, and lowland tropical forest the highest diversity for rare TreMs. Our extended typology of tree-related microhabitats can support studies of forest-dwelling biodiversity in tropical forests. Specifically, given the ongoing threat to tropical forests, TreMs can serve as an additional tool allowing rapid assessments of biodiversity in these hyperdiverse ecosystems.