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The opportunistic human pathogen Acinetobacter baumannii is one of the leading causes of nosocomial infections. The high prevalence of multidrug‐resistant strains, a high adaptability to changing environments and an overall pronounced stress resistance contribute to persistence and spread of the bacteria in hospitals and thereby promote repeated outbreaks. Altogether, the success of A. baumannii is mainly built on adaptation and stress resistance mechanisms, rather than relying on ‘true’ virulence factors. One of the stress factors that pathogens must cope with is osmolarity, which can differ between the external environment and different body parts of the human host. A. baumannii ATCC 19606T accumulates the compatible solutes glutamate, mannitol and trehalose in response to high salinities. In this work, it was found that most of the solutes vanish immediately after reaching stationary phase, a very unusual phenomenon. While glutamate can be metabolized, mannitol produced by MtlD is excreted to the medium in high amounts. First results indicate that A. baumannii ATCC 19606T undergoes a rapid switch to a dormant state (viable but non‐culturable) after disappearance of the compatible solutes. Resuscitation from this state could easily be achieved in PBS or fresh medium.
The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD‐glucose and glucose‐6‐phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose‐6‐phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose‐6‐phosphate phosphatase activity accumulated trehalose‐6‐phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose‐6‐phosphate. Our results demonstrate that trehalose‐6‐phosphate affects multiple physiological activities in A. baumannii ATCC 19606.
Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H2 + CO2 by the only pathway that combines CO2 fixation with ATP synthesis, the Wood–Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP+, an electron carrier in the Wood–Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP+ oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe–Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.
The Rnf complex is a Na+ coupled respiratory enzyme in a fermenting bacterium, Thermotoga maritima
(2020)
rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.
Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar‐based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2‐dependent CO2 reductase and furthermore belong to different classes of ‘Rnf’‐ and ‘Ech‐acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g−1 h−1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO‐based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.
Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2 via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.
Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."
Background: Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study.
Results: We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis.
Discussion: We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.
The hydrogen-dependent carbon dioxide reductase is a soluble enzyme complex that directly utilizes hydrogen (H2) for the reduction of carbon dioxide (CO2) to formate in the first step of the acetyl-coenzyme A- or Wood-Ljungdahl pathway (WLP). HDCR consists of 2 catalytic subunits, a hydrogenase and a formate dehydrogenase (FDH) and two small subunits carrying iron-sulfur clusters. The enzyme complex has been purified and characterized from two acetogenic bacteria, from the mesophile Acetobacterium woodii and, recently, from the thermophile Thermoanaerobacter kivui. Physiological studies toward the importance of the HDCR for growth and formate metabolism in acetogens have not been carried out yet, due to the lack of genetic tools. Here, we deleted the genes encoding HDCR in T. kivui taking advantage of the recently developed genetic system. As expected, the deletion mutant (strain TKV_MB013) did not grow with formate as single substrate or under autotrophic conditions with H2 + CO2. Surprisingly, the strain did also not grow on any other substrate (sugars, mannitol or pyruvate), except for when formate was added. Concentrated cell suspensions quickly consumed formate in the presence of glucose only. In conclusion, HDCR provides formate which was essential for growth of the T. kivui mutant. Alternatively, extracellularly added formate served as terminal electron acceptor in addition to CO2, complementing the growth deficiency. The results show a tight coupling of multi-carbon substrate oxidation to the WLP. The metabolism in the mutant can be viewed as a coupled formate + CO2 respiration, which may be an ancient metabolic trait.
The strictly anaerobic acetogenic bacterium Acetobacterium woodii is metabolically diverse and grows on variety of substrates which includes H2 + CO2, sugars, alcohols and diols. It is unique in producing bacterial microcompartments (BMC) during growth on different substrates such as 1,2-propanediol, 2,3-butanediol, ethanol or fructose. In this study, we analyzed the genetic organization and expression of the BMC genes within the A. woodii genome, the previously described 18 gene pdu cluster as well as four other cluster potentially encoding one or two shell proteins. Expression analysis of respective gene clusters revealed that the pdu gene cluster is highly expressed during growth on 1,2-PD, 2,3-BD, ethanol and ethylene glycol. The promoter region upstream of the pduA gene was identified and used to establish a reporter gene assay based on chloramphenicol acetyl transferase as a reporter protein. The reporter gene assay confirmed the qPCR data and demonstrated that 1,2-PD is superior over ethanol and ethylene glycol as inducer. BMCs were enriched from cells grown on 2,3- BD and 1,2-PD and shown to have typical structure in electron micrographs. Biochemical analyses revealed several of the protein encoded by the pdu cluster to be part of the isolated BMCs. These data demonstrate a very unique situation in A. woodii in which apparently one BMC gene cluster in expressed during growth on different substrates.