Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C₄-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO₂ in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides cells

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate pathway, is studied. It is found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate pathway. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.     

Enzymes of the citramalate cycle in <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis>

Rhodospirillum rubrum is among the bacteria that can assimilate acetate in the absence of isocitrate lyase, the key enzyme of glyoxylate shunt. Previously we have suggested the functioning of a new anaplerotic cycle of acetate assimilation in this bacterium: citramalate cycle, where acetyl-CoA is oxidized to glyoxylate. This work has demonstrated the presence of all the key enzymes of this cycle in R. rubrum extracts: citramalate synthase catalyzing condensation of acetyl-CoA and pyruvate with the formation of citramalate, mesaconase forming mesaconate from L-citramalate, and the enzymes catalyzing transformation of propionyl-CoA + glyoxylate 3-methylmalyl-CoA ? mesaconyl-CoA. At the same time, R. rubrum synthesizes crotonyl-CoA carboxylase/reductase, which is the key enzyme of ethylmalonyl-CoA pathway discovered recently in Rhodobacter sphaeroides. Physiological differences between the citramalate cycle and the ethylmalonyl-CoA pathway are discussed.     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Acetate Assimilation in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. It has been found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate shunt. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 313–318.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Tsygankov, Laurinavichene, Ivanovsky.     

The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: enzymes of the citramalate cycle in Rhodobacter sphaeroides

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shortcut, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shortcut, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl-CoA and occurs via the following reaction sequence: propionyl-CoA (+ CO2) --> methylmalonyl-CoA --> succinyl-CoA --> succinate --> fumarate --> malate --> oxalacetate (- CO2) --> phosphoenolpyruvate --> pyruvate. The independence of the cell growth and the acetate assimilation of CO2 is due to the accumulation of CO2/HCO3- (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Enzymes of the Citramalate Cycle in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shunt, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl- CoA and occurs via the following reaction sequence: propionyl-CoA (+CO₂) å methylmalonyl-CoA å succinyl-CoA å succinate å fumarate malate å oxaloacetate (−CO₂) å phosphoenolpyruvate å pyruvate. The independence of the cell growth and the acetate assimilation of CO₂ is due to the accumulation of CO₂/HCO ₃ ⁻ (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 319–328.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Ivanovsky.     

Microbiological oxidation of the branched C5-dicarboxylic acids

Summary The oxidation of branched C₅-dicarboxylates has been studied with microorganisms obtained from enrichement cultures with each of these acids. Bacteria grown on itaconate, mesaconate or methylsuccinate oxidize most of these compounds rapidly; (+) citramalate is oxidized 2–4 times as rapidly as (-) citramalate; and citraconate only feebly. Citraconate-grown cells do not oxidize itaconate and oxidize feebly mesaconate and methylsuccinate; they oxidize (-) citramalate 3–4 times faster than (+) citramalate. Cell-free extracts of various strains grown on different C₅-dicarboxylates contain pronounced mesaconase activity, varying amounts of citraconase and hardly any itaconase activity.Communication number 633 from National Chemical Laboratory.     

A bicyclic autotrophic CO2 fixation pathway in Chloroflexus aurantiacus

Phototrophic CO(2) assimilation by the primitive, green eubacterium Chloroflexus aurantiacus has been shown earlier to proceed in a cyclic mode via 3-hydroxypropionate, propionyl-CoA, succinyl-CoA, and malyl-CoA. The metabolic cycle could be closed by cleavage of malyl-CoA affording glyoxylate (the primary CO(2) fixation product) with regeneration of acetyl-CoA serving as the starter unit of the cycle. The pathway of glyoxylate assimilation to form gluconeogenic precursors has not been elucidated to date. We could now show that the incubation of cell extract with a mixture of glyoxylate and [1,2,3-(13)C(3)]propionyl-CoA afforded erythro-beta-[1,2,2'-(13)C(3)]methylmalate and [1,2,2'-(13)C(3)]citramalate. Similar experiments using a partially purified protein fraction afforded erythro-beta-[1,2,2'-(13)C(3)]methylmalyl-CoA and [1,2,2'-(13)C(3)]mesaconyl-CoA. Cell extracts of C. aurantiacus were also shown to catalyze the conversion of citramalate into pyruvate and acetyl-CoA in a succinyl-CoA-dependent reaction. The data suggest that glyoxylate obtained by the cleavage of malyl-CoA can be utilized by condensation with propionyl-CoA affording erythro-beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. This reaction sequence regenerates acetyl-CoA, which serves as the precursor of propionyl-CoA in the 3-hydroxypropionate cycle. Autotrophic CO(2) fixation proceeds by combination of the 3-hydroxypropionate cycle with the methylmalyl-CoA cycle. The net product of that bicyclic autotrophic CO(2) fixation pathway is pyruvate serving as an universal building block for anabolic reactions.     

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.     

Features of structural organization and expression regulation of malate dehydrogenase isoforms from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> strain 2R

Two isoforms of malate dehydrogenase (MDH), dimeric and tetrameric, have been found in the purple non-sulfur bacterium Rhodobacter sphaeroides strain 2R, devoid of the glyoxylate shunt, which assimilate acetate via the citramalate cycle. Inhibitory analysis showed that the 74-kDa protein is involved in tricarboxylic acid cycle, while the 148-kDa MDH takes part in the citramalate pathway. A single gene encoding synthesis of the isologous subunits of the MDH isoforms was found during molecular-biological investigations. The appearance in the studied bacterium of the tetrameric MDH isoform during growth in the presence of acetate is probably due to the increased level of mdh gene expression, revealed by the real-time PCR, the product of which in cooperation with the citramalate cycle enzymes plays an important role in acetate assimilation.     

A novel pathway for L-citramalate synthesis in Rhodospirillum rubrum.

When [14C]propionate was incubated with a cell-free extract of Rhodospirillum rubrum in the presence of glyoxylate, ATP, CoA, Mg2+, and Mn2+, radioactivity was incorporated into mesaconate (MSA) as well as into beta-methylmalate (MMA) and citramalate (CMA). MSA was suggested to be an intermediate of the conversion of MMA to CMA based on the following observations. (i) When non-labeled MSA was added to the CMA-forming reaction system, radioactivity was trapped in MSA. (ii) When MSA was incubated with the cell-free extract, CMA was formed. (iii) The alpha-carboxyl group of CMA was shown to be derived from the beta-carboxyl group of MMA, implying that CMA was formed from MMA via MSA through successive dehydration and hydration. From the results of Sephadex G-10 column chromatography of the reaction products, beta-methylmalyl-CoA and mesaconyl-CoA were presumed to be involved in the reaction. A new CMA-forming metabolic pathway is proposed as follows: erythro-beta-methylamalyl-CoA leads to mesaconyl-CoA leads to MSA leads to L-CMA.     

Pyruvate formate lyase inRhodospirillum rubrum Ha adapted to anaerobic dark conditions

The fermatation metabolism ofRhodospirillum rubrum Ha was studied after adaptation of both light-anaerobic and dark aerobic to dark anaerobic conditions.Pyruvate was metabolized to acetate, formate, CO₂ and propionate by suspensions of cells adapted to anaerobiosis. Pyruvate cleavage to formate accounted for about two-thirds of the pyruvate decomposed. This process was catalyzed by a coenzyme A dependent pyruvate formate lyase. In carboxylate- and nucleotide-free extracts, the substrate concentrations for half-maximal velocity [S]_0.5V were found to be 1.5 mM for pyruvate and 75 M for coenzyme A.Pyruvate formate lyase could practically not be demonstrated in light-anaerobic photosynthesizing cells. Lyase activity was low at a basic level in darkaerobic respiring cells. After adaptation of both types of cells under growth conditions to dark anaerobiosis lyase activity increased about 10-fold. Highest levels could be observed in cells grown aerobically in the dark on pyruvate after transition to dark anaerobic conditions. It is concluded that pyruvate formate lyase is the characteristic key enzyme of the dark-anaerobic fermentative metabolism ofR. rubrum Ha.     

PATHWAY FOR THE DISSIMILATION OF ITACONIC AND MESACONIC ACIDS.

Brightman, Vernon (The University of Chicago, Chicago), and William R. Martin. Pathway for the dissimilation of itaconic and mesaconic acids. J. Bacteriol. 82:376-382. 1961.-Studies on the oxidation of itaconic and mesaconic acids by a Pseudomonas sp., adapted to utilize either of these acids as a sole carbon source, have provided evidence for a pathway converting both itaconate and mesaconate to succinate. A metabolic interconversion of itaconate, mesaconate, and citramalate has also been demonstrated by whole cell and cell-free enzyme studies.Succinate derived from methylene-labeled itaconate was found to be labeled in the inside carbon atoms, a fact which indicates that the branched chain compound was converted into a straight chain molecule by a shift of the methylene carbon (C-5) from the side chain of itaconate to a position between C-2 and C-3 in an, as yet, unknown straight chain intermediate prior to its conversion to succinate.     

Metabolism of the thermophilic bacterium Oceanithermus profundus

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurs via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.     

L-malyl-coenzyme A/beta-methylmalyl-coenzyme A lyase is involved in acetate assimilation of the isocitrate lyase-negative bacterium Rhodobacter capsulatus

Cell extracts of Rhodobacter capsulatus grown on acetate contained an apparent malate synthase activity but lacked isocitrate lyase activity. Therefore, R. capsulatus cannot use the glyoxylate cycle for acetate assimilation, and a different pathway must exist. It is shown that the apparent malate synthase activity is due to the combination of a malyl-coenzyme A (CoA) lyase and a malyl-CoA-hydrolyzing enzyme. Malyl-CoA lyase activity was 20-fold up-regulated in acetate-grown cells versus glucose-grown cells. Malyl-CoA lyase was purified 250-fold with a recovery of 6%. The enzyme catalyzed not only the reversible condensation of glyoxylate and acetyl-CoA to L-malyl-CoA but also the reversible condensation of glyoxylate and propionyl-CoA to beta-methylmalyl-CoA. Enzyme activity was stimulated by divalent ions with preference for Mn(2+) and was inhibited by EDTA. The N-terminal amino acid sequence was determined, and a corresponding gene coding for a 34.2-kDa protein was identified and designated mcl1. The native molecular mass of the purified protein was 195 +/- 20 kDa, indicating a homohexameric composition. A homologous mcl1 gene was found in the genomes of the isocitrate lyase-negative bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum in similar genomic environments. For Streptomyces coelicolor and Methylobacterium extorquens, mcl1 homologs are located within gene clusters implicated in acetate metabolism. We therefore propose that L-malyl-CoA/beta-methylmalyl-CoA lyase encoded by mcl1 is involved in acetate assimilation by R. capsulatus and possibly other glyoxylate cycle-negative bacteria.     

Investigation of the catabolism of acetate and peptides in the new anaerobic thermophilic bacterium Caldithrix abyssi

This work is concerned with the metabolism of Caldithrix abyssi-an anaerobic, moderately thermophilic bacterium isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge and representing a new, deeply deviated branch within the domain Bacteria. Cells of C. abyssi grown on acetate and nitrate, which was reduced to ammonium, possessed nitrate reductase activity and contained cytochromes of the b and c types. Utilization of acetate occurred as a result of the operation of the TCA and glyoxylate cycles. During growth of C. abyssi on yeast extract, fermentation with the formation of acetate, propionate, hydrogen, and CO2 occurred. In extracts of cells grown on yeast extract, acetate was produced from pyruvate with the involvement of the following enzymes: pyruvate:ferredoxin oxidoreductase (2.6 micromol/(min mg protein)), phosphate acetyltransferase (0.46 micromol/(min mg protein)), and acetate kinase (0.3 micromol/(min mg protein)). The activity of fumarate reductase (0.14 micromol/(min mg protein)), malate dehydrogenase (0.17 micromol/(min mg protein)), and fumarate hydratase (1.2 micromol/(min mg protein)), as well as the presence of cytochrome b, points to the formation of propionate via the methyl-malonyl-CoA pathway. The activity of antioxidant enzymes (catalase and superoxide dismutase) was detected. Thus, enzymatic mechanisms have been elucidated that allow C. abyssi to switch from fermentation to anaerobic respiration and to exist in the gradient of redox conditions characteristic of deep-sea hydrothermal vents.     

Acetate metabolism in purple non-sulfur bacteria

Abstract The photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus E1F1 can grow on acetate or dl -malate photoheterotrophically under anerobic conditions or chemoheterotrophically in the dark in the presence of dioxygen. Bacterial cells grown under both anaerobic and aerobic conditions exhibited high amounts of the tricarboxylic acid cycle enzymes especially in dark-aerobic cultures. A high activity of isocitrate lyase was found in cells of R. capsulatus E1F1 and, to a lesser extent, in those of R. capsulatus IP2, Rhodobacter sphaeroides and Rhodospirillum rubrum grown photoheterotrophically on acetate under anaerobic conditions. The second enzyme of the glyoxylate shunt, malate synthase, appears to be constitutive. Itaconate, a powerful inhibitor of isocitrate lyase, severely inhibited growth of R. capsulatus, R. rubrum and R. sphaeroides on acetate, thus corroborating a physiological role of the enzyme in acetate metabolism by Rhodospirillaceae.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

Mesaconase Activity of Class I Fumarase Contributes to Mesaconate Utilization by Burkholderia xenovorans

Pseudomonas aeruginosa, Yersinia pestis, and many other bacteria are able to utilize the C₅-dicarboxylic acid itaconate (methylenesuccinate). Itaconate degradation starts with its activation to itaconyl coenzyme A (itaconyl-CoA), which is further hydrated to (S)-citramalyl-CoA, and citramalyl-CoA is finally cleaved into acetyl-CoA and pyruvate. The xenobiotic-degrading betaproteobacterium Burkholderia xenovorans possesses a P. aeruginosa-like itaconate degradation gene cluster and is able to grow on itaconate and its isomer mesaconate (methylfumarate). Although itaconate degradation proceeds in B. xenovorans in the same way as in P. aeruginosa, the pathway of mesaconate utilization is not known. Here, we show that mesaconate is metabolized through its hydration to (S)-citramalate. The latter compound is then metabolized to acetyl-CoA and pyruvate with the participation of two enzymes of the itaconate degradation pathway, a promiscuous itaconate-CoA transferase able to activate (S)-citramalate in addition to itaconate and (S)-citramalyl-CoA lyase. The first reaction of the pathway, the mesaconate hydratase (mesaconase) reaction, is catalyzed by a class I fumarase. As this enzyme (Bxe_A3136) has similar efficiencies (k_cat/K_m) for both fumarate and mesaconate hydration, we conclude that B. xenovorans class I fumarase is in fact a promiscuous fumarase/mesaconase. This promiscuity is physiologically relevant, as it allows the growth of this bacterium on mesaconate as a sole carbon and energy source.     

Facultative methanotrophy: false leads, true results, and suggestions for future research

Methanotrophs are a group of phylogenetically diverse microorganisms characterized by their ability to utilize methane as their sole source of carbon and energy. Early studies suggested that growth on methane could be stimulated with the addition of some small organic acids, but initial efforts to find facultative methanotrophs, i.e., methanotrophs able to utilize compounds with carbon-carbon bonds as sole growth substrates were inconclusive. Recently, however, facultative methanotrophs in the genera Methylocella, Methylocapsa, and Methylocystis have been reported that can grow on acetate, as well as on larger organic acids or ethanol for some species. All identified facultative methanotrophs group within the Alphaproteobacteria and utilize the serine cycle for carbon assimilation from formaldehyde. It is possible that facultative methanotrophs are able to convert acetate into intermediates of the serine cycle (e.g. malate and glyoxylate), because a variety of acetate assimilation pathways convert acetate into these compounds (e.g. the glyoxylate shunt of the tricarboxylic acid cycle, the ethylmalonyl-CoA pathway, the citramalate cycle, and the methylaspartate cycle). In this review, we summarize the history of facultative methanotrophy, describe scenarios for the basis of facultative methanotrophy, and pose several topics for future research in this area.