Genetic and proteomic analyses of CO utilization by <Emphasis Type="Italic">Methanosarcina acetivorans</Emphasis>

Methanosarcina acetivorans, a member of the methanogenic archaea, can grow with carbon monoxide (CO) as the sole energy source and generates, unlike other methanogens, substantial amounts of acetate and formate in addition to methane. Phenotypic analyses of mutant strains lacking the cooS1F operon and the cooS2 gene suggest that the monofunctional carbon monoxide dehydrogenase (CODH) system contributes to, but is not required for, carboxidotrophic growth of M. acetivorans. Further, qualitative proteomic analyses confirm a recent report (Lessner et al., Proc Natl Acad Sci USA, 103:17921–17926, 2006) in showing that the bifunctional CODH/acetyl-CoA synthase (ACS) system, two enzymes involved in CO₂-reduction, and a peculiar protein homologous to both corrinoid proteins and methyltransferases are synthesized at elevated levels in response to CO; however, the finding that the latter protein is also abundant when trimethylamine serves as growth substrate questions its proposed involvement in the reduction of methyl-groups to methane. Potential catabolic mechanisms and metabolic adaptations employed by M. acetivorans to effectively utilize CO are discussed.     

Trace Metal Imaging of Sulfate-Reducing Bacteria and Methanogenic Archaea at Single-Cell Resolution by Synchrotron X-Ray Fluorescence Imaging

Metal cofactors are required for many enzymes in anaerobic microbial respiration. This study examined iron, cobalt, nickel, copper, and zinc in cellular and abiotic phases at the single-cell scale for a sulfate-reducing bacterium (Desulfococcus multivorans) and a methanogenic archaeon (Methanosarcina acetivorans) using synchrotron X-ray fluorescence microscopy. Relative abundances of cellular metals were also measured by inductively coupled plasma mass spectrometry. For both species, zinc and iron were consistently the most abundant cellular metals. M. acetivorans contained higher nickel and cobalt content than D. multivorans, likely due to elevated metal requirements for methylotrophic methanogenesis. Cocultures contained spheroid zinc sulfides and cobalt/copper sulfides.     

Methyl Sulfide Production by a Novel Carbon Monoxide Metabolism in Methanosarcina acetivorans

We observed dimethyl sulfide and methanthiol production in pure incubations of the methanogen Methanosarcina acetivorans when carbon monoxide (CO) served as the only electron donor. Energy conservation likely uses sodium ion gradients for ATP synthesis. This novel metabolism permits utilization of CO by the methanogen, resulting in quantitative sulfide methylation.     

A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans

Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor).     

Metabolic reconstruction of the archaeon methanogen <Emphasis Type="Italic">Methanosarcina Acetivorans</Emphasis>

Background  

Methanogens are ancient organisms that are key players in the carbon cycle accounting for about one billion tones of biological methane produced annually. Methanosarcina acetivorans, with a genome size of ~5.7 mb, is the largest sequenced archaeon methanogen and unique amongst the methanogens in its biochemical characteristics. By following a systematic workflow we reconstruct a genome-scale metabolic model for M. acetivorans. This process relies on previously developed computational tools developed in our group to correct growth prediction inconsistencies with in vivo data sets and rectify topological inconsistencies in the model.     

Stable Carbon Isotope Fractionation by Methylotrophic Methanogenic Archaea

In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C₁ compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (−83‰ to −72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.     

Different mechanisms of acetate activation in Desulfurella acetivorans and Desulfuromonas acetoxidans

Desulfurella acetivorans and Desulfuromonas acetoxidans are both acetate oxidizing sulfur reducing eubacteria. The two organisms differ in G+C content of DNA (31.4% versus 50–52%) and in growth temperature optimum (55°C versus 30°C) and in that D. acetivorans does not contain cytochromes. Both organisms are shown to be similar in that they metabolize acetate via the citric acid cycle rather than via the carbon monoxide dehydrogenase pathway. They were found to differ, however, in the mechanism of acetate activation and of succinate formation. In D. acetoxidans acetyl-CoA and succinate are formed from acetate and succinyl-CoA involving only one enzyme, succinyl-CoA: acetate CoA-transferase. In D. acetivorans acetyl-CoA is generated from acetate via acetyl phosphate involving acetate kinase and phosphate acetyltransferase; succinate is formed from succinyl-CoA via succinyl-CoA synthetase. Both sulfur reducers were found to contain menaquinone.Abbreviations HPLC high performance liquid chromatography - acetyl-P acetyl phosphate     

Inhibition of stomatal opening in sunflower leaves by carbon monoxide,and reversal of inhibition by light

When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO₂, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm·s^-1. The CO effect on stomatal aperture required a CO/O₂ ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibitio of cytochrome-c oxidase by CO is competitive in respect to O₂. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO₂ was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus.In contrast to red light, very weak blue light (405 nm) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a ₃-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis.     

The effect of CreA in glucose and xylose catabolism in<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis>

The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars. In the cultivations on single carbon sources, it was demonstrated that xylose acted as a carbon catabolite repressor (xylose cultivations), while the enzymes in the xylose utilisation pathway were also subject to repression in the presence of glucose (glucose cultivations). In the wild type strain growing on the sugar mixture, glucose repression of xylose utilisation was observed; with xylose utilisation occurring only after glucose was depleted. This phenomenon was not seen in the creA deleted strain, where glucose and xylose were catabolised simultaneously. Measurement of key metabolites and the activities of key enzymes in the xylose utilisation pathway revealed that xylose metabolism was occurring in the creA deleted strain, even at high glucose concentrations. Conversely, in the wild type strain, activities of the key enzymes for xylose metabolism increased only when the effects of glucose repression had been relieved. Xylose was both a repressor and an inducer of xylanases at the same time. The creA mutation seemed to have pleiotropic effects on carbohydratases and carbon catabolism.     

Cloning and characterization of indolepyruvate decarboxylase from <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> AM1

For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k _cat value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k _cat/K _m). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown.     

Cell growth and P(3HB) accumulation from CO<Subscript>2</Subscript> of a carbon monoxide-tolerant hydrogen-oxidizing bacterium, <Emphasis Type="Italic">Ideonella</Emphasis> sp. O-1

Cell growth and accumulation of polyhydroxybutyric acid, P(3HB), from CO₂ in autotrophic condition of a newly isolated hydrogen-oxidizing bacterium, the strain O-1, was investigated. The bacterium, which was deposited in the Japan Collection of Microorganisms as JCM17105, autotrophically grows by assimilating H₂, O₂, and CO₂ as substrate. 16S rRNA gene sequence of the bacterium was the closest to Ideonella dechloratans (99%). Specific growth rate of the strain O-1 was faster than a hydrogen-oxidizing bacterium, Ralstonia eutropha, which is well-known P(3HB)-producing microorganism. The strain O-1 is tolerant to high O₂ concentration and it can grow above 30% (v/v) O₂, while the growth of R. eutropha and Alcaligenes latus was seriously inhibited. In culture medium containing 1 g/L (NH₄)₂SO₄, cell concentration of the strain O-1 and P(3HB) increased to 6.75 and 5.26 g/L, respectively. The content of P(3HB) in the cells was 77.9% (w/w). The strain O-1 was very tolerant to carbon monoxide (CO) and it grew even at 70% (v/v) CO, while the growth of R. eutropha and A. latus were seriously inhibited at 5% (v/v) CO. From these results, it is expected that the strain O-1 will be useful in the manufacture of P(3HB) because the industrial exhaust gas containing CO₂, H₂, and CO can be directly used as the substrate in the fermentation process.     

Methylotrophic Metabolism Is Advantageous for Methylobacterium extorquens during Colonization of Medicago truncatula under Competitive Conditions

Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO₂ (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.     

Defective formation and/or utilization of carbon monoxide in H2/CO2 fermenting methanogens dependent on acetate as carbon source

Autotrophic methanogens reduce CO₂ to CO and assimilate CO in a carbonylation reaction. Heterotrophic species were found not to form CO and/or to incorporate CO into cell matiral. The absence of CO formation correlated with the absence of carbon monoxide dehydrogenase activity. The heterotrophic Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanococcus voltae and Methanospirillum hungatei (strain GP 1) were investigated.     

Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions

Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO2 (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.     

In vivo role of three fused corrinoid/methyl transfer proteins in Methanosarcina acetivorans

Methanosarcina acetivorans is able to use carbon monoxide (CO) as the sole source of energy for growth. Its carboxidotrophic growth is peculiar as it involves formation of acetate, formate and methylated thiols, besides methane. Under this condition three proteins homologous to both corrinoid proteins and methyltransferases (MA0859, MA4384 and MA4558) are highly abundant. To address their role in M. acetivorans , a set of single and double mutants, and the triple mutant, was constructed by deletion/disruption of the encoding genes. Phenotypic analysis of the mutants rules out an important role of the methyltransferase homologues in the CO₂ reduction pathway of methanogenesis. Instead, the single and double mutants were affected to various degrees in their capacity to generate dimethylsulphide (DMS) from CO and to form methane from DMS. The triple mutant was unable to produce or metabolize DMS, and could not grow with DMS as the sole energy source, which demonstrates that MA0859, MA4384 and MA4558 are involved in, and required for, methylsulphide metabolism of M. acetivorans . Based on these findings we propose to designate MA0859, MA4384 and MA4558 as m ethyl t ransferases specific for methyl s ulphides, MtsD, MtsF and MtsH respectively.     

Regulation of methanol metabolism in the yeast Hansenula polymorpha

A study of enzyme profiles in Hansenula polymorpha grown on various carbon substrates revealed that the synthesis of the methanol dissimilatory and assimilatory enzymes is regulated in the same way, namely by catabolite repression and induction by methanol. Mutants of H. polymorpha blocked in dihydroxyacetone (DHA) synthase (strain 70 M) or DHA kinase (strain 17 B) were unable to grow on methanol which confirmed the important role attributed to these enzymes in the biosynthetic xylulose monophosphate (XuMP) cycle. Both mutant strains were still able to metabolize methanol. In the DNA kinase-negative strain 17 B this resulted in accumulation of DHA. Although DHA kinase is thought to be involved in DHA and glycerol metabolism in methylotrophic yeasts, strain 17 B was still able to grow on glycerol at a rate similar to that of the wild type. DHA on the other hand only supported slow growth of this mutant when relatively high concentrations of this compound were provided in the medium. This slow but definite growth of strain 17 B on DHA was not based on the reversible DHA synthase reaction but on conversion of DHA into glycerol, a reaction catalyzed by DNA reductase. The subsequent metabolism of glycerol in strain 17 B and in wild type H. polymorpha, however, remains to be elucidated.Abbreviations XuMP xylulose monophosphate - DHA dihydroxyacetone - EMS ethyl methanesulphonate     

Fermentation parameters of Peptostreptococcus productus on gaseous substrates (CO,H2/CO2)

Intrinsic growth and substrate uptake parameters were obtained for Peptostreptococcus productus, strain U-1, using carbon monoxide as the limiting substrate. A modified Monod model with substrate inhibition was used for modeling. In addition, a product yield of 0.25 mol acetate/mol CO and a cell yield of 0.034 g cells/g CO were obtained. While CO was found to be the primary substrate, P. productus is able to produce acetate from CO₂ and H₂, although this substrate could not sustain growth. Yeast extract was found to also be a growth substrate. A yield of 0.017 g cell/g yeast extract and a product yield of 0.14 g acetate/g yeast extract were obtained. In the presence of acetate, the maximum specific CO uptake rate was increased by 40% compared to the maximum without acetate present. Cell replication was inhibited at acetate concentrations of 30 g/l. Methionine was found to be an essential nutrient for growth and CO uptake by P. productus. A minimum amount of a complex medium such as yeast extract (0.01%) is, however, required.