Modified pathway to synthesize ribulose 1,5-bisphosphate in methanogenic archaea

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO2 assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO2 fixation reaction, and it was recently shown that Methanocaldococcus (Methanococcus) jannaschii and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with M. jannaschii, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of M. jannsachii provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-D-ribose-1-pyrophosphate (PRPP) in M. jannaschii and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in M. jannaschii (Mj0601) and Methanosarcina acetivorans (Ma2851), and recombinant Ma2851 was active in extracts of Escherichia coli. Thus, in this work we identified a novel means to synthesize the CO2 acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.     

The growth-promoting effect of Beijerinckia and Clostridium sp. cultures on some agricultural crops

New strains of Beijerinckia mobilis and Clostridium sp. isolated from the pea rhizosphere were studied with respect to their promoting effect on the growth and development of some agricultural crops. Seed soaking in bacterial suspensions followed by the soil application of the suspensions or their application by means of foliar spraying was found to be the most efficient method of bacterization. The application of B. mobilis and Clostridium sp. cultures in combination with mineral fertilizers increased the crop production by 1.5-2.5 times. The study of the population dynamics of B. mobilis by the method of genetic marking showed that this bacterium quickly colonized the rhizoplane of plants and, therefore, had characteristics of an r-strategist. At the same time, Clostridium sp. was closer to K-strategists, since this bacterium slowly colonized the econiches studied. The introduction of the bacteria into soil did not affect the indigenous soil bacterial complex. The presence of Clostridium sp. slowed down the colonization of roots by the fungal mycelium. The possible mechanisms of the plant growth-promoting activity of B. mobilis and Clostridium sp. are discussed.     

Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses the key reaction by which inorganic carbon may be assimilated into organic carbon. Phylogenetic analyses indicate that there are three classes of bona fide RubisCO proteins, forms I, II and III, which all catalyse the same reactions. In addition, there exists another form of RubisCO, form IV, which does not catalyse RuBP carboxylation or oxygenation. Form IV is actually a homologue of RubisCO and is called the RubisCO-like protein (RLP). Both RubisCO and RLP appear to have evolved from an ancestor protein in a methanogenic archaeon, and comprehensive analyses indicate that the different forms (I, II, III and IV) contain various subgroups, with individual sequences derived from representatives of all three kingdoms of life. The diversity of RubisCO molecules, many of which function in distinct milieus, has provided convenient model systems to study the ways in which the active site of this protein has evolved to accommodate necessary molecular adaptations. Such studies have proven useful to help provide a framework for understanding the molecular basis for many important aspects of RubisCO catalysis, including the elucidation of factors or functional groups that impinge on RubisCO carbon dioxide/oxygen substrate discrimination.     

Genomics and Biochemistry of Metabolic Pathways for the C<Subscript>1</Subscript> Compounds Utilization in Colorless Sulfur Bacterium <Emphasis Type="Italic">Beggiatoa leptomitoformis</Emphasis> D-402

The metabolic pathways of one-carbon compounds utilized by colorless sulfur bacterium Beggiatoa leptomitoformis D-402 were revealed based on comprehensive analysis of its genomic organization, together with physiological, biochemical and molecular biological approaches. Strain D-402 was capable of aerobic methylotrophic growth with methanol as a sole source of carbon and energy and was not capable of methanotrophic growth because of the absence of genes of methane monooxygenases. It was established that methanol can be oxidized to CO₂ in three consecutive stages. On the first stage methanol was oxidized to formaldehyde by the two PQQ (pyrroloquinolinequinone)-dependent methanol dehydrogenases (MDH): XoxF and Mdh2. Formaldehyde was further oxidized to formate via the tetrahydromethanopterin (H₄MPT) pathway. And on the third stage formate was converted to CO₂ by NAD⁺-dependent formate dehydrogenase Fdh2. Finally, it was established that endogenous CO₂, formed as a result of methanol oxidation, was subsequently assimilated for anabolism through the Calvin–Benson–Bassham cycle. The similar way of one-carbon compounds utilization also exists in representatives of another freshwater Beggiatoa species—B. alba.     

A hybrid ribulosebisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor.

Two hybrid ribulose-1,5 bisphosphate carboxylase/oxygenase (RubisCO) enzymes were constructed using RubisCO small subunit genes (rbcS) from two eucaryotic marine organisms, Cylindrotheca sp. N1 and Olisthodiscus luteus, cloned downstream of the RubisCO large subunit gene (rbcL) of the cyanobacterium Synechococcus PCC 6301. The expression products synthesized by Escherichia coli JM107 (pVTAC223 and pANOLI) were purified and examined by polyacrylamide gel electrophoresis and compared to the purified products generated by E. coli MV1190 (pBGL710), containing cyanobacterial rbcL and rbcS genes. Both Cylindrotheca and Olisthodiscus small subunits were able to assemble in vivo with the Synechococcus large subunit octamer to form heterologous hexadecameric L8S8 enzymes, the pVTAC223 and pANOLI hybrid enzymes, respectively. Like the Synechococcus RubisCO, the hybrid enzymes were rapidly activated by Mg2+ plus HCO3-, even in the presence of RuBP. The hybrid enzymes, however, were considerably more sensitive to the competitive inhibitor 6-phosphogluconate. Detailed kinetic analysis indicated that while the carboxylase activity of both chimeric enzymes was severely reduced, in the case of the pVTAC223 hybrid enzyme, the degree of partitioning between carboxylation and oxygenation was increased nearly 60% relative to the Synechococcus RubisCO. Other kinetic properties, including the Michaelis constants for the gaseous substrates and RuBP, were altered in the hybrid proteins. These studies also led to the finding that the substrate specificity factor of the Cylindrotheca RubisCO is unusually high.     

Bacterial and fungal oxidation of dibenzofuran.

Cunninghamella elegans and a mutant strain (B8/36) of Beijerinckia both oxidized dibenzofuran to 2,3-dihydroxy-2,3-dihydrodibenzofuran. The bacterial metabolite was extremely unstable and, in the presence of acid, was rapidly converted into a mixture of 2- and 3-hydroxydibenzofuran. In contrast, the 2,3-dihydroxy-2,3-dihydrodibenzofuran formed by C. elegans was stable and only yielded 2- and 3-hydroxydibenzofuran when heated under acidic conditions. The results suggest that Beijerinckia B8/36 and C. elegans form the respective cis- and trans-isomers of 2,3-dihydroxy-2,3-dihydrodibenzofuran. C. elegans also oxidized dibenzofuran to 2- and 3-hydroxydibenzofuran under conditions that would not lead to the dehydration of the trans-dihydrodiol. These observations implicate the initial formation of dibenzofuran- 2,3-epoxide in the fungal oxidation of dibenzofuran. Beijerinckia B8/36 also produced a second unstable dihydrodiol that was tentatively identified as cis-1,2-dihydroxy-1,2-dihydrodibenzofuran. This compound gave 2-hydroxydibenzofuran as the major dehydration product and the cis relative stereochemistry was suggested by the isolation and characterization of an isopropylidine derivative. A preparation of cis-naphthalene dihydrodiol dehydrogenase and cell extracts of the parent strain of Beijerinckia oxidized both bacterial dihydrodiols to catechols. Cell extracts prepared from C. elegans catalysed an analogous oxidation of trans-2,3-dihydroxy-2,3-dihydrodibenzofuran to 2,3-dihydroxydibenzofuran. The latter product was also isolated and identified from culture filtrates. The results suggest that bacteria and fungi utilize different mechanisms to initiate the oxidation of dibenzofuran.     

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).     

Assessment of active methanogenic archaea in a methanol-fed upflow anaerobic sludge blanket reactor

Methanogenic archaea enrichment of a granular sludge was undertaken in an upflow anaerobic sludge blanket (UASB) reactor fed with methanol in order to enrich methylotrophic and hydrogenotrophic methanogenic populations. A microbial community assessment, in terms of microbial composition and activity—throughout the different stages of the feeding process with methanol and acetate—was performed using specific methanogenic activity (SMA) assays, quantitative real-time polymerase chain reaction (qPCR), and high-throughput sequencing of 16S ribosomal RNA (rRNA) genes from DNA and complementary DNA (cDNA). Distinct methanogenic enrichment was revealed by qPCR of mcrA gene in the methanol-fed community, being two orders of magnitude higher with respect to the initial inoculum, achieving a final mcrA/16S rRNA ratio of 0.25. High-throughput sequencing analysis revealed that the resulting methanogenic population was mainly composed by methylotrophic archaea (Methanomethylovorans and Methanolobus genus), being also highly active according to the RNA-based assessment. SMA confirmed that the methylotrophic pathway, with a direct conversion of methanol to CH₄, was the main step of methanol degradation in the UASB. The biomass from the UASB, enriched in methanogenic archaea, may bear great potential as additional inoculum for bioreactors to carry out biogas production and other related processes.     

How overproduction of foreign proteins affects physiology of the recombinant strains ofHansenula polymorpha

Changes in the activity of key enzymes of the methanol utilization pathway of the recombinant strains of methylotrophic yeastHansenula polymorpha R22-2B and LAC-56 were studied at different rates of chemostat growth on methanol containing mineral media. It was shown that the strain R22-2B, initially having a 10-fold increased activity of dihydroxyacetone kinase (DHAK, a key enzyme of formaldehyde assimilation) acquired increased activity of formaldehyde dehydrogenase (FADH, a key enzyme of formaldehyde dissimilation) which resulted in the enhanced oxidation of formaldehyde to CO₂. Strain LAC-56, overproducingEscherichia coli β-galactosidase, acquired the decreased intracellular concentration of ATP which resulted in the decrease of the efficiency of formaldehyde assimilation catalyzed by DHAK and resulted in accumulation of toxic formaldehyde. As a consequence some biochemical responses occurred in cells that were directed to a diminishing of the toxic effect of accumulated formaldehyde, namely, the decreasing of methanol oxidase activity (to reduce the rate of formaldehyde synthesis), and the increasing of FADH activity (to increase the rate of formaldehyde oxidation).     

Novel function of Wsc proteins as a methanol‐sensing machinery in the yeast Pichia pastoris

Wsc family proteins are plasma membrane spanning sensor proteins conserved from yeasts to mammalian cells. We studied the functional roles of Wsc family proteins in the methylotrophic yeast Pichia pastoris, and found that PpWsc1 and PpWsc3 function as methanol‐sensors during growth on methanol. PpWsc1 responds to a lower range of methanol concentrations than PpWsc3. PpWsc1, but not PpWsc3, also functions during high temperature stress, but PpWsc1 senses methanol as a signal that is distinct from high‐temperature stress. We also found that PpRom2, which is known to function downstream of the Wsc family proteins in the cell wall integrity pathway, was also involved in sensing methanol. Based on these results, these PpWsc family proteins were demonstrated to be involved in sensing methanol and transmitting the signal via their cytoplasmic tail to the nucleus via PpRom2, which plays a critical role in regulating expression of a subset of methanol‐inducible genes to coordinate well‐balanced methanol metabolism.     

A novel plant-associated thermotolerant alkalophilic methylotroph of the genus Paracoccus

Strain GB isolated from the maize rhizosphere is a gram-negative, aerobic, non-spore-forming, nonpigmented, nonmotile, chemolithotrophic, facultatively methylotrophic bacterium. Cells are cocci or short rods. The strain does not require vitamins. Optimum growth in a medium with methanol occurs at 38-42 degrees C at pH 8.0-9.2. The doubling time is 12 h. In addition to methanol, the bacterium can grow on methylamine, dimethylformamide, acetone, thiosulfate + NaHCO3, and in an atmosphere of H2 + CO2 + O2. Methanol and methylamine are oxidized by the respective dehydrogenases to CO2 via formaldehyde and formate, respectively. The CO2 produced is assimilated via the ribulose bisphosphate pathway. Fatty acids are dominated by cyclopropanoic (58-61%), palmitic (24-26%), and octadecanoic (8-9%) acids. The main phospholipids are phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The major ubiquinone is Q10. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The culture liquid exhibits cytokinin activity. The G + C content of DNA is 62.5 mol %, as determined from the DNA thermal denaturation temperature (Tm). Strain GB shows a moderate degree of DNA-DNA homology (< 40%) with the type representatives of the genus Paracoccus. Based on the data obtained, the bacterium was classified as a new species of this genus, named P. kondratievae.     

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using ¹³C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.     

1株甲基杆菌Methylobacterium sp.WGM16的鉴定及降解甲醇的最佳培养条件

从水稻根部土壤中筛选到1株粉红色、需氧的兼性甲基营养型菌株WGM16,该菌为革兰阴性杆菌。根据菌株16S rRNA基因序列比对分析及结合菌株常规形态特征、生理生化性状的鉴定,将该菌初步鉴定为Methylobacterium sp.PCR扩增到菌株WGM16编码甲醇脱氢酶α-亚基的mxaF基因,表明菌株WGM16中存在甲基营养代谢途径。在培养温度为32℃、以1%的甲醇作为碳源、pH值为8.0的培养条件下,其甲醇降解率可达75%。     

Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth

Mutants of the methylotrophic yeast Hansenula polymorpha deficient in NAD-dependent formaldehyde or formate dehydrogenases have been isolated. They were more sensitive for exogenous methanol but retained the ability for methylotrophic growth. In the medium with methanol the growth yields of the mutant 356–83 deficient in formaldehyde dehydrogenase and of the wild-type strain were identical (0.34 g cells/g methanol) under chemostat cultivation. These results indicate that enzymes of direct formaldehyde oxidation are not indispensable for methylotrophic growth. At the same time inhibition of tricarboxylic acid cycle has resulted in suppression of growth in the media with multicarbon nonfermentable substrates such as glycerol, succinate, ethanol and dihydroxyacetone as well as with methanol, but not with glucose. In the experiments with the wild-type strain H. polymorpha it has been shown that citrate and dihydroxyacetone inhibit the radioactivity incorporation from ¹⁴C-methanol into CO₂. All obtained data indicate that for the dissimilation of methanol and the supplying of energy for methylotrophic growth, the functioning of tricarboxylic acid cycle reactions as oppossed to those of direct formaldehyde oxidation is essential.     

Phylogenetic analysis of aerobic methylotrophic bacteria, using dichloromethane

The phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different C1-assimilation pathways was determined based on 16S ribosomal RNA sequences and DNA-DNA hybridization data. The restricted facultative methylotroph "Methylophilus leisingerii" DM11 with the ribulose monophosphate pathway was found to belong to the genus Methylophilus cluster of the beta subdivision of the phylogenetic kingdom Proteobacteria. The facultative methylotroph Methylorhabdus multivorans DM13 was assigned to a separate branch of the alpha-2 group of Proteobacteria. Paracoccus methylutens DM12, which utilizes C1-compounds via the Calvin cycle was found to belong to the alpha-3 group of the Proteobacteria (more precisely, to the genus Paracoccus cluster). Thus, phylogenetic analysis confirmed the taxonomic status of these recently characterized bacteria. According to the degree of DNA homology, several novel strains of methylotrophic bacteria were divided into three genotypic groups within the alpha-2 group of the Proteobacteria. Genotypic group 1, comprising strains DM1, DM3, and DM5 through DM9, and genotypic group 3, comprising strain DM10, were phylogenetically close to the methylotrophic bacteria of the genus Methylopila, whereas genotypic group 2 (strain DM4) was close to bacteria of the genus Methylobacterium. The genotypic groups obviously represent distinct taxa of methylotrophic bacteria, whose status should be confirmed by phenotypic analysis.     

L-lysine production at 50 degrees C by mutants of a newly isolated and characterized methylotrophic Bacillus sp.

The amino acid L-lysine was produced from homoserine auxotrophic and S-(2-aminoethyl)-L-cysteine-resistant mutants of a newly isolated gram-positive methylotrophic bacterium, capable of growth on methanol at 60 degrees C. The temperature optimum for growth was between 50 and 53 degrees C. These aerobic, gram-positive, endospore-forming, rod-shaped bacteria required biotin and vitamin B12 for growth. Extracts of the bacteria grown on methanol lacked hydroxypyruvate reductase and contained hexulose 6-phosphate synthase activity. Therefore, these bacteria were considered to be type I methylotrophic bacteria of the genus Bacillus. Fed-batch fermentations resulted in cell densities of 50 g of cell dry weight per liter. Biomass yields on carbon, nitrogen, phosphate, and sulfate were determined. Generation of homoserine auxotrophic and amino acid analog-resistant mutants resulted in L-lysine concentrations of nearly 20 g/liter in fed-batch fermentations.     

New unified nomenclature for genes involved in the oxidation of methanol in Gram-negative bacteria

Abstract The system involving the oxidation of methanol to formaldehyde in Gram-negative methylotrophic bacteria is complex. A total of 32 genes have been reported, termed mox , for methanol oxidation, and it is possible that more will be identified. Some mox genes carrying out completely different functions have been given the same designations by different laboratories and others have been given separate designations that were later discovered to be the same. It is now important to change the mox nomenclature to remedy this confusing situation. This communication proposes a new nomenclature for genes involved in methanol oxidation based on currently known linkage groups.     

Comparison of the proteome of Methylobacterium extorquens AM1 grown under methylotrophic and nonmethylotrophic conditions

Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that is capable of growing in the presence of methanol as the sole carbon and energy source, but is also able to grow on a limited number of C(2), C(3), and C(4) compounds, for example succinate. This study provides a proteomic view of the cellular adaptation of M. extorquens AM1 to growth on methanol and succinate, respectively. Cytosolic proteins were separated by two-dimensional gel electrophoresis employing overlapping pH ranges and visualized by silver nitrate or fluorescence staining. A proteomic reference map containing 229 different proteins identified by peptide mass fingerprinting of tryptic fragments was established. Comparative proteome profiling of methanol- and succinate-grown cells led to the identification of 68 proteins that are induced under methylotrophic growth conditions in comparison to growth on succinate. This group includes most proteins known to be directly involved in methanol oxidation to CO(2) and in assimilation of one carbon units by the serine cycle as well as 18 proteins without any assigned function and two proteins with a predicted regulatory function. Furthermore, the proteome analysis revealed putative isoenzymes for formaldehyde-activating enzyme Fae, malyl-CoA lyase, malate-dehydrogenase, and fumarase, that need to be characterized functionally in future studies.     

Initial microbial adhesion is a determinant for the strength of biofilm adhesion

Abstract A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts. Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway. Through investigations with Pichia methanolica , methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase. When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased. How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.