The oxidative photosynthetic carbon cycle or C2 cycle

The oxidative photosynthetic carbon cycle (or C₂ cycle) is the metabolic pathway responsible for photosynthetic oxygen uptake and the light‐dependent production of carbon dioxide that is termed photorespiration. The C₂ and reductive C₃ cycles coexist, and combined, represent total photosynthetic carbon metabolism. A brief historical review is presented beginning with the early observations of the oxygen inhibition of photosynthesis up to the discovery of the oxygenase activity associated with ribulose 1,5‐bisphosphate carboxylase/oxygenase. The properties and the role of the compartmentalization of the enzymes involved with the pathway and the transport of C₂ cycle intermediates are reviewed. The relationship of the C₂ cycle to photorespiratory nitrogen metabolism and other associated metabolic pathways and the properties and regulation of the C₂ cycle in diverse photosynthetic organisms are discussed.     

Biochemical properties and phylogeny of hydroxypyruvate reductases from methanotrophic bacteria with different c<Subscript>1</Subscript>-assimilation pathways

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C₁-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C₁-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.     

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

Background

Methylobacterium extorquens AM1 is an aerobic facultative methylotrophic α-proteobacterium that can use reduced one-carbon compounds such as methanol, but also multi-carbon substrates like acetate (C₂) or succinate (C₄) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock.

Methodology/Principal Findings

We present a comprehensive study of all postulated enzymes for the assimilation of methanol and their regulation in response to the carbon source. Formaldehyde, which is derived from methanol oxidation, is assimilated via the serine cycle, which starts with glyoxylate and forms acetyl-CoA. Acetyl-CoA is assimilated via the proposed ethylmalonyl-CoA pathway, which thereby regenerates glyoxylate. To further the understanding of the central carbon metabolism we identified and quantified all enzymes of the pathways involved in methanol assimilation. We observed a strict differential regulation of their activity level depending on whether C₁, C₂ or C₄ compounds are used. The enzymes, which are specifically required for the utilization of the individual substrates, were several-fold up-regulated and those not required were down-regulated. The enzymes of the ethylmalonyl-CoA pathway showed specific activities, which were higher than the calculated minimal values that can account for the observed growth rate. Yet, some enzymes of the serine cycle, notably its first and last enzymes serine hydroxymethyl transferase and malate thiokinase, exhibit much lower values and probably are rate limiting during methylotrophic growth. We identified the natural C₁ carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.

Conclusion/Significance

This study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps.     

Methanol-Assimilation durch ein acidophiles Bacterium der Gattung Acetobacter

Generally, methylotrophic bacteria grow optimally in a pH range between 6 and 7.2. The assimilation of methanol can take place via several pathways. Acetobacter methanolicus preiers an acidic pH range for growth, the pH optimum is about 4, and it uses the FBP variant for methanol assimilation. The latter is interesting from a regulatory point of view because phosphofructokinase disappears during growth on glucose, which is assimilated via the hexosemonophosphate pathway. Since Entner-Doudoroff enzymes and phosphoketolase are absent in A. `ethanolicus as well as in non-methylotrophic Acetobacter and Gluconobacter species phosphofructokinase becomes a key enzyme of the assimilation of methanol. Although A. methanolicus uses the hexulosephosphate pathway the growth yield on methanol is smaller than with other “hexulosephosphate pathway bacteria” e. g. with obligate methanol assimilating bacteria. At first sight it may appear that the acidic optimum pH is responsible for the smaller growth yield and the discrepancy between the experimental and predicted values. The relationship between the dependence on and the protection from, high external proton concentration on the one hand and the causes of the low growth yield on the other are discussed. Accordingly, A. methanolicus and another heterotrophic acidophiles seem to be acidoresistant above all, their machinery guaranteeing the protection from the high proton concentration is responsible for the acidophily and the low growth efficiency is caused by a simple respiratory chain.     

Comparative proteomic analysis reveals insights into anoxic growth of Methyloversatilis universalis FAM5 on methanol and ethanol

Methyloversatilis universalis FAM5 is a facultative methylotrophic bacterium that has been found in a variety of natural and engineered ecosystems. The goal of this study was to investigate M. universalis FAM5 responses to different electron/carbon donors, e.g. methanol or ethanol, during anoxic growth in chemostats with nitrate as the electron acceptor. During steady‐state anoxic growth on either methanol or ethanol, over 90% of the influent nitrate was reduced primarily to nitrite. The cell yield on methanol was lower, possibly due to high energy requirements for C₁ assimilation. Label‐free proteomics further revealed that methanol‐grown cells displayed elevated concentrations of the enzymes involved in C₁ metabolism (H₄MPT/H₄F pathways, formate oxidation and serine cycle). In contrast, C₂ metabolism (glyoxylate shunt and tri‐carboxylic acid cycle) and polyhydroxy‐β‐butyrate (PHB) synthesis related proteins were overrepresented during subsequent growth on ethanol. Notably, the expression of respiratory nitrate reductase was not affected by the carbon sources applied. Furthermore, the changes in the proteome upon switching back to methanol were mostly reversible. Therefore, M. universalis displays wide‐ranging responses to adapt between growth on methanol and ethanol. Such metabolic versatility could be particularly useful in wastewater treatment systems, which need to switch between different electron donors, while still reliably meeting effluent nitrogen discharge goals.     

A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli

Most central metabolic pathways such as glycolysis, fatty acid synthesis, and the TCA cycle have complementary pathways that run in the reverse direction to allow flexible storage and utilization of resources. However, the glyoxylate shunt, which allows for the synthesis of four-carbon TCA cycle intermediates from acetyl-CoA, has not been found to be reversible to date. As a result, glucose can only be converted to acetyl-CoA via the decarboxylation of the three-carbon molecule pyruvate in heterotrophs. A reverse glyoxylate shunt (rGS) could be extended into a pathway that converts C₄ carboxylates into two molecules of acetyl-CoA without loss of CO₂. Here, as a proof of concept, we engineered in Escherichia coli such a pathway to convert malate and succinate to oxaloacetate and two molecules of acetyl-CoA. We introduced ATP-coupled heterologous enzymes at the thermodynamically unfavorable steps to drive the pathway in the desired direction. This synthetic pathway in essence reverses the glyoxylate shunt at the expense of ATP. When integrated with central metabolism, this pathway has the potential to increase the carbon yield of acetate and biofuels from many carbon sources in heterotrophic microorganisms, and could be the basis of novel carbon fixation cycles.     

Formate as the main branch point for methylotrophic metabolism in Methylobacterium extorquens AM1

In serine cycle methylotrophs, methylene tetrahydrofolate (H4F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H4F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H4F to generate methylene H4F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H4F via H4F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H4F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H4F and formate are unable to grow on methanol, suggesting that this route for methylene H4F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in Methylobacterium extorquens AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H4F synthesis pathway through formate. These results suggested that the methylene H4F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in M. extorquens AM1.     

A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens.

Cell extracts were used to determine the enzymes involved in the intermediary carbon metabolism of several strains of Shewanella putrefaciens. Enzymes of the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) were detected, but those of the Embden-Meyerhof-Parnas pathway were not. While several tricarboxylic acid cycle enzymes were present under both aerobic and anaerobic conditions, two key enzymes (2-oxoglutarate dehydrogenase and pyruvate dehydrogenase) were greatly diminished under anaerobic conditions. Extracts of cell grown anaerobically on formate as the sole source of carbon and energy were positive for hydroxypyruvate reductase, the key enzyme of the serine pathway in other methylotrophs, while no hexulose synthase activity was seen.     

Regulation of metabolic pathways in sulfobacilli under different aeration regimes

A comparative study of the activities of the enzymes of carbon metabolism from the cells of moderately thermophilic chemolithotrophic bacteria Sulfobacillus sibiricus (strains N1 and SSO) and Sulfobacillus thermosulfidooxidans subsp. asporogenes (strain 41) was carried out grown in a high layer of medium without forced aeration and cells grown with intense aeration. Limited air access to the growing S. sibiricus N1 cells resulted in switching from the pentose phosphate pathway of glucose metabolism to the Entner-Doudoroff pathway while the Embden-Meyerhof-Parnas pathway persisted. Irrespective of the level of the aeration, in the cells of S. sibiricus SSO and S. thermosulfidooxidans subsp. asporogenes 41, degradation of the glucose occurred via the Entner-Doudoroff and pentose phosphate metabolic pathways, respectively, as well as via the Embden-Meyerhof-Parnas pathway. Prolonged growth of S. sibiricus, strains N1 and SSO, in a high layer of the medium without forced aeration led to the repression of synthesis of most of the tricarboxylic acid cycle (TCA cycle) enzymes, in particular dehydrogenases, as well as of some carboxylases including RuBisCO. The traits of carbon metabolism in various strains of Sulfobacillus under conditions of oxygen deficiency are discussed.     

Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium''s ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.     

Constraint-Based Model of Shewanella oneidensis MR-1 Metabolism: A Tool for Data Analysis and Hypothesis Generation

Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level.     

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and O-12, growing in the temperature range from 30 to 61°C with a optimum at 55°C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes and genes of membrane-bound methane monooxygenase, as well as on phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulosephosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55°C than in cells grown at 37°C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH₄ ⁺ assimilation occurred through reductive amination of -ketoglutarate and via the glutamate cycle. The relationship between the physiological and biochemical peculiarities of the isolates and their thermophilic nature is discussed.     

Effects of Exogenous Factors on Enzymes of Carbon Metabolism in Thermoacidophilic Bacteria of the GenusSulfobacillus

Aerobic thermoacidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans1269^Tand Sulfobacillus thermosulfidooxidanssubsp. asporogenes41 were shown to be resistant to stress factors, including high concentrations of Zn²⁺(0.8 M) and supraoptimal concentrations of H⁺(pH 1.2). The growth and biomass gain rates decreased, but bacteria retained their functions. The activity of nearly all enzymes involved in carbon metabolism decreased. Glucose was primarily metabolized via the Entner–Doudoroff pathway. The activity of tricarboxylic acid cycle enzymes decreased compared to that in cells grown under normal conditions. After saturation of the growth medium with 5 vol % CO₂, sulfobacteria utilized glucose by the Embden–Meyerhof and pentose phosphate pathways under mixotrophic conditions.     

Bacterial yields on methanol,methylamine, formaldehyde,and formate

Several bacteria utilizing C₁-compounds as sole carbon sources were grown on these substrates in continuous culture. The molar yield values (g of cell dry wt/mol of substrate utilized) of bacteria which utilize C₁-compounds via the ribulose monophosphate pathway were between 15.7 to 17.3 when grown on methanol; while the molar yield values of bacteria which use the serine pathway for the assimilation of C₁-compounds varied between 9.8 and 13.1. The molar yield values of different bacteria which use the serine pathway decreased as the oxidation levels of the C₁-growth substrates increased. On formaldehyde the values were between 7.2 to 9.6, whereas on formate the values varied from 3.3 to 6.9. It appears that bacteria utilize C_l-compounds more efficiently via the ribulose monophosphate pathway than via the serine pathway. The oxidation step from methanol to formaldehyde (and from methylamine to formaldehyde) in the bacteria studied may be energy yielding. A comparison has been made between the experimental yield values obtained and theoretical values.     

The anaerobic degradation of l-serine and l-threonine in enterobacteria: networks of pathways and regulatory signals

The mechanisms controlling the biosynthesis and degradation of l-serine and l-threonine are remarkably complex. Their metabolism forms a network of pathways linking several amino acids, central primary metabolites such as pyruvate, oxaloacetate and 3-phosphoglycerate, and C₁ metabolism. Studies on the degradation of these amino acids in Escherichia coli have revealed the involvement of fascinating enzymes that utilise quite diverse catalytic mechanisms. Moreover, it is emerging that both environmental and metabolic signals have a major impact in controlling enzyme synthesis. This is exemplified by the anaerobically regulated tdc operon, which encodes a metabolic pathway for the degradation of serine and threonine. Studies on this pathway are beginning to provide insights into how an organism adapts its genetic makeup to meet the physiological demands of the cell. Received: 30 August 1998 / Accepted: 9 October 1998     

Multiphyletic origins of methylotrophy in Alphaproteobacteria,exemplified by comparative genomics of Lake Washington isolates

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the Alphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N‐methylglutamate pathway, the two types of methanol dehydrogenase (MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl‐CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of Alphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions.     

Photosynthetic carbon metabolism of the cool-temperate C4 grass Spartina anglica Hubb.

The aim of this work was to describe the photosynthetic carbon metabolism of the cooltemperate C₄ grass Spartina anglica. With the exception of pyruvate, phosphate dikinase and pyruvate kinase, the maximum catalytic activities in leaves of putative enzymes of the C₄ cycle of a phosphoenolpyruvate-carboxykinase C₄ plant were considerably in excess of the observed, steady-state rate of photosynthesis, and were comparable with the maximum catalytic activities of key enzymes of the reductive pentose-phosphate pathway. Radioactive carbon from ¹⁴CO₂ supplied to attached leaves during steady-state photosynthesis appeared first in malate and aspartate from which it moved to intermediates of the reductive pentose-phosphate pathway, and then to sucrose. These experiments show that photosynthetic carbon metabolism in this cool-temperate C₄ plant is similar to that of C₄ plants of hotter climates.