Mannitol biosynthesis is required for plant pathogenicity by Alternaria alternata

Mannitol has been hypothesized to play a role in antioxidant defense. In previous work, we confirmed the presence of the two mannitol biosynthetic enzymes, mannitol dehydrogenase (MtDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH), in the fungus Alternaria alternata and created disruption mutants for both enzymes. These mutants were used to investigate the role of mannitol in pathogenicity of A. alternata on its host, tobacco. Conidia of all mutants were viable and germinated normally. GC-MS analysis demonstrated elevated levels of trehalose in the mutants, suggesting that trehalose may substitute for mannitol as a storage compound for germination. Tobacco inoculation showed no reduction in lesion severity caused by the MtDH mutant as compared with wild type; however, the MPDH mutant and a mutant in both enzymes caused significantly less disease. Microscopy analysis indicated that the double mutant was unaffected in the ability to germinate and produce appressoria on tobacco leaves and elicited a defense response from the host, indicating that it was able to penetrate and infect the host. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for spore germination either in vitro or in planta or for initial infection.     

Mannitol Metabolism in Lentinus edodes, the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.     

The crystallographic structure of the mannitol 2-dehydrogenase NADP+ binary complex from Agaricus bisporus

Mannitol, an acyclic six-carbon polyol, is one of the most abundant sugar alcohols occurring in nature. In the button mushroom, Agaricus bisporus, it is synthesized from fructose by the enzyme mannitol 2-dehydrogenase (MtDH; EC ) using NADPH as a cofactor. Mannitol serves as the main storage carbon (up to 50% of the fruit body dry weight) and plays a critical role in growth, fruit body development, osmoregulation, and salt tolerance. Furthermore, mannitol dehydrogenases are being evaluated for commercial mannitol production as alternatives to the less efficient chemical reduction of fructose. Given the importance of mannitol metabolism and mannitol dehydrogenases, MtDH was cloned into the pET28 expression system and overexpressed in Escherichia coli. Kinetic and physicochemical properties of the recombinant enzyme are indistinguishable from the natural enzyme. The crystal structure of its binary complex with NADP was solved at 1.5-A resolution and refined to an R value of 19.3%. It shows MtDH to be a tetramer and a member of the short chain dehydrogenase/reductase family of enzymes. The catalytic residues forming the so-called catalytic triad can be assigned to Ser(149), Tyr(169), and Lys(173).     

Mannitol and Fructose Catabolic Pathways of Pseudomonas aeruginosa Carbohydrate-Negative Mutants and Pleiotropic Effects of Certain Enzyme Deficiencies

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

Mannitol oxidation in two Micromonospora isolates and in representative species of other actinomycetes.

Mannitol kinase and mannitol-1-phosphate dehydrogenase activities were detected in two Micromonospora isolates. The presence of these enzyme activities indicates that mannitol is catabolized first to mannitol-1-phosphate and then to fructose-6-phosphate. Mannitol-oxidizing enzymes were also surveyed in representative species of four other genera of actinomycetes. Mannitol-1-phosphate dehydrogenase was detected in cell-free extracts of Streptomyces lactamdurans. In contrast, cell-free extracts of Mycobacterium smegmatis, Nocardia erythrophila, Streptomyces lavendulae, and Actinoplanes missouriensis contained mannitol dehydrogenase activity but no detectable mannitol-1-phosphate dehydrogenase activity. The mannitol dehydrogenase activities in the latter species support the operation of a pathway for catabolism of mannitol that involves the oxidation of mannitol to fructose, followed by phosphorylation to fructose-6-phosphate.     

Mannitol oxidation in two Micromonospora isolates and in representative species of other actinomycetes.

Mannitol kinase and mannitol-1-phosphate dehydrogenase activities were detected in two Micromonospora isolates. The presence of these enzyme activities indicates that mannitol is catabolized first to mannitol-1-phosphate and then to fructose-6-phosphate. Mannitol-oxidizing enzymes were also surveyed in representative species of four other genera of actinomycetes. Mannitol-1-phosphate dehydrogenase was detected in cell-free extracts of Streptomyces lactamdurans. In contrast, cell-free extracts of Mycobacterium smegmatis, Nocardia erythrophila, Streptomyces lavendulae, and Actinoplanes missouriensis contained mannitol dehydrogenase activity but no detectable mannitol-1-phosphate dehydrogenase activity. The mannitol dehydrogenase activities in the latter species support the operation of a pathway for catabolism of mannitol that involves the oxidation of mannitol to fructose, followed by phosphorylation to fructose-6-phosphate.     

Enzymes related to fructose utilization in Pseudomonas cepacia

Growth of Pseudomonas cepacia on fructose, mannitol, or sorbitol depended on formation of an inducible fructokinase (forming fructose-6-phosphate) and the presence of enzymes of the Entner-Doudoroff pathway. Mutants deficient in any of these enzymes failed to utilize the aforementioned carbohydrates. Fructokinase deficiency did not affect growth of the bacteria on glucose. Fructose was accumulated intracellularly by active transport. Mutants blocked in transport of fructose grew normally on mannitol or sorbitol despite their inability to utilize fructose. Growth on either of these hexitols or on galactitol was accompanied by induction of two hexitol dehydrogenases, one active primarily with mannitol and the other active with sorbitol and galactitol. As expected, a mutant deficient in mannitol dehydrogenase failed to utilize mannitol as a carbon and energy source but grew normally on sorbitol and galactitol. Extracts of bacteria grown on fructose, mannitol, or sorbitol and higher levels of phosphoglucose isomerase than extracts of bacteria grown on alternate carbon sources such as citrate or phthalate. The higher levels were due to appearance of a second phosphoglucose isomerase species not present in cells with the lower activity. The results indicate that the initial steps in fructose utilization by P. cepacia differ from those of most other pseudomonads, which transport fructose by phosphoenolpyruvate-dependent translocation, forming fructose-1-phosphate, and suggest that degradation of fructose, mannitol, and sorbitol occurs primarily via the Entner-Doudoroff pathway.     

Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism.

Mutant cells of mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined for their ability to synthesize alginic acid in resting cell suspensions. Unlike the wild-type strain which synthesizes alginic acid from glycerol, fructose, mannitol, glucose, gluconate, glutamate, or succinate, mutants lacking specific enzymes of carbohydrate metabolism are uniquely impaired. A phosphoglucose isomerase mutant did not synthesize the polysaccharide from mannitol, nor did a glucose 6-phosphate dehydrogenase mutant synthesize the polysaccharide from mannitol or glucose. Mutants lacking the Entner-Doudoroff pathway dehydrase or aldolase failed to produce alginate from mannitol, glucose, or gluconate, as a 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase mutant failed to produce from glutamate or succinate. These results demonstrate the primary role of the Entner-Doudoroff pathway enzymes in the synthesis of alginate from glucose, mannitol, or gluconate and the role of glyceraldehyde 3-phosphate dehydrogenase reaction for the synthesis from gluconeogenic precursors such as glutamate. The virtual absence of any activity of phosphomannose isomerase in cell extracts of several independent mucoid bacteria and the impairment of alginate synthesis from mannitol in mutants lacking phosphoglucose isomerase or glucose 6-phosphate dehydrogenase rule out free mannose 6-phosphate as an intermediate in alginate biosynthesis.     

Production of NADPH in the mannitol cycle and its relation to polyketide formation in Alternaria alternata.

The enzymes mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternaria alternata. One turn of the cycle gives the net result: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A. alternata shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.     

Glucose-1-Phosphate-Negative Mutant of Agrobacterium tumefaciens

Glucose-1-phosphate-negative mutants that are unable to grow in a synthetic medium containing glucose-1-phosphate (G-1-P) as a sole carbon source were isolated by treatment of Agrobacterium tumefaciens IAM 1525 with N-methyl-N'-nitro-N-nitrosoguanidine. All of the enzymes involved in G-1-P metabolism (glucoside-3-dehydrogenase, 3-ketoglucose-1-phosphate-degrading enzyme, alpha-glucosidase, and phosphatases) were detected in the sonic extract prepared from resting cells of one of the mutants, strain M-24, in approximately equal levels to those in the parent strain. Resting cells of the mutant oxidized G-1-P to 3-ketoglucose-1-phosphate (3KG-1-P), the first product in G-1-P metabolism by the bacterium, with little subsequent degradation, whereas the parent showed further degradation of G-1-P via 3KG-1-P. Glucoside-3-dehydrogenase catalyzing 3-ketoglucoside formation was readily released from cells by osmotic shock, whereas the 3KG-1-P-degrading enzyme was not released. Thus, the former and the latter enzymes might be at different intracellular loci, such as periplasm and cytoplasm, respectively. It is suggested that the mutant strain M-24 is a G-1-P-negative mutant deficient in a 3KG-1-P transport system located on the cytoplasmic membrane.     

Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.

The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.     

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.     

Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch)

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant-Microbe Interact. 18, 110-115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.     

Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana

Knockout and complement mutants of mannitol-1-phosphate dehydrogenase (MPD) and mannitol dehydrogenase (MTD) were constructed to probe the roles of both enzymes in the mannitol metabolism and multi-stress tolerances of entomopathogenic fungus Beauveria bassiana. Compared with wild-type and complement mutants, ΔBbMPD lost 99.5% MPD activity for reducing fructose-6-phosphate to mannitol-1-phosphate while ΔBbMTD lost 78.9% MTD activity for oxidizing mannitol to fructose. Consequently, mannitol contents in mycelia and conidia decreased 68% and 83% for ΔBbMPD, and 16% and 38% for ΔBbMTD, accompanied by greatly enhanced trehalose accumulations due to 81-87% decrease in their neutral trehalase expression. Mannitol as mere carbon source in a nitrate-based minimal medium suppressed the colony growth of ΔBbMTD instead of ΔBbMPD, and delayed more conidial germination of ΔBbMTD than ΔBbMPD. Based on median lethal responses, conidial tolerances to H(2) O(2) oxidation, UV-B irradiation and heat stress at 45°C decreased 38%, 39% and 22% in ΔBbMPD, and 18%, 16% and 11% in ΔBbMTD respectively. Moreover, ΔBbMPD and ΔBbMTD lost 14% and 7% of their virulence against Spodoptera litura larvae respectively. Our findings highlight the primary roles of MPD and MTD in mannitol metabolism and their significant contributions to multi-stress tolerances and virulence influential on the biocontrol potential of B.bassiana.     

Catabolism of Fructose and Mannitol in Clostridium thermocellum: Presence of Phosphoenolpyruvate: Fructose Phosphotransferase, Fructose l-Phosphate Kinase, Phosphoenolpyruvate: Mannitol Phosphotransferase, and Mannitol l-Phosphate Dehydrogenase in Cell Extracts

Fructose and mannitol are fermented by Clostridium thermocellum in a medium containing salts and 0.5% yeast extract. The initial reaction in the catabolism of fructose was found to be the formation of fructose l-phosphate by phosphoenolpyruvate (PEP):fructose phosphotransferase which resembles the Kundig-Roseman phosphotransferase system. The phosphorylation of fructose l-phosphate to form fructose-1, 6-diphosphate is catalyzed by fructose l-phosphate kinase. Fructose-1, 6-diphosphate can be further metabolized by the Embden-Meyerhof pathway. The formation of both PEP:fructose phosphotransferase and fructose l-phosphate kinase is induced by growth in fructose medium. Mannitol catabolism was found to proceed by the phosphorylation of mannitol by PEP:mannitol phosphotransferase to form mannitol l-phosphate. Mannitol l-phosphate is converted to fructose 6-phosphate by a nicotinamide adenine dinucleotide-specific mannitol l-phosphate dehydrogenase. The fructose 6-phosphate formed in the reaction can enter the glycolytic scheme. The formation of both PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase is induced by growth in mannitol medium. Evidence is presented for the induction by mannitol of PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase in suspensions of fructose-grown cells.     

Mutations Affecting the Dissimilation of Mannitol by Escherichia coli K-12

Mutants of Escherichia coli K-12 defective in the mannitol-specific enzyme II complex of the phosphoenolpyruvate phosphotransferase system (PTS) or lacking mannitol-1-phosphate dehydrogenase have been isolated. These mutants fail only to grow on mannitol. Growth of the dehydrogenase-negative mutant on casein hydrolysate can be abruptly inhibited by exposure to mannitol. A mutant with constitutive expression of both of these enzymes has also been isolated. All three mutations are clustered in a region represented at min 71 of the Taylor map. In a mutant with less than 5% of the activity of enzyme I of the PTS, both the enzyme II complex and the dehydrogenase remain inducible by mannitol. In the mutant defective in the enzyme II complex, mannitol is able to induce the dehydrogenase. Thus, mannitol, rather than its phosphorylated product, seems to be the inducer.     

The mannitol cycle in Pleurotus ostreatus (Florida)

In mushroom, presence of the mannitol cycle has not been reported so far although the polyol is supposed to be generated by the reduction of fructose by mannitol dehydrogenase. This study submits evidence for the presence of the mannitol cycle in Pleurotus ostreatus. The key enzyme of the cycle, mannitol-1-phosphate dehydrogenase (M1PDH), was present appreciably in all the developmental stages of the mushroom. However, the enzyme level dropped significantly at the onset of sporulation. The presence of M1DPH was confirmed by isozyme analysis and RT-PCR mediated amplification of a approximately 400 bp DNA fragment.     

Biochemical characterization of mannitol metabolism in the unicellular red alga Dixoniella grisea (Rhodellophyceae)

The mannitol cycle has been verified in a unicellular red alga (Rhodellophyceae) for the first time. All four enzymes involved in the cycle (mannitol-1-phosphate dehydrogenase, Mt1PDH: EC 1.1.1.17; mannitol-1-phosphatase, Mt1Pase: EC 3.1.3.22; mannitol dehydrogenase, MtDH: 1.1.1.67; hexokinase, HK: 2.7.1.1.) were detected and characterized in crude algal extracts from Dixoniella grisea. These enzymes, with the exception of Mt1Pase, were specific to their corresponding substrates and nucleotides. The activities of enzymes in the anabolic pathway (fructose-6-P reduction by Mt1PDH and mannitol-6-P reduction by Mt1Pase) were at least 2- to 4-fold greater than those of the catabolic pathway (mannitol oxidation by MtDH and fructose oxidation by HK). There appears to be, therefore, a net carbon flow in D. grisea towards a high intracellular mannitol pool. The mannitol cycle guarantees a rapid accumulation or degradation of mannitol within algal cells in response to changing salinity in natural habitats. Moreover, the demonstration of the mannitol cycle within the Rhodellophyceae provides evidence that this metabolic pathway is of ancient origin in the red algal lineage.     

Mannitol in six autotrophic stramenopiles and Micromonas

Mannitol plays a central role in brown algal physiology since it represents an important pathway used to store photoassimilate. Several specific enzymes are directly involved in the synthesis and recycling of mannitol, altogether forming the mannitol cycle. The recent analysis of algal genomes has allowed tracing back the origin of this cycle in brown seaweeds to a horizontal gene transfer from bacteria, and furthermore suggested a subsequent transfer to the green micro-alga Micromonas. Interestingly, genes of the mannitol cycle were not found in any of the currently sequenced diatoms, but were recently discovered in pelagophytes and dictyochophytes. In this study, we quantified the mannitol content in a number of ochrophytes (autotrophic stramenopiles) from different classes, as well as in Micromonas. Our results show that, in accordance with recent observations from EST libraries and genome analyses, this polyol is produced by most ochrophytes, as well as the green alga tested, although it was found at a wide range of concentrations. Thus, the mannitol cycle was probably acquired by a common ancestor of most ochrophytes, possibly after the separation from diatoms, and may play different physiological roles in different classes.Key words: algae, stramenopiles, mannitol cycle, primary metabolism, osmotic stress, evolutionBrown algae produce mannitol directly from the photoassimilate fructose-6-phosphate. Its metabolism occurs through the mannitol cycle, which involves four enzymatic reactions: (1) the reduction of fructose-6-phosphate (F6P) to mannitol-1-phosphate (M1P) via the activity of an M1P dehydrogenase (M1PDH); (2) the production of mannitol from M1P via an M1P phosphatase (M1Pase); (3) the oxidation of mannitol via the activity of a mannitol-2-dehydrogenase (M2DH) yielding fructose; and (4) the phosphorylation of fructose yielding F6P and involving a hexokinase (HK).^1,2 The first completed draft of a brown algal genome enabled the identification of candidate genes for each of these steps.³ As these genes were not found in the genomes of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, mannitol metabolism in stramenopiles was considered a trait typical for brown algae. The corresponding genes were thought to have been acquired horizontally from bacteria and subsequently transferred to some green algae.⁴ Recently, however, homologs of several genes of the cycle were also found in the genome of the pelagophyte Aureococcus anophagefferens⁵ and an EST library produced for the dictyochophyte Pseudochattonella farcimen (Dittami et al. personal communication). These observations prompted us to examine the presence of mannitol in a range of strains covering different classes of autotrophic stramenopiles (ochrophytes). In addition, because of the identification of genes encoding enzymes for the production of mannitol through the mannitol cycle in the green alga Micromonas, one strain of this genus was also included in our analysis.