DHA system mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418.

In Klebsiella pneumoniae NCIB 418, the pathways normally responsible for aerobic growth on glycerol and sn-glycerol 3-phosphate (the glp system) are superrepressed. However, aerobic growth on glycerol can take place by the intervention of the NAD-linked glycerol dehydrogenase and the ATP-dependent dihydroxyacetone kinase of the dha system normally inducible only anaerobically by glycerol or dihydroxyacetone. Conclusive evidence that the dha system is responsible for both aerobic and anaerobic dissimilation of glycerol was provided by a Tn5 insertion mutant lacking dihydroxyacetone kinase. An enzymatically coupled assay specific for this enzyme was devised. Spontaneous reactivation of the glp system was achieved by selection for aerobic growth on sn-glycerol 3-phosphate or on limiting glycerol as the sole carbon and energy source. However, the expression of this system became constitutive. Aerobic operation of the glp system highly represses synthesis of the dha system enzymes by catabolite repression.     

Anaerobic growth of Escherichia coli on glycerol by importing genes of the dha regulon from Klebsiella pneumoniae

The dha regulon of Klebsiella pneumoniae specifying fermentative dissimilation of glycerol was mobilized by the broad-host-range plasmid RP4:mini Mu and introduced conjugatively into Escherichia coli. The recipient E. coli was enabled to grow anaerobically on glycerol without added hydrogen acceptors, although its cell yield was less than that of K. pneumoniae. The reduced cell yield was probably due to the lack of the coenzyme-B12-dependent glycerol dehydratase of the dha system. This enzyme initiates the first step in an auxiliary pathway for disposal of the extra reducing equivalents from glycerol. The lack of this enzyme would also account for the absence of 1,3-propanediol (a hallmark fermentation product of glycerol) in the spent culture medium. In a control experiment, a large quantity of this compound was detected in a similar culture medium following the growth of K. pneumoniae. The other three known enzymes of the dha system, glycerol dehydrogenase, dihydroxyacetone kinase and 1,3-propanediol oxidoreductase, however, were synthesized at levels comparable to those found in K. pneumoniae. Regulation of the dha system in E. coli appeared to follow the same pattern as in K. pneumoniae: the three acquired enzymes were induced by glycerol, catabolite repressed by glucose, and glycerol dehydrogenase was post-translationally inactivated during the shift from anaerobic to aerobic growth. The means by which the E. coli recipient can achieve redox balance without formation of 1,3-propanediol during anaerobic growth on glycerol remains to be discovered.     

Insights into the genome of the enteric bacterium Escherichia blattae: cobalamin (B12) biosynthesis, B12-dependent reactions, and inactivation of the gene region encoding B12-dependent glycerol dehydratase by a new mu-like prophage

The enteric bacterium Escherichia blattae has been analyzed for the presence of cobalamin (B12) biosynthesis and B12-dependent pathways. Biochemical studies revealed that E. blattae synthesizes B12 de novo aerobically and anaerobically. Genes exhibiting high similarity to all genes of Salmonella enterica serovar Typhimurium, which are involved in the oxygen-independent route of B12 biosynthesis, were present in the genome of E. blattae DSM 4481. The dha regulon encodes the key enzymes for the anaerobic conversion of glycerol to 1,3-propanediol, including coenzyme B12-dependent glycerol dehydratase. E. blattae DSM 4481 lacked glycerol dehydratase activity and showed no anaerobic growth with glycerol, but the genome of E. blattae DSM 4481 contained a dha regulon. The E. blattaedha regulon is unusual, since it harbors genes for two types of dihydroxyacetone kinases. The major difference to dha regulons of other enteric bacteria is the inactivation of the dehydratase-encoding gene region by insertion of a 33,339-bp prophage (MuEb). Sequence analysis revealed that MuEb belongs to the Mu family of bacteriophages. The E. blattae strains ATCC 33429 and ATCC 33430 did not contain MuEb. Accordingly, both strains harbored an intact dehydratase-encoding gene region and fermented glycerol. The properties of the glycerol dehydratases and the correlating genes (dhaBCE) of both strains were similar to other B12-dependent glycerol and diol dehydratases, but both dehydratases exhibited the highest affinity for glycerol of all B12-dependent dehydratases characterized so far. In addition to the non-functional genes encoding B12-dependent glycerol dehydratase, the genome of E. blattae DSM 4481 contained the genes for only one other B12-dependent enzyme, the methylcobalamin-dependent methionine synthase.     

Glycerol kinase as a substitute for dihydroxyacetone kinase in a mutant of Klebsiella pneumoniae.

With dihydroxyacetone as the sole source of carbon and energy, constitutively synthesized glycerol kinase of the glp system supported aerobic growth of Klebsiella pneumoniae mutants lacking the inducible dihydroxyacetone kinase of the dha system. Glycerol kinase had an apparent Km of 0.01 mM for its physiological substrate and 1 mM for its surrogate substrate. However, the growth rate on dihydroxyacetone of cells relying on glycerol kinase increased with the concentration of the carbon and energy source up to 50 mM, suggesting that permeation is rate limiting.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

Hydrogen peroxide mediates the oxidative inactivation of enzymes following the switch from anaerobic to aerobic metabolism in Klebsiella pneumoniae

Klebsiella pneumoniae utilizes distinct pathways for the anaerobic and aerobic metabolism of glycerol. During anaerobic growth, glycerol is first converted to dihydroxyacetone by glycerol dehydrogenase; subsequent phosphorylation yields dihydroxyacetone phosphate. During aerobic growth, glycerol is initially phosphorylated to yield glycerol 3-phosphate; subsequent reduction then gives dihydroxyacetone phosphate. A coordinated response occurs when anaerobically growing cells are switched to aerobic conditions. Synthesis of glycerol dehydrogenase is repressed, glycerol dehydrogenase is inactivated, and the protein is degraded. Ethanol dehydrogenase and propanediol oxidoreductase are also inactivated when cells are exposed to oxygen (Johnson, E. A., Levine, R. L., and Lin, E. C. C. (1985) J. Bacteriol. 164, 479-483). Exposure of anaerobically growing cells to low concentrations of hydrogen peroxide also inactivated these three enzymes and led to rapid degradation of glycerol dehydrogenase. Glycerol dehydrogenase was purified and characterized after in vivo oxidative modification initiated by hydrogen peroxide. No differences in molecular weight, amino acid composition, or Km were detected between the native and oxidatively modified forms, although the modified enzyme had only 10% of the catalytic activity of the native form. The oxidatively modified enzyme was very susceptible to degradation by subtilisin while the native enzyme was resistant. Chloramphenicol prevented the inactivation and degradation of glycerol dehydrogenase caused by exposure to oxygen but did not block that caused by hydrogen peroxide. Thus, protein synthesis appears necessary for in vivo oxidative modification caused by exposure to oxygen but is not necessary when the process is initiated by exposure to hydrogen peroxide. The newly synthesized protein(s) presumably catalyzes the production of hydrogen peroxide which is required for the metal-catalyzed oxidative modification of susceptible enzymes.     

Biosynthesis and catabolism of 6-deoxy L-talitol by Klebsiella aerogenes mutants.

A mutant strain of Klebsiella aerogenes was constructed and, when incubated anaerobically with L-fucose and glycerol, synthesized and excreted a novel methyl pentitol, 6-deoxy L-talitol. The mutant was constitutive for the synthesis of L-fucose isomerase but unable to synthesize L-fuculokinase activity. Thus, it could convert the L-fucose to L-fuculose but was incapable of phosphorylating L-fuculose to L-fuculose 1-phosphate. The mutant was also constitutive for the synthesis of ribitol dehydrogenase, and in the presence of sufficient reducing power this latter enzyme catalyzed the reduction of the L-fuculose to 6-deoxy L-talitol. The reducing equivalents required for this reaction were generated by the oxidation of glycerol to dihydroxyacetone with an anaerobic glycerol dehydrogenase. The parent strain of K. aerogenes was unable to utilize the purified 6-deoxy L-talitol as a sole source of carbon and energy for growth; however, mutant could be isolated which had gained this ability. Such mutants were found to be constitutive for the synthesis of ribitol dehydrogenase and were thus capable of oxidizing 6-deoxy L-talitol to L-fuculose. Further metabolism of L-fuculose was shown by mutant analysis to be mediated by the enzymes of the L-fucose catabolic pathway.     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

Physiologic Mechanisms Involved in Accumulation of 3-Hydroxypropionaldehyde during Fermentation of Glycerol by Enterobacter agglomerans

When grown in 700 mM glycerol within the pH range 6.0 to 7.5, anaerobic pH-regulated cultures of Enterobacter agglomerans exhibited an extracellular accumulation of 3-hydroxypropionaldehyde (3-HPA). This phenomenon, which causes fermentation cessation, occurred earlier when pH was low. In contrast, substrate consumption was complete at pH 8. Levels of glycerol-catabolizing enzymes, i.e., glycerol dehydrogenase and dihydroxyacetone kinase for the oxidative route and glycerol dehydratase and 1,3-propanediol dehydrogenase for the reductive route, as well as the nucleotide pools were determined periodically in the pH 7- and pH 8-regulated cultures. A NAD/NADH ratio of 1.7 was correlated with the beginning of the production of the inhibitory metabolite. Further accumulation was dependent on the ratio of glycerol dehydratase activity to 1,3-propanediol dehydrogenase activity. For a ratio higher than 1, 3-HPA was produced until fermentation ceased, which occurred for the pH 7-regulated culture. At pH 8, a value below 1 was noticed and 3-HPA accumulation was transient, while the NAD/NADH ratio decreased. The low rate of glycerol dissimilation following the appearance of 3-HPA in the culture medium was attributed to the strong inhibitory effect exerted by 3-HPA on glycerol dehydrogenase activity.     

Independent constitutive expression of the aerobic and anaerobic pathways of glycerol catabolism in Klebsiella aerogenes.

Klebsiella aerogenes dissimilates glycerol aerobically via an inducible pathway initiated by an adenosine triphosphate-linked kinase that converts the substrate to sn-glycerol 3-phosphate. Phosphorylated glycerol is then dehydrogenated to dihydroxyacetone phosphate by an enzyme characteristic of a flavoprotein. Anaerobically, the organism dissimilates glycerol via an inducible pathway initiated by a nicotinamide adenine dinucleotide-linked dehydrogenase that converts the substrate to dihydroxyacetone. The keto product is then phosphorylated by another adenosine triphosphate-linked kinase. Two kinds of constitutive mutants have been isolated: one affecting the aerobic and the other the anaerobic pathway.     

Immunochemical properties of NAD+-linked glycerol dehydrogenases from Escherichia coli and Klebsiella pneumoniae.

An NAD+-linked glycerol dehydrogenase hyperproduced by a mutant of Escherichia coli K-12 was found to be immunochemically homologous to a minor glycerol dehydrogenase of unknown physiological function in Klebsiella pneumoniae 1033, but not to the glycerol dehydrogenase of the dha system responsible for anaerobic dissimilation of glycerol or to the 2,3-butanediol dehydrogenase of K. pneumoniae.     

共表达甘油脱氢酶和二羟丙酮激酶对大肠杆菌生长及甘油代谢的影响

考察共表达甘油脱氢酶(GldA)和二羟丙酮激酶(DhaKLM)对大肠杆菌生长及甘油代谢的影响。结果表明:在好氧条件下,共表达甘油脱氢酶及二羟丙酮激酶可以提高大肠杆菌利用甘油合成菌体的效率,利用等量的甘油,重组菌最高菌密度比对照菌提高了70%,细胞干质量为3.54 g(以每升发酵液计)。在厌氧条件下,仅共表达甘油脱氢酶并不能促进大肠杆菌的甘油代谢,而同时共表达甘油脱氢酶和二羟丙酮激酶可以明显提高大肠杆菌代谢甘油的能力,每克菌体消耗的甘油量提高了42%,每克干细胞中达11.08 g,代谢产物组成也发生显著变化,乙酸成为主要产物。这说明共表达gldA及dhaKLM基因能有效促进大肠杆菌好氧利用甘油生长及厌氧甘油代谢的能力。     

Comparative genomic analysis of dha regulon and related genes for anaerobic glycerol metabolism in bacteria

The dihydroxyacetone (dha) regulon of bacteria encodes genes for the anaerobic metabolism of glycerol. In this work, genomic data are used to analyze and compare the dha regulon and related genes in different organisms in silico with respect to gene organization, sequence similarity, and possible functions. Database searches showed that among the organisms, the genomes of which have been sequenced so far, only two, i.e., Klebsiella pneumoniae MGH 78578 and Clostridium perfringens contain a complete dha regulon bearing all known enzymes. The components and their organization in the dha regulon of these two organisms differ considerably from each other and also from the previously partially sequenced dha regulons in Citrobacter freundii, Clostridium pasteurianum, and Clostridium butyricum. Unlike all of the other organisms, genes for the oxidative and reductive pathways of anaerobic glycerol metabolism in C. perfringens are located in two separate organization units on the chromosome. Comparisons of deduced protein sequences of genes with similar functions showed that the dha regulon components in K. pneumoniae and C. freundii have high similarities (80-95%) but lower similarities to those of the Clostridium species (30-80%). Interestingly, the protein sequence similarities among the dha genes of the Clostridium species are in many cases even lower than those between the Clostridium species and K. pneumoniae or C. freundii, suggesting two different types of dha regulon in the Clostridium species studied. The in silico reconstruction and comparison of dha regulons revealed several new genes in the microorganisms studied. In particular, a novel dha kinase that is phosphoenolpyruvate-dependent is identified and experimentally confirmed for K. pneumoniae in addition to the known ATP-dependent dha kinase. This finding gives new insights into the regulation of glycerol metabolism in K. pneumoniae and explains some hitherto not well understood experimental observations.     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

Uptake and dissimilation of glycerol by wild type and glycerol nonutilizing strains of Neurospora crassa

Summary Seven mutant strains defective for utilization of glycerol, glyceraldehyde or dihydroxyacetone were isolated. One strain was deficient for NAD-linked glycerol-3-phosphate dehydrogenase, two for glycerol kinase, and four had no detected enzymatic deficiency, although one of the latter strains was deficient in glycerol uptake. Glycerol uptake was increased by incubation in glycerol, glycerol-3-phosphate, erythritol, and propanediol, and was protein-mediated below 0.14 mM glycerol, but at higher concentrations free diffusion predominated. Glycerol uptake was decreased by cycloheximide and was more sensitive to sodium azide than to iodoacetate.     

Glycerol catabolism in Aspergillus nidulans

Glycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol 3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific induction. Biochemical and genetical analyses show that dihydroxyacetone and D-glyceraldehyde are converted into glycerol and then catabolized by the same pathway. D-Glyceraldehyde can be reduced by NADP(+)-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.     

Isolation and characterization of mutants from Schyzosaccharomyces pombe defective in glycerol catabolism

Mutants unable to grow on glycerol were isolated from the fission yeast Schyzosaccharomyces pombe. Two types of mutants were obtained: one type was able to grow on dihydroxyacetone while the other one did not grow on this compound. The first type of mutants was defective in glycerol dehydrogenase while the second one was affected both in the glycerol dehydrogenase and in dihydroxyacetone kinase. It was found that the second type was defective in the derepression of several enzymes. The mutations were nuclear and monogenic and defined two complementation groups. Spontaneous revertants, able to grow on glycerol, were obtained from the first type of mutants. They have regained the glycerol dehydrogenase activity. The results presented provide genetic evidence for a pathway of glycerol catabolism in Sch. pombe involving dehydrogenation of glycerol as the first step followed by phosphorylation of the dihydroxyacetone formed.     

Relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives by Pseudomonas aeruginosa.

The relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives in Pseudomonas aeruginosa was studied by comparing the growth on glycerol and enzymatic constitution of strain PAO with these characteristics of glucose-catabolic mutants and revertants. Growth of strain PAO on glycerol induced a catabolic oxidized nicotinamide adenine dinucleotide-linked glyceraldehyde-phosphate dehydrogenase and seven glucose-catabolic enzymes. The results indicated that these enzymes were induced by a six-carbon metabolite of glucose. All strains possessed a constitutive anabolic Embden-Meyerhof-Parnas pathway allowing limited conversion of glycerol-derived triosephosphate to hexosephosphate derivatives, which was consistent with induction of these enzymes by glycerol. Phosphogluconate dehydratase-deficient mutants grew on glycerol. However, mutants lacking both phosphogluconate dehydrogenase and phosphogluconate dehydratase were unable to grow on glycerol, although these strains possessed all of the enzymes needed for degradation of glycerol. These mutants apparently were inhibited by hexosephosphate derivatives, which originated from glycerol-derived triosephosphate and could not be dissimilated. This conclusion was supported by the fact that revertants regaining only a limited capacity to degrade 6-phosphogluconate were glycerol positive but remained glucose negative.