Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.     

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.     

Disruption of the fucose pathway as a consequence of genetic adaptation to propanediol as a carbon source in Escherichia coli.

In Escherichia coli, L-fucose is dissimilated via an inducible pathway mediated by L-fucose permease, L-fucose isomerase, L-fucose kinase, and L-fuculose 1-phosphate aldolase. The last enzyme cleaves the six-carbon substrate into dihydroxyacetone phosphate and L-lactaldehyde. Aerobically, lactaldehyde is oxidized to L-lactate by a nicotinamide adenine dinucleotide (NAD)-linked dehydrogenase. Anaerobically, lactaldehyde is reduced by an NADH-COUPLED REDUCTASE TO L-1,2-propanediol, which is lost into the medium irretrievably, even when oxygen is subsequently introduced. Propanediol excretion is thus the end result of a dismutation that permits further anaerobic metabolism of dihydroxy-acetone phosphate. A mutant selected for its ability to grow aerobically on propanediol as a carbon and energy source was reported to produce lactaldehyde reductase constitutively and at high levels, even aerobically. Under the new situation, this enzyme serves as a propanediol dehydrogenase. It was also reported that the mutant had lost the ability to grow on fucose. In the present study, it is shown that in wild-type cells the full synthesis of lactaldehyde dehydrogenase requires the presence of both molecular oxygen and a small molecule effector, and the full synthesis of lactaldehyde reductase requires anaerobiosis and the presence of a small molecule effector. The failure of mutant cells to grow on fucose reflects the impairment of a regulatory element in the fucose system that prevents the induction of the permease, the isomerase, and the kinase. The aldolase, on the other hand, is constitutively synthesized. Three independent fucose-utilizing revertants of the mutant all produce the permease, the isomerase, the kinase, as well as the aldolase, constitutively. These strains grow less well than the parental mutant on propanediol.     

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol.

L-1,2-Propanediol is an irretrievable end product of L-fucose fermentation by Escherichia coli. Selection for increased aerobic growth rate on propanediol results in the escalation of basal synthesis of the NAD+-linked oxidoreductase encoded by fucO, a member of the fuc regulon for the utilization of L-fucose. In general, when fucO becomes constitutively expressed, two other simultaneous changes occur: the fucA gene encoding fuculose-1-phosphate aldolase becomes constitutively expressed and the fucPIK operon encoding fucose permease, fucose isomerase, and fuculose kinase becomes noninducible. In the present study, we show that fucO and fucA form an operon which is divergently transcribed from the adjacent fucPIK operon. In propanediol-positive and fucose-negative mutants the cis-controlling region shared by the operons fucAO and fucPIK is lengthened by 1.2 kilobases. DNA hybridization identified the insertion element to be IS5. This element, always oriented in the same direction with the left end (the BglII end) proximal to fucA, apparently causes constitutive expression of fucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.     

Loss of aldehyde dehydrogenase in an Escherichia coli mutant selected for growth on the rare sugar L-galactose.

Escherichia coli K-12 converts L-fucose to dihydroxyacetone phosphate (C-1 to C-3) and L-lactaldehyde (C-4 to C-6) by a pathway specified by the fuc regulon. Aerobically, L-lactaldehyde serves as a carbon and energy source by the action of an aldehyde dehydrogenase of broad specificity; the product, L-lactate, is then converted to pyruvate. Anaerobically, L-lactaldehyde serves as an electron acceptor to regenerate NAD from NADH by the action of an oxidoreductase; the reduced product, L-12-propanediol, is excreted. A strain selected for growth on L-galactose (a structural analog of L-fucose) acquired a broadened inducer specificity because of an altered fucR gene encoding the activator protein for the fuc regulon (Y. Zhu and E. C. C. Lin, J. Mol. Evol. 23:259-266, 1986). In this study, a second mutation that abolished aldehyde dehydrogenase activity was discovered. The L-fucose pathway converts L-galactose to dihydroxyacetone phosphate and L-glyceraldehyde. Aldehyde dehydrogenase then converts L-glyceraldehyde to L-glycerate, which is toxic. Loss of the dehydrogenase averts the toxicity during growth on L-galactose, but reduces by one-half the aerobic growth yield on L-fucose. When mutant cells induced in the L-fucose system were incubated with radioactive L-fucose, accumulation of radioactivity occurred if the substrate was labeled at C-1 but not if it was labeled C-6. Complete aerobic utilization of carbons 4 through 6 of L-fucose depends not only on an adequate activity of aldehyde dehydrogenase to trap L-lactaldehyde as its anionic acid but also on the lack of L-1,2-propanediol oxidoreductase activity, which converts L-lactaldehyde to a readily excreted alcohol.     

NAD-linked aldehyde dehydrogenase for aerobic utilization of L-fucose and L-rhamnose by Escherichia coli.

Mutant analysis revealed that complete utilization of L-fucose and L-rhamnose by Escherichia coli requires the activity of a common NAD-linked aldehyde dehydrogenase which converts L-lactaldehyde to L-lactate. Mutations affecting this activity mapped to the ald locus at min 31, well apart from the fuc genes (min 60) encoding the trunk pathway for L-fucose dissimilation (as well as L-1,2-propanediol oxidoreductase) and the rha genes (min 88) encoding the trunk pathway for L-rhamnose dissimilation. Mutants that grow on L-1,2-propanediol as a carbon and energy source also depend on the ald gene product for the conversion of L-lactaldehyde to L-lactate.     

Isolation of a mutation resulting in constitutive synthesis of L-fucose catabolic enzymes.

A ribitol-positive transductant of Escherichia coli K-12, JM2112, was used to facilitate the isolation and identification of mutations affecting the L-fucose catabolic pathway. Analysis of L-fucose-negative mutants of JM2112 enabled us to confirm that L-fucose-1-phosphate is the apparent inducer of the fucose catabolic enzymes. Plating of an L-fuculokinase-negative mutant of JM2112 on D-arabinose yielded an isolate containing a second fucose mutation which resulted in the constitutive synthesis of L-fucose permease, isomerase, and kinase. This constitutive mutation differs from the constitutive mutation described by Chen et al. (J. Bacteriol. 159:725-729, 1984) in that it is tightly linked to the fucose genes and appears to be located in the gene believed to code for the positive activator of the L-fucose genes.     

Evolution of propanediol utilization in Escherichia coli: mutant with improved substrate-scavenging power.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. A series of mutants, able to grow on this compound at progressively faster rates, had been isolated by repeated transfers to a medium containing 20 mM L-1,2-propanediol. These strains synthesize at high constitutive levels a propanediolmicotinamide adenine dinucleotide oxidoreductase, an enzyme serving as a lactaldehyde during L-fucose fermentation by wild type cells. In this study, a mutant that can grow rapidly on the novel carbon source was subjected to further selection in a medium containing L-1,2-propanediol never exceeding 0.5 mM to obtain a derivative that has an increased power to extract the substrate from the medium. The emerging mutant exhibited four changes at the enzymatic level: (i) fuculose 1-phosphate aldolase activity is lost; (ii) the constitutive propanediol oxidoreductase activity is increased in its level; (iii) lactaldehyde dehydrogenase becomes constitutive and shows an elevated specific activity in crude extracts; and (iv) at low concentrations of propanediol, the facilitated diffusion across the cell membrane is enhanced. Changes two to four seem to act in concert in the trapping of propanediol by hastening its rate of entry and conversion to an ionized metabolite, lactate.     

Regulatory changes in the fucose system associated with the evolution of a catabolic pathway for propanediol in Escherichia coli.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. Strain 3, a mutant selected for the ability to grow on this compound at progressively more rapid rates, synthesizes constitutively a nicotinamide adenine dinucleotide-linked propanediol oxidoreductase. This enzyme is normally synthesized during anaerobic growth on L-fucose when it functions as a lactaldehyde reductase. Propanediol, the end product of this fermentation process, escapes irretrievably into the medium. The propanediol-utilizing mutant can no longer grow on fucose in either the presence or absence of molecular oxygen. In the present study nine independent lines of propanediol-positive mutants were characterized. One mutant, strain 418, attained a propanediol growth rate close to that of strain 3 without loss of the ability to grow on fucose. In all cases examined, however, prolonged selection on propanediol did result in the emergence of fucose-negative mutants. All of these mutants had enzyme patterns similar to that of strain 3; namely, fucose permease, fucose isomerase, and fuculose kinase were noninducible, whereas fuculose 1-phosphate aldolase was constitutive. In strain 418 and in the fucose-positive predecessors of the other mutants, the first four enzymes in the pathway remained inducible, as in the wild-type strain. Improvements in the growth rate on propanediol appeared to reflect principally the increased activity level of the oxidoreductase during the early stages of evolution. According to transductional analysis, the mutations affecting the ability to grow on propanediol and those that affect the expression of the first enzymes in the fucose pathway were very closely linked. The loss of the ability to grow on fucose is thought to be a mechanistic consequence incidental to the remodeling of the regulatory system in favor of the utilization of the novel carbon source.     

Natural and altered induction of the L-fucose catabolic enzymes in Klebsiella aerogenes.

Mutants of Klebsiella aerogenes W70 were isolated that had gained the ability to utilize the uncommon pentose D-arabinose as their sole source of carbon and energy. In contrast to the D-arabinose-negative, parent strain, these mutants were found to be either constitutive for certain enzymes of the L-fucose catabolic pathway or inducible for such enzymes when incubated in the presence of D-arabinose. The mutants used L-fucose isomerase to convert D-arabinose to D-ribulose, which is an intermediate and inducer of the ribitol catabolic pathway. The D-ribulokinase of the ribitol pathway was then induced. This enzyme catalyzed the phosphorylation of D-ribulose at the 5-carbon position. Mutants that were negative for D-ribulokinase could still dissimilate D-arabinose slowly by using all three enzymes, the isomerase, kinase, and aldolase, of the L-fucose pathway. Using condition negative mutants, we were able to demonstrate that the natural induction of the L-fucose pathway enzymes by L-fucose required the activity of a functional L-fucose isomerase and a functional L-fuculokinase but not an L-fuculose-1-phosphate aldolase. A metabolic intermediate, L-fuculose-1-phosphate, was thereby shown to be a probable inducer of at least the isomerase and kinase of the L-fucose catabolic pathway. Similar experiments, with D-arabinose-positive mutants, which were induced for the L-fucose pathway enzymes upon incubation with D-arabinose, revealed that the activities of the L-fucose isomerase and the L-fuculokinase were also required for the induction of the L-fucose enzymes. These D-arabinose-positive mutants apparently produced an altered regulatory protein that accepted both L-fuculose-1-phosphate and D-ribulose-1-phosphate as inducers. Examination of constitutive mutants revealed that L-fucose isomerase and L-fuculokinase were both synthesized constitutively, with the aldolase apparently under separate control.     

Clustering of genes for L-fucose dissimilation by Escherichia coli.

Aerobic and anaerobic L-fucose utilization by Escherichia coli involves an inducible trunk pathway mediated by a permease, an isomerase, a kinase, and an aldolase. Tn5 insertion mutants of a parental strain expressing this pathway constitutively were used to map the positions of the structural genes by transduction. Results from this and previous studies show that all of the structural genes of the L-fucose trunk pathway map between eno and argA at minute 60.2 of the chromosome.     

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.     

Construction of an improved D-arabinose pathway in Escherichia coli K-12.

A ribitol catabolic pathway was transduced into Escherichia coli K-12 in an effort to determine whether the ribitol pathway would confer an advantage to D-arabinose-positive mutants growing on D-arabinose as the sole carbon source. Competition studies in chemostats showed that ribitol-positive strains, with a selection coefficient of 9%/h, have a significant competitive advantage over ribitol-negative strains. Ribitol-positive strains grown in batch culture also exhibited a shorter lag period than did ribitol-negative strains when transferred from glucose to D-arabinose. Repeated transfer of a ribitol-positive strain of E. coli K-12 on D-arabinose yielded a strain with further improved growth on D-arabinose. This "evolved" strain was found to constitutively synthesize L-fucose permease, isomerase, and kinase but had lost the ability to grow on L-fucose, apparently owing to the loss of a functional aldolase. This constitutive mutation is not linked to the fucose gene cluster and may be similar to an unlinked constitutive mutation described by Chen et al. (J. Bacteriol. 159:725-729, 1984).     

Metabolism of L-fucose and L-rhamnose in Escherichia coli: aerobic-anaerobic regulation of L-lactaldehyde dissimilation.

L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization. Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase. Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions. In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio. The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions. In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change. Experimental evidence in the presence of rhamnose with mutants unable to produce L-lactaldehyde and mutants capable of producing but not further metabolizing it points toward L-lactaldehyde as the effector molecule in the induction of lactaldehyde dehydrogenase. Analysis of a temperature-sensitive mutation affecting the synthesis of lactaldehyde dehydrogenase permitted us to locate an apparently single regulator gene linked to the ald locus at 31 min and probably acting as a positive control element on the expression of the structural gene.     

A comparison of alternate metabolic strategies for the utilization of D-arabinose

Summary Mutants ofKlebsiella aerogenes W70 that metabolize the uncommon pentose D-arabinose were isolated. These mutants were found to be either constitutive or indicible by D-arabinose for the synthesis of enzymes in the L-fucose pathway. Such mutants could then utilize L-fucose isomerase to convert the structurally similar D-arabinose molecule to D-ribulose. D-Ribulose is an inter-mediate and the inducer of an existing ribitol pathway and could thus be metabolized. In those D-arabinose-positive mutants where the ribitol pathway was blocked by mutation, D-ribulose could alternatively be metabolized by using the remaining L-fucose pathway enzymes. When the two D-arabinose catabolic routes were compared, catabolism of D-arabinose via the ribitol pathway was found to be more efficient. Catabolism of D-arabinose using the L-fucose pathway per-mitted D-ribulose to escape into the media and produced an unmetabolizable end product, L-glycolic acid. A comparison of growth using constitutive versus inducible control of the borrowed L-fucose isomerase did not reveal an advantage for one control type over the other. Several differences were observed,however, when we determined the degree to which these control mutations perturbed the normal functioning of the L-fucose and associated pathways. Growth of the constitutive mutant was impaired with L-fucose as substrate. The inducible-control mutant had altered growth characteristics on ribitol and L-rhamnose.     

Fermentation mechanism of fucose and rhamnose in Salmonella typhimurium and Klebsiella pneumoniae.

An equimolar amount of 1,2-propanediol was detected in the medium when Salmonella typhimurium or Klebsiella pneumoniae fermented L-fucose or L-rhamnose. These metabolic conditions induced a propanediol oxidoreductase that converted the lactaldehyde formed in the dissimilation of either sugar into the diol. The enzyme was further identified by cross-reaction with antibodies against Escherichia coli propanediol oxidoreductase. This indicates that L-fucose and L-rhamnose fermentation takes place in these species by 1,2-propanediol production and excretion.     

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression.

Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.     

A Suppressor of snf1 Mutations Causes Constitutive High-Level Invertase Synthesis in Yeast

The SNF1 gene product of Saccharomyces cerevisiae is required to derepress expression of many glucose-repressible genes, including the SUC2 structural gene for invertase. Strains carrying a recessive snf1 mutation are unable to ferment sucrose. We have isolated 30 partial phenotypic revertants of a snf1 mutant that were able to ferment sucrose. Genetic characterization of these revertants showed that the suppressor mutations were all recessive and defined eight complementation groups, designated ssn1 through ssn8 (suppressor of snf1 ). The revertants were assayed for secreted invertase activity, and although activity was detected in members of each complementation group, only the ssn6 strains contained wild-type levels. Synthesis of secreted invertase in ssn6 strains was found to be constitutive, that is, insensitive to glucose repression; moreover, the ssn6 mutations also conferred constitutivity in a wild-type ( SNF1 ) genetic background and are, therefore, not merely suppressors of snf1 . Pleiotropic defects were observed in ssn6 mutants. Genetic analysis suggested that the ssn6 mutations are allelic to the cyc8 mutation isolated by R. J. Rothstein and F. Sherman, which causes increased production of iso-2-cytochrome c. The data suggest a regulatory function for SSN6 .