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1.
Regulation of pentitol metabolism by aerobacter aerogenes. II. Induction of the ribitol pathway 总被引:1,自引:1,他引:0
The incubation of Aerobacter aerogenes PRL-R3 with ribitol resulted in the induction of ribitol dehydrogenase and d-ribulokinase, coordinately controlled enzymes of the pathway of ribitol catabolism. A dehydrogenase-negative mutant was unable to induce d-ribulokinase activity following incubation with ribitol. Similar experiments using a kinase-negative mutant resulted in normal induction of ribitol dehydrogenase, as compared to the wild-type PRL-R3 strain. Constitutive or induced cells for l-fucose isomerase were capable of catalyzing the isomerization of d-arabinose to d-ribulose. In contrast to the experiments using ribitol as the substrate, the isomerization of d-arabinose resulted in the induction of d-ribulokinase with dehydrogenase-negative cells. These data indicated that d-ribulose, rather than ribitol, acts as the inducer of the enzymes for ribitol degradation. 相似文献
2.
Induction studies on Aerobacter aerogenes strain PRL-R3, using ribitol as the inducer-substrate, indicated that two enzymes of ribitol catabolism, ribitol dehydrogenase and d-ribulokinase, are coordinately induced. The utilization of d-arabinose as a substrate resulted in the induction of ribitol dehydrogenase as well as d-ribulokinase. Mutants which were constitutive for ribitol dehydrogenase were also constitutive for d-ribulokinase. In contrast, d-xylulokinase and d-arabitol dehydrogenase did not appear to be coordinately controlled. Induction studies and examination of d-arabitol dehydrogenase constitutive mutants indicated that the three enzymes of the converging pathways for d-arabitol and d-xylose catabolism are under separate control. 相似文献
3.
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. 相似文献
4.
Mutants of Aerobacter aerogenes capable of utilizing xylitol as a novel carbon 总被引:16,自引:10,他引:6
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Wild-type Aerobacter aerogenes 1033 is unable to utilize xylitol. A succession of mutants was isolated capable of growth on this compound (0.2%) at progressively faster rates. Whereas the ability to utilize xylitol was achieved in the first-stage mutant (X1) by constitutive production of ribitol dehydrogenase (for which xylitol is a substrate but not an inducer), the basis for enhanced utilization of xylitol in the second-stage mutant (X2) was an alteration of ribitol dehydrogenase. This enzyme was purified from the various mutants. The apparent K(m) for xylitol was 0.12 m with X2 enzyme and 0.29 m with X1 enzyme. The X2 enzyme was also less heat stable and, at 0.05 m substrate concentration, had a higher ratio of activity with xylitol compared to ribitol than did the X1 enzyme. The third mutant (X3), with an even faster growth rate on xylitol, produced a ribitol dehydrogenase indistinguishable physically or kinetically from that of X2. However, X3 produced constitutively an active transport system which accepts xylitol. The usual function of this system is apparently for the transport of d-arabitol since the latter is not only a substrate but also an inducer of the transport system in parental strains of X3. The sequence of mutations described herein illustrates how genes belonging to different metabolic systems can be mobilized to serve a new biochemical pathway. 相似文献
5.
Klebsiella aerogenes strain W70 has an inducible pathway for the degradation of d-arabitol which is comparable to the one found in Aerobacter aerogenes strain PRL-R3. The pathway is also similar to the pathway of ribitol catabolism in that it is composed of a pentitol dehydrogenase, d-arabitol dehydrogenase (ADH), and a pentulokinase, d-xylulokinase (DXK). These two enzymes are coordinately controlled and induced in response to d-arabitol, the apparent inducer of synthesis of these enzymes. We obtained mutants which lacked a functional d-xylose pathway and were constitutive for the ribitol catabolic pathway. These mutants were able to grow on the unusual pentitol, xylitol, only if they contained the functional DXK of the d-arabitol pathway. This provided us with a specific selection technique for DXK(+) transductants. As in A. aerogenes, mutants constitutive for ADH were able to use this enzyme to convert the hexitol d-mannitol to d-fructose. With mutants blocked in the normal d-mannitol catabolic pathway, growth on d-mannitol became a test for ADH constitutivity. Growth of such mutants on xylitol, d-arabitol, and d-mannitol was utilized to classify transductants in mapping, by transductional analysis, the loci involved in d-arabitol utilization. Three-point crosses gave the order dalK-dalD-dalC, where dalK is the DXK structural gene, dalD is the ADH structural gene, and dalC is a regulatory site controlling synthesis of both enzymes. 相似文献
6.
Natural and altered induction of the L-fucose catabolic enzymes in Klebsiella aerogenes. 总被引:9,自引:9,他引:0
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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. 相似文献
7.
In Klebsiella aerogenes W70, there is an inducible pathway for the catabolism of ribitol consisting of at least two enzymes, ribitol dehydrogenase (RDH) and d-ribulokinase (DRK). These two enzymes are coordinately controlled and induced in response to d-ribulose, an intermediate of the pathway. Whereas wild-type K. aerogenes W70 are unable to utilize xylitol as a carbon and energy source, mutants constitutive for the ribitol pathway are able to utilize RDH to oxidize the unusual pentitol, xylitol, to d-xylulose. These mutants are able to grow on xylitol, presumably by utilization of the d-xylulose produced. Mutants constitutive for l-fucose isomerase can utilize the isomerase to convert d-arabinose to d-ribulose. In the presence of d-ribulose, RDH and DRK are induced, and such mutants are thus able to phosphorylate the d-ribulose by using the DRK of the ribitol pathway. Derivatives of an l-fucose isomerase-constitutive mutant were plated on d-arabinose, ribitol, and xylitol to select and identify mutations in the ribitol pathway. Using the transducing phage PW52, we were able to demonstrate genetic linkage of the loci involved. Three-point crosses, using constitutive mutants as donors and RDH(-), DRK(-) double mutants as recipients and selecting for DRK(+) transductants on d-arabinose, resulted in DRK(+)RDH(+)-constitutive, DRK(+)RDH(+)-inducible, and DRK(+)RDH(-)-inducible transductants but no detectable DRK(+)RDH(-) constitutive transductants, data consistent with the order rbtC-rbtD-rbtK, where rbtC is a control site and rbtD and rbtK correspond to the sites for the sites for the enzymes RDH and DRK, respectively. 相似文献
8.
Constitutive activation of L-fucose genes by an unlinked mutation in Escherichia coli. 总被引:8,自引:8,他引:0
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Wild-type Escherichia coli cannot grow on L-1,2-propanediol; mutants that can do so have increased basal activity of an NAD-linked L-1,2-propanediol oxidoreductase. This enzyme belongs to the L-fucose system and functions normally as L-lactaldehyde reductase during fermentation of the methylpentose. In wild-type cells, the activity of this enzyme is fully induced only anaerobically. Continued aerobic selection for mutants with an improved growth rate on L-1,2-propanediol inevitably leads to full constitutive expression of the oxidoreductase activity. When this occurs, L-fuculose 1-phosphate aldolase concomitantly becomes constitutive, whereas L-fucose permease, L-fucose isomerase, and L-fuculose kinase become noninducible. It is shown in this study that the noninducibility of the three proteins can be changed by two different kinds of suppressor mutations: one mapping external to and the other within the fuc gene cluster. Both mutations result in constitutive synthesis of the permease, the isomerase, and the kinase, without affecting synthesis of the oxidoreductase and the aldolase. Since expression of the fuc structural genes is activated by a protein specified by the regulator gene fucR, and since all the known genes of the fuc system are clustered at minute 60.2 of the chromosome, the external gene in which the suppressor mutation can occur probably has an unrelated function in the wild-type strain. The internal suppressor mutation might be either in fucR or in the promoter region of the genes encoding the permease, the isomerase, and the kinase, if these genes belong to the same operon. 相似文献
9.
A comparison of wild-type and mutant ribitol dehydrogenases from Klebsiella aerogenes 总被引:6,自引:1,他引:5
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Bruce D. Burleigh Jr. Peter W. J. Rigby Brian S. Hartley 《The Biochemical journal》1974,143(2):341-352
A ribitol dehydrogenase (ribitol-NAD(+) oxidoreductase, EC. 1.1.1.56) having increased specificity and catalytic efficiency toward xylitol was isolated from mutant strains of Klebsiella aerogenes, which were selected for increased growth rate on xylitol over the ribitol dehydrogenase constitutive wild-type organism. 2. The mutant enzyme was purified to homogeneity and its general characteristics were compared with those of the previously purified wild-type enzyme. 3. Initial-velocity steady-state kinetic parameters were determined for both wild-type and mutant enzymes and the results compared. 4. The results are interpreted in terms of a model in which the mutant enzyme results from a small change of amino acid sequence, which affects both the stability and conformational equilibria of the molecule. 相似文献
10.
11.
Disruption of the fucose pathway as a consequence of genetic adaptation to propanediol as a carbon source in Escherichia coli. 总被引:19,自引:19,他引:0
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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. 相似文献
12.
Summary The growth of the strain Escherichia coli K12 1EA in a chemostat was limited by ribitol as the source of carbon and energy. Specific activities of ribitol dehydrogenase and D-arabinitol dehydrogenase were assayed to measure expression of the two closely linked catabolic operons rbt and dal. Population changes occuring in a chemostat were analyzed by testing single colony isolates in batch cultures: double constitutive mutant 1EA-A was first selected while later hyperproducing strains of the type 11EA and 111EA synthesizing constitutively 4 times and 8 times more ribitol dehydrogenase, respectively, prevail in the chemostat. 相似文献
13.
Human placental 17 beta-hydroxysteroid dehydrogenase is homologous to NodG protein of Rhizobium meliloti 总被引:3,自引:0,他引:3
M E Baker 《Molecular endocrinology (Baltimore, Md.)》1989,3(5):881-884
The amino acid sequence of human placental 17 beta-hydroxysteroid dehydrogenase (17 beta-OH-steroid dehydrogenase) was found to be similar to that of the NodG protein of Rhizobium meliloti. The computer-based comparison score is 11.5 SD higher than that obtained with 2500 comparisons of randomized sequences of these proteins. The probability of getting such a score by chance is 6 x 10(-31). 17 beta-OH-steroid dehydrogenase is also similar to Klebsiella aerogenes ribitol dehydrogenase and Escherichia coli glucitol-6-phosphate dehydrogenase. We propose that the steroid recognition site on 17 beta-OH-steroid dehydrogenase evolved from an ancestral recognition site for polyols such as ribitol and glucitol-6-phosphate. 相似文献
14.
Selection of an interspecific hybridEscherichia coli K 12 1EA in a chemostat on xylitol yielded a stable mutant synthesizing a four-fold amount of ribitol dehydrogenase (EC 1.1.1.56).
Subsequent cultivation of the mutant under increased selection pressure resulted in an accumulation of a mutant with 12-fold
higher level of ribitol dehydrogenase relative to the parent strain 1EA. A selection during which a UV-mutagenized population
of the 1EA mutant was cultivated in a chemostat on xylitol was accompanied by monitoring the activities of ribitol dehydrogenase
andD-arabinitol dehydrogenase (EC 1.1.1.11) of two adjacent catabolite operons. A several-fold increase in the activity of the
two enzymes was followed by further increase in the activity of ribitol dehydrogenase and a concomitant drop in the activity
ofD-arabinitol dehydrogenase. The two hyperproducing strains are compared with the parent mutant as to the rate of synthesis
of the two dehydrogenases and growth parameters under the conditions of batch cultivation. 相似文献
15.
Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase. 总被引:9,自引:0,他引:9
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An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization. 相似文献
16.
Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli.
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Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol. 相似文献
17.
Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli. 总被引:2,自引:2,他引:0
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Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed. 相似文献
18.
Directed evolution of a second xylitol catabolic pathway in Klebsiella pneumoniae. 总被引:2,自引:2,他引:0
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Klebsiella pneumoniae PRL-R3 has inducible catabolic pathways for the degradation of ribitol and D-arabitol but cannot utilize xylitol as a growth substrate. A mutation in the rbtB regulatory gene of the ribitol operon permits the constitutive synthesis of the ribitol catabolic enzymes and allows growth on xylitol. The evolved xylitol catabolic pathway consists of an induced D-arabitol permease system that also transports xylitol, a constitutively synthesized ribitol dehydrogenase that oxidizes xylitol at the C-2 position to produce D-xylulose, and an induced D-xylulokinase from either the D-arabitol or D-xylose catabolic pathway. To investigate the potential of K. pneumoniae to evolve a different xylitol catabolic pathway, strains were constructed which were unable to synthesize ribitol dehydrogenase or either type of D-xylulokinase but constitutively synthesized the D-arabitol permease system. These strains had an inducible L-xylulokinase; therefore, the evolution of an enzyme which oxidized xylitol at the C-4 position to L-xylulose would establish a new xylitol catabolic pathway. Four independent xylitol-utilizing mutants were isolated, each of which had evolved a xylitol-4-dehydrogenase activity. The four dehydrogenases appeared to be identical because they comigrated during nondenaturing polyacrylamide gel electrophoresis. This novel xylitol dehydrogenase was constitutively synthesized, whereas L-xylulokinase remained inducible. Transductional analysis showed that the evolved dehydrogenase was not an altered ribitol or D-arabitol dehydrogenase and that the evolved dehydrogenase structural gene was not linked to the pentitol gene cluster. This evolved dehydrogenase had the highest activity with xylitol as a substrate, a Km for xylitol of 1.4 M, and a molecular weight of 43,000. 相似文献
19.
Glycerol uptake, glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) activities are specifically induced during growth ofPseudomonas aeruginosa PAO on either glycerol or glycerol-3-phosphate. Mutants of strain PAO unable to grow on both glycerol and glycerol-3-phosphate were isolated. Mutant PFB 121 was deficient in an inducible, membrane-bound, pyridine nucleotide-independent, glycerol-3-phosphate dehydrogenase activity and PFB 82 was deficient in glycerol uptake and glycerol kinase and glycerol-3-phosphate dehydrogenase activities. Each mutant spontaneously reverted to wild phenotype, which indicates that each contained a single genetic lesion. These results demonstrate that membrane-bound, inducible glycerol-3-phosphate dehydrogenase is required for catabolism of both glycerol and glycerol-3-phosphate and provide suggestive evidence for a single regulatory locus that controls the synthesis of glycerol uptake, glycerol kinase, and glycerol-3-phosphate dehydrogenase inP. aeruginosa. 相似文献
20.
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). 相似文献