Induction and multi-sensitive end-product repression in the enzymic pathway degrading mandelate in Pseudomonas fluorescens

1. The first five enzymes involved in the degradation of mandelate in Pseudomonas fluorescens have been examined. 2. Induction is not significantly affected by glucose. 3. The first three enzymes form a group inducible by mandelate and repressible by benzoate, catechol and succinate. 4. The possibility that benzoate and catechol act as repressors only after they have been degraded to succinate is unlikely since mutants blocked at suitable points in the pathway have the same repression pattern as the wild type. 5. It is concluded that synthesis of the enzymes is subject to a multi-sensitive repression mechanism that can be independently activated by benzoate or catechol or succinate. 6. In each case the repression can be largely overcome by increasing the concentration of the inducer. 7. The enzymes of the first group are thus controlled by a dual system in which induction by the first substrate is opposed by repression exerted by the end product of the first group and by the products of succeeding groups.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.     

Catechol oxygenase induction in Pseudomonas aeruginosa

1. The transfer from benzenesulphonate to benzoate as a growth substrate for Pseudomonas aeruginosa strain A resulted in a change in the enzymic route by which catechol was degraded; at intermediate stages it was possible to obtain cells containing the enzymes of both the ;ortho' and ;meta' metabolic pathways. 2. A similar result was effected by the reverse transfer, benzoate to benzenesulphonate. 3. Catechol itself always elicited a catechol 2,3-oxygenase in uninduced cells, but the product of this reaction, 2-hydroxymuconic semialdehyde, and biochemically related compounds such as 4-hydroxy-2-oxovalerate, unexpectedly induced a catechol 1,2-oxygenase. 4. Both types of catechol oxygenase are strongly repressed by the metabolic end products of both the ;ortho' and ;meta' pathways, but there was no inhibition of enzymic activity by these end products.     

Regulation of pathways degrading aromatic substrates in Pseudomonas putida. Enzymic response to binary mixtures of substrates

1. Induction constants (K(ind)) and repression constants (K(rep)), which are a measure of the affinity of the inducers or repressors for the induction systems, were measured for mandelate, benzoate and p-hydroxybenzoate in Pseudomonas putida. 2. From these results, the enzymic response of the organism to media containing pairs of these substrates was predicted. Nitrogen-limited chemostats, operated at high growth rates, were used to investigate these predictions in cells grown first on one aromatic substrate with the second added later. 3. In general, the values of K(ind) and K(rep) predicted quite accurately the response to substrate mixtures. Thus, in the presence of mandelate and either benzoate or p-hydroxybenzoate, the enzymes of mandelate metabolism were repressed almost completely, and the bacteria were fully induced for the alternative substrate (benzoate or p-hydroxybenzoate), which was preferentially utilized for growth. When benzoate and p-hydroxybenzoate were the two substrates in the mixture, the enzymes for metabolism of the latter were strongly repressed and growth took place mainly on benzoate. 4. The enzymic response to mixed substrates did not result in the metabolism of the better growth substrate, but in the substrate requiring the synthesis of fewer enzymes. Thus benzoate is used in preference to mandelate although the latter supports a faster growth rate. It is nevertheless considered that, with our present knowledge of the natural habitat of the organism, it is impossible to decide whether protein economy or growth rate was the factor determining the evolution of this control system.     

Degradation of mandelate and 4-hydroxymandelate by Rhizobium leguminosarum biovar trifolii TA1

Rhizobium leguminosarum biovar trifolii TA1 grows on 4-hydroxymandelate and enzymes involved in its catabolism are inducible. Strain TA1 does not grown on mandelate or cis, cis-muconate, but spontaneous mutants capable of growth on these substrates were isolated. Enzymes involved in mandelate degradation were also inducible. The presence of intermediates of the mandelate and hydroxymandelate pathways resulted in a significant decrease in some of the enzymes involved in their degradation. Succinate and acetate, end products of the pathways, and glucose caused reductions in the levels of enzymes in the mandelate and hydroxymandelate pathways.     

Regulation of synthesis of benzyl alcohol dehydrogenase in Acinetobacter calcoaceticus NCIB8250.

Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a maximum at a specific growth rate of 0.2 h-1, but fell off at lower and higher growth rates. The specific activity in nitrogen-limited cultures was always lower and was inversely proportional to growth rate. There was severe repression of benzyl alcohol dehydrogenase during metabolism of L(+)-mandelate or phenylglyoxylate in batch cultures. Synthesis of benzyl alcohol dehydrogenase was followed in experiments where various compounds, including a gratuitous inducer and an anti-inducer of the mandelate enzymes, were added to uninduced or pre-induced cultures and to constitutive and blocked mutants. The results led to the conclusion that there were at least two types of repression. One was caused by phenylglyoxylate carbon-lyase (or a compound synthesized co-ordinately with it), but not by the other mandelate enzymes or by L(+)-mandelate, phenylglyoxylate, benzaldehyde or benzoate. A second type of repression was observed during rapid growth or after the addition of compound such as succinate which are rapidly and completely metabolized.     

Glucose represses the lactose-galactose regulon in Kluyveromyces lactis through a SNF1 and MIG1- dependent pathway that modulates galactokinase (GAL1) gene expression.

Expression of the lactose-galactose regulon in Kluyveromyces lactis is induced by lactose or galactose and repressed by glucose. Some components of the induction and glucose repression pathways have been identified but many remain unknown. We examined the role of the SNF1 (KlSNF1) and MIG1 (KlMIG1) genes in the induction and repression pathways. Our data show that full induction of the regulon requires SNF1; partial induction occurs in a Klsnf1 -deleted strain, indicating that a KlSNF1 -independent pathway(s) also regulates induction. MIG1 is required for full glucose repression of the regulon, but there must be a KlMIG1 -independent repression pathway also. The KlMig1 protein appears to act downstream of the KlSnf1 protein in the glucose repression pathway. Most importantly, the KlSnf1-KIMig repression pathway operates by modulating KlGAL1 expression. Regulating KlGAL1 expression in this manner enables the cell to switch the regulon off in the presence of glucose. Overall, our data show that, while the Snf1 and Mig1 proteins play similar roles in regulating the galactose regulon in Saccharomyces cerevisiae and K.lactis , the way in which these proteins are integrated into the regulatory circuits are unique to each regulon, as is the degree to which each regulon is controlled by the two proteins.     

Control of catechol meta-cleavage pathway in Alcaligenes eutrophus

Alcaligenes eutrophus 335 (ATCC 17697) metabolizes phenol and p-cresol via a catechol meta-cleavage pathway. Studies with mutant strains, each defective in an enzyme of the pathway, showed that the six enzymes assayed are induced by the primary substrate. Studies with a putative polarity mutant defective in the expression of aldehyde dehydrogenase suggested that the structural genes encoding this and subsequent enzymes of the pathway exist in the same operon. From studies with mutant strains that constitutively synthesize catechol 2,3-oxygenase and subsequent enzymes and from the coordination of repression of these enzymes by p-toluate, benzoate, and acetate, it is proposed the catechol 2,3-oxygenase structural gene is situated in this operon (2,3-oxygenase operon). Studies with regulatory mutant strains suggest that the 2,3-oxygenase operon is under negative control.     

Regulation of ugp, the sn-glycerol-3-phosphate transport system of Escherichia coli K-12 that is part of the pho regulon.

The expression of the ugp-dependent sn-glycerol-3-phosphate transport system that is part of the pho regulon was studied in mutants of Escherichia coli K-12 containing regulatory mutations of the pho regulon. The phoR and phoST gene products exerted a negative control on the expression of ugp. Induction of the system was positively controlled by the phoB, phoM, and phoR gene products. Using a ugp-lacZ operon fusion, we showed that the ugp and phoA genes were coordinately derepressed and repressed.     

Phosphate and carbon source regulation of alkaline phosphatase and phospholipase in Vibrio vulnificus

In this study, the effects of phosphate concentration and carbon source on the patterns of alkaline phosphatase (APase) and phospholipase (PLase) expression in Vibrio vulnificus ATCC 29307 were assessed under various conditions. The activities of these enzymes were repressed by excess phosphate (4 mM) in the culture medium, but this repression was reversed upon the onset of phosphate starvation in low phosphate defined medium (LPDM) containing 0.2 mM of phosphate at approximately the end of the exponential growth phase. The expressions of the two enzymes were also influenced by different carbon sources, including glucose, fructose, maltose, glycerol, and sodium acetate at different levels. The APase activity was derepressed most profoundly in LPDM containing fructose as a sole carbon source. However, the repression/derepression of the enzyme by phosphate was not observed in media containing glycerol or sodium acetate. In LPDM-glycerol or sodium acetate, the growth rate was quite low. The highest levels of PLase activity were detected in LPDMsodium acetate, followed by LPDM-fructose. PLase was not fully repressed by high phosphate concentrations when sodium acetate was utilized as the sole carbon source. These results showed that multiple regulatory systems, including the phosphate regulon, may perform a function in the expression of both or either APase and PLC, in the broader context of the survival of V. vulnificus.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91:1140-1154. 1966.-The control of synthesis of the five enzymes responsible for the conversion of d(-)-mandelate to benzoate by Pseudomonas putida was investigated. The first three compounds occurring in the pathway, d(-)-mandelate, l(+)-mandelate, and benzoylformate, are equipotent inducers of all five enzymes. A nonmetabolizable inducer, phenoxyacetate, also induces synthesis of these enzymes; but, unlike the metabolizable inducer-substrates, it does not elicit synthesis of enzymes that mediate steps in the pathway beyond benzoate. Under conditions of semigratuity, dl-mandelate elicits immediate synthesis at a steady rate of the first two enzymes of the pathway, but two enzymes which act below the level of benzoate are synthesized only after a considerable lag. Succinate and asparagine do not significantly repress the synthesis of the enzymes responsible for mandelate oxidation.     

Metabolism of benzyl alcohol via catechol ortho-pathway in methylnaphthalene-degrading Pseudomonas putida CSV86

Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways. In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes. However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol. Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway. Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons. Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid. The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway. CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.     

Metabolism of aromatic compounds by Caulobacter crescentus.

Cultures of Caulobacter crescentus were found to grow on a variety of aromatic compounds. Degradation of benzoate, p-hydroxybenzoate, and phenol was found to occur via beta-ketoadipate. The induction of degradative enzymes such as benzoate 1,2-dioxygenase, the ring cleavage enzyme catechol 1,2-dioxygenase, and cis, cis-muconate lactonizing enzyme appeared similar to the control mechanism present in Pseudomonas spp. Both benzoate 1,2-dioxygenase and catechol 1,2-dioxygenase had stringent specificities, as revealed by their action toward substituted benzoates and substituted catechols, respectively.     

Regulation of arginine and proline catabolism in Bacillus licheniformis

The enzymes in the arginine breakdown pathway (arginase, ornithine-delta-transaminase, and Delta'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Delta'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Delta'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.     

Microbial metabolism of mandelate: a microcosm of diversity

This review highlights the diversity of prokaryotic and eukaryotic microorganisms that can metabolise mandelate and it describes how a wide range of compounds related to mandelate is formed in many environments. The chief aspects that are summarised include the various pathways whereby mandelate and its structural analogues are converted into catechol or protocatechuate, the properties of the enzymes that are involved in the pathways, and the regulation and genetics of the pathways. The review incorporates the idea that the study of peripheral metabolic pathways is particularly useful for illuminating evolutionary speculations and it concludes with a list of questions that need to be answered.     

Microbial metabolism of mandelate: a microcosm of diversity

Abstract This review highlights the diversity of prokaryotic and eukaryotic microorganisms that can metabolise mandelate and it describes how a wide range of compounds related to mandelate is formed in many environments. The chief aspects that are summarised include the various pathways whereby mandelate and its structural analogues are converted into catechol or protocatechuate, the properties of the enzymes that are involved in the pathways, and the regulation and genetics of the pathways. The review incorporates the idea that the study of peripheral metabolic pathways is particularly useful for illuminating evolutionary speculations and it concludes with a list of questions that need to be answered.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. 3. Isolation and properties of constitutive mutants

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. III. Isolation and properties of constitutive mutants. J. Bacteriol. 91:1161-1167. 1966.-Mutants of Pseudomonas putida constitutive for the synthesis of l(+)-mandelate dehydrogenase were obtained after mandelate- or benzoylformate-limited growth in a chemostat. When grown in media noninducing for the wild type, the mutants are capable of coordinate, constitutive synthesis of the first five enzymes of the mandelate pathway. Later enzymes of the pathway that were examined are normally repressed. The constitutive mutants have two other noteworthy properties: they are superinducible by some compounds which induce the mandelate group enzymes in the wild type, or as a result of exhaustion of the carbon and energy source of the medium in which they are grown; and they exhibit a decreased specificity of induction, being inducible by a wide range of compounds devoid of inductive function for the wild type. These results, together with other evidence indicating that the five mandelate group enzymes comprise a regulatory unit, are discussed and evaluated in the context of the general problem of the regulation of complex dissimilatory pathways.     

Catabolite repression of Pseudomonas aeruginosa amidase: the effect of carbon source on amidase synthesis.

Synthesis of the Pseudomonas aeruginosa aliphatic amidase was repressed severely by succinate and malate and less severely by glucose, acetate or lactate. Amidase synthesis in inducible and constitutive strains was stimulated by cyclic AMP, which also gave partial relief to catabolite repression produced by the addition of lactate to cultures growing in pyruvate medium. Mutants which were resistant to catabolite repression were isolated from succinate+lactamide medium.