Catabolite repression and derepression of arylsulfatase synthesis in Klebsiella aerogenes

When a mutant (Mao(-)) of Klebsiella aerogenes lacking an enzyme for tyramine degradation (monoamine oxidase) was grown with d-xylose as a carbon source, arylsulfatase was repressed by inorganic sulfate and repression was relieved by tyramine. When the cells were grown on glucose, tyramine failed to derepress the arylsulfatase synthesis. When grown with methionine as the sole sulfur source, the enzyme was synthesized irrespective of the carbon source used. Addition of cyclic adenosine monophosphate overcame the catabolite repression of synthesis of the derepressed enzyme caused by tyramine. Uptake of tyramine was not affected by the carbon source. We isolated a mutant strain in which derepression of arylsulfatase synthesis by tyramine occurred even in the presence of glucose and inorganic sulfate. This strain also produced beta-galactosidase in the presence of an inducer and glucose. These results, and those on other mutant strains in which tyramine cannot derepress enzyme synthesis, strongly suggest that a protein factor regulated by catabolite repression is involved in the derepression of arylsulfatase synthesis by tyramine.     

Tyramine oxidase and regulation of arylsulfatase synthesis in Klebsiella aerogenes.

The participation of tyramine oxidase in the regulation of arylsulfatase synthesis in Klebsiella aerogenes was studied. Arylsulfatase was synthesized when this organism was grown with methionine or taurine as the sulfur source (nonrepressing conditions) and was repressed by inorganic sulfate or cysteine; this repression was relieved by tyramine and related compounds (derepressing conditions). Under nonrepressing conditions, arylsulfatase synthesis was not regulated by tyramine oxidase synthesis. However, derepression of arylsulfatase and induction of tyramine oxidase synthesis by tyramine were both antagonized by glucose and other carbohydrate compounds. The derepressed synthesis of arylsulfatase, like that of tyramine oxidase, was released from catabolite repression by use of tyramine as the sole source of nitrogen. A mutant strain that exhibits constitutive synthesis of glutamine synthetase and high levels of histidase when grown in glucose-ammonium medium was subject to the catabolite repression of both tyramine oxidase and arylsulfatase syntheses. Mutants in which repression of arylsulfatase could not be relieved by tyramine could not utilize tyramine as the sole source of nitrogen and were defective in the gene for tyramine oxidase.     

Regulation of derepressed synthesis of arylsulfatase by tyramine oxidase in Salmonella typhimurium.

The participation of tyramine oxidase in the regulation of arylsulfatase synthesis in Salmonella typhimurium was studied. Arylsulfatase synthesis was repressed by inorganic sulfate, cysteine, methionine, or taurine. This repression was relieved by tyramine, octopamine, or dopamine, which induced tyramine oxidase synthesis, although the level of arylsulfatase activity was very low. The induction of tyramine oxidase and derepression of arylsulfatase by tyramine were strongly inhibited by glucose and ammonium chloride, and the repression of both enzymes was relieved by use of xylose as a carbon source after consumption of glucose or by use of tyramine as the sole source of nitrogen, irrespective of the carbon source used. The initial rates of tyramine uptake by cells grown with glucose and xylose were similar. Results with tyramine oxidase-constitutive mutants showed that constitutive expression of the tyramine oxidase gene resulted in derepression of arylsulfatase synthesis in the absence of tyramine. Thus, catabolite and ammonium repressions of arylsulfatase synthesis and the induction of the enzyme by tyramine seem to reflect the levels of tyramine oxidase synthesis. These results in S. typhimurium support our previous finding that the specific regulation system of arylsulfatase synthesis by tyramine oxidase is conserved in enteric bacteria.     

Regulation of arylsulfatase synthesis by sulfur compounds in Klebsiella aerogenes.

In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys minus), and mutants (AtsS minus, AtsR minus) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS minus mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS minus mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR minus mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.     

Immunological study of the regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes.

Regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes was analyzed by immunological techniques. Antibody directed against the purified arylsulfatase from K. aerogenes W70 was obtained from rabbits and characterized by immunoelectrophoresis, double-diffusion, quantitative precipitation, and enzyme neutralization tests. Arylsulfatase was located in the periplasmic space when the wild-type strain was cultured with methionine or with inorganic sulfate plus tyramine, but not with inorganic sulfate without tyramine, as the sole sulfur source. Tyramine oxidase was retained in the membrane fraction prepared from cells grown in the presence of tyramine. Arylsulfatase protein was not synthesized in the presence of tyramine and inorganic sulfate by mutant K611, which is deficient in tyramine oxidase (tynA). We conclude that the expression of the arylsulfatase gene (atsA) is regulated by the expression of tynA and that inorganic sulfate serves as a corepressor. In addition, strains mutated in the atsA gene were analyzed by using antibody.     

Formation and Purification of Serratia marcescens Arylsulfatase

The effects of culture conditions on arylsulfatase production by six strains of the genus Serratia were studied. Synthesis of arylsulfatases in all six strains was repressed in media with inorganic sulfate or methionine as the sole source of sulfur and derepressed by the addition of tyramine. Serratia marcescens IFO 3046 grew most rapidly and produced a high level of arylsulfatase when cultured on mannitol with inorganic sulfate and tyramine. The derepressed synthesis of arylsulfatase in S. marcescens was not subject to strong catabolite repression. The molecular weight of purified arylsulfatase was determined to be between 46,000 and 49,000. Arylsulfatase from S. marcescens differed in K_m and V_max values, substrate specificities, fluoride inhibition, and electrophoretic mobility from the enzyme from K. aerogenes, but had the same molecular weight as the latter.     

Genetic control of arylsulfatase synthesis in Klebsiella aerogenes.

It was shown that at least four genes are specifically responsible for arylsulfatase synthesis in Klebsiella aerogenes. Mutations at chromosome site atsA result in enzymatically inactive arylsulfatase. Mutants showing constitutive synthesis of arylsulfatase (atsR) were isolated by using inorganic sulfate or cysteine as the sulfur source. Another mutation in which repression of arylsulfatase by inorganic sulfate or cysteine could not be relieved by tyramine was determined by genetic analysis to be on the tyramine oxidase gene (tyn). This site was distinguished from the atsC mutation site, which is probably concerned with the action or synthesis of corepressors of arylsulfatase synthesis. Genetic analysis with transducing phage PW52 showed that the order of mutation sites was atsC-atsR-atsA-tynA-tynB. On the basis of these results and previous physiological findings, we propose a new model for regulation of arylsulfatase synthesis.     

Arylsulfatase in Salmonella typhimurium: detection and influence of carbon source and tyramine on its synthesis.

Arylsulfatase synthesis was shown to occur in Salmonella typhimurium LT2. The enzyme had a molecular weight of approximately 50,000 and was separated into five forms by isoelectrofocusing. The optimal pH for substrate hydrolysis was pH 6.7, with Michaelis constants for nitrocatechol sulfate and nitrophenyl sulfate being 4.1 and 7.9 mM, respectively. Enzyme synthesis was strongly influenced by the presence of tyramine in the growth medium. The uptake of [14C]tyramine and arylsulfatase synthesis were initiated during the second phase of a diauxie growth response, when the organism was cultured with different carbon sources. Adenosine 3',5'-cyclic monophosphoric acid enhanced the uptake of tyramine and the levels of arylsulfatase synthesized. However, the addition of glucose and glycerol to organisms actively transporting tyramine and synthesizing enzyme caused a rapid inhibition of both of these processes. This inhibition was not reversed by adding adenosine 3',5'-cyclic monophosphoric acid. The results suggest that the effect of the carbon source on tyramine transport and arylsulfatase synthesis may be explained in terms of inducer exclusion.     

Novel Mutation Causing Derepression of Several Enzymes of Sulfur Metabolism in Neurospora crassa

A group of enzymes of sulfur metabolism (arylsulfatase, cholinesulfatase, and a number of others) are normally repressed in Neurospora crassa by an abundant supply of a "favored" sulfur source such as methionine or inorganic sulfate. A mutant called scon(c) was isolated in which the formation of each of these enzymes is largely or completely nonrepressible. The structural genes for three of these enzymes have been mapped; scon(c) is not linked to any of them. It is also not linked to cys-3, another gene which is involved in control of the same group of enzymes. Two alleles of the structural gene for arylsulfatase [ars(+) and ars(UFC-220)] produce electrophoretically distinguishable forms of arylsulfatase. Heterokaryons with the constitution scon(c) ars(+) + scon(+)ars(UFC-220) were prepared. These heterokaryons produce both forms of arylsulfatase under conditions of sulfur limitation, but produce only the wild-type (ars(+)) form under conditions of sulfur abundance. When the alleles of ars and scon are in the opposite relationship, only the ars(UFC-220) form of arylsulfatase can be detected under conditions of sulfur abundance. Thus the effect of the scon(c) mutation seems to be limited to its own nucleus. The implications of these findings are discussed.     

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.     

Quantitative Analysis of the Sex Pheromone of Several Phycitid Moths by Electron-capture Gas Chromatography

β-Phenetyl alcohol and procaine hydrochloride are known to alter membrane structure. Their effects on the syntheses of tyramine oxidase and arylsulfatase were studied in Klebsiella aerogenes. β-Phenetyl alcohol inhibited the syntheses of membrane-bound tyramine oxidase and arylsulfatase, located in the periplasm, under non-repressing and derepressing conditions, but did not affect the syntheses of β-galactosidase and histidase, which are located internally. In contrast, procaine hydrochloride stimulated the synthesis of tyramine oxidase and derepressed the synthesis of arylsulfatase, but inhibited non-repressed synthesis of arylsulfatase. Thus, derepressed synthesis of cellular arylsulfatase was affected by the level of tyramine oxidase synthesis. Structural alterations in the cell membrane seem to impair the formation of active-arylsulfatase protein in the periplasmic space.     

Characterization of arylsulfatase formed by derepressed synthesis in Citrobacter braakii

Arylsulfatase activity was detected in a bacterial strain, Citrobacter braakii 69-b, isolated from soil by enrichment cultivation using porcine gastric mucin. The production of arylsulfatase was derepressed markedly in a synthetic medium by the addition of tyramine. The purified enzyme hydrolyzed 4-nitrophenyl sulfate, 4-nitrocatechol sulfate, and 3-indoxyl sulfate, and was classified as type I arylsulfatase.     

Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source.

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.     

Multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme.

Isolation of multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme is reported. Three enzyme forms were isolated from cells with derepressed synthesis, and one form was isolated from cells with repressed enzyme synthesis. The multiple enzyme forms did not differ in pH optimum, thermostability, or the degree of inhibition with orthophosphate; however, they did differ in the relative rate of hydrolysis of different substrates. The addition of substrates to the cells during enzyme derepression resulted in changes of the ratio of the multiple forms.     

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.     

Compartmentalization and regulation of arylsulfatase activities in Streptomyces sp., Microbacterium sp. and Rhodococcus sp. soil isolates in response to inorganic sulfate limitation

Arylsulfatases allow microorganisms to satisfy their sulfur (S) requirements as inorganic sulfate after sulfate ester hydrolysis. Our objectives were to investigate the arylsulfatase activities among soil isolates, especially Streptomyces sp., Microbacterium sp. and Rhodococcus sp., because such investigations are limited for these bacteria, which often live in sulfate-limited conditions. Physiological and biochemical analyses indicated that these isolates possessed strong specific arylsulfatase activities ranging from 6 to 8 U. Moreover, for Streptomyces sp., an arylsulfatase localization study revealed 2 forms of arylsulfatases. A first form was located in the membrane, and a second form was located in the intracellular compartment. Both arylsulfatases had different patterns of induction. Indeed, the intracellular arylsulfatase was strictly induced by inorganic sulfate limitation, whereas the membrane arylsulfatase was induced both by substrate presence or S demand independently. For Microbacterium and Rhodococcus isolates, only a membrane arylsulfatase was found. Consequently, our results suggest the presence of a previously undescribed arylsulfatase in these microorganisms that allows them to develop an alternative strategy to fulfill their S requirements compared to bacteria previously studied in the literature.     

Use of lac Gene Fusions to Study Regulation of Tyramine Oxidase,Which is Involved in Derepression of Latent Arylsulfatase in Escherichia coli

Strains with lac fused to each of the arylsulfatase (ats) and tyramine oxidase (tyn) operons in Escherichia coli were isolated. Synthesis of β-galactosidase in strains with tyn:: lac fusions was induced by tyramine, histamine, tryptamine, dopamine and octopamine, and the induction of the tyn operon was subject to catabolite and ammonium repressions. These repressions were relieved when the cells were grown with a poor carbon or nitrogen source. No arylsulfatase activity is detected in E. coli strains. Synthesis of β-galactosidase in strains with ats:: lac fusions was repressed by sulfur compounds. The repression was relieved by monoamine compounds, which induced tyramine oxidase synthesis. The inhibition of tyramine oxidase activity by cysteine resulted in a decrease of the derepressed synthesis of β-galactosidase in the ats:: lac fusion. Repressing and derepressing conditions for the tyn operon prevented and stimulated, respectively, expression of the ats operon. Thus, the expression of latent arylsulfatase in E. coli seems to be regulated by expression of the tyn operon.     

Effect of inorganic phosphate and benzyl thiocyanate on the activity of anhydrotetracycline oxygenase inStreptomyces aureofaciens

The level of anhydrotetracycline oxygenase (an enzyme catalyzing the penultimate reaction in the biosynthesis of tetracycline) inStreptomyces aureofaciens was substantially influenced by the amount of inorganic phosphate and by the presence of benzyl thiocyanate in the cultivation medium. Phosphate decreased the specific activity of the enzyme, particularly when added to a growing culture. On the other hand, benzyl thiocyanate increased the specific activity of the enzyme. Its effect was most conspicuous in the growth phase. The effect of benzyl thiocyanate was more pronounced in the low-production strain than in the producing variant. Inorganic phosphate and benzyl thiocyanate did not influence the enzyme activityin vitro. Phosphate added to the growing cultures was readily absorbed by the cells. During this time the enzyme synthesis was repressed, derepression occurred only after exhaustion of phosphate from the medium. The stimulatory efect of benzyl thiocyanate on the enzyme synthesis was not reversed by the inorganic phosphate added.