Agricultural and Biological Chemistry Use of lac Gene ...

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

b

Mitsuo Yamashita & Yoshikatsu Murooka a

The Insitute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 565, Japan b

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Saijo, Higashi-hiroshima 724, Japan Published online: 09 Sep 2014.

To cite this article: Mitsuo Yamashita & Yoshikatsu Murooka (1984) Use of lac Gene Fusions to Study Regulation of Tyramine Oxidase, Which is Involved in Derepression of Latent Arylsulfatase in Escherichia coli, Agricultural and Biological Chemistry, 48:6, 1459-1470 To link to this article: http://dx.doi.org/10.1080/00021369.1984.10866354

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Agric. Bioi. Chern., 48 (6), 1459"" 1470, 1984

1459

Use of lac Gene Fusions to Study Regulation of Tyramine Oxidase, Which is Involved in Derepression of Latent Arylsulfatase in Escherichia coli Mitsuo YAMASHITA and Yoshikatsu MUROOKA * The Insitute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 565, Japan *Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Saijo, Higashi-hiroshima 724, Japan


Received November 24, 1983 Strains with lac fused to each of the arylsulfatase (ats) and tyramine oxidase (tyn) operons in Escherichia coli were isolated. Synthesis of p-galactosidase in strains withtyn: : 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 fJ-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 fJ-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.

Monoamine oxidase in bacteria, which is found in the cell membrane, is induced by tyramine and related compounds and is highly specific for tyramine, octopamjne, dopamine and norepinephrine. Thus, the enzyme is classified as a tyramine oxidase. 1 ,2) The enzyme oxidizes tyramine and catecholamines to hydroxyphenylacetoaldehyde compounds and ammonium ions. Tyramine oxidase in Klebsiella aerogenes is specified by the tynA gene and subject to catabolite repression by glucose in the presence of ammonium salts. This repression is relieved when the cells are grown with a poor nitrogen source. 2 ,3) Arylsulfatase synthesis in K. aerogenes is repressed by a repressor molecule (atsR) that has been activated by a corepressor, which comes from inorganic sulfate, cysteine or related compounds and is governed by atsC. 3 ,4) This repression is relieved by· addition of

*

To whom reprint requests should be addressed.

tyramine.5 -7) By using tyramine oxidase deficient (tynA) and tyramine oxidaseconstitutive (tynP tynR) mutants, we showed that the expression of tyramine oxidase induced by tyramine resulted in· derepression of the atsA gene specifying arylsufatase, which is closely linked· to tynA 3 ,8) (Fig. 1). In some strains of E. coli and Citrobacter freundii, no arylsulfatase activity has been found. However, an enzymatically inactive protein that crossreacts with antibody to arylsulfatase of K. aerogenes is synthesized in these strains under derepressing conditions with tyramine. 9 ,lO) Furthermore, intergeneric hybrid strains, formed by transfer of the ats and tyn genes between Salmonella typhimurium, E. coli and C. freundii, which produce little or no arylsulfatase, suggested that the system of regulation of arylsulfatase synthesis in enteric bacteria was conserved more than the structure of

M. YAMASHITA and Y. MURaoKA

1460

0%

FIG. 1.

78-85%

0%

Model for the Regulation ~f Arylsulfatase and Tyramine Oxidase Syntheses in Enteric Bacteria.

-+-,


negative control. Percentages represent cotransduction frequencies with Symbols: < o

~

~ >....

10

10

20

30 KLETT UNITS

40

50

(B)

FIG. 3. Effect of Ammonium Chloride on the Induction of Tyramine Oxidase and Derepressed Synthesis of {J-Galactosidase in the ats: : lac Fusion Strain. Cells from an overnight culture of strain M63 were washed with saline and suspended in medium containing 0.5% succinate, 0.1 % NH4 Cl and 3 mM tyramine with the indicated amounts of glucose. Symbols: (A) 0, growth with 0.005% NH4 Cl; f:1, growth with 0.01 % NH4 Cl; (B) 0, tyramine oxidase activity with 0.005% NH4 Cl; 1:::., tyramine oxidase activity with 0.01 % NH4 Cl; e, {J-galactosidase activity with 0.05% NH4 Cl, . , {J-galactosidase activity with 0.01 % NH4 Cl.

The relief of syntheses of tyramine oxidase and J3-galactosidase repressed by cysteine was· significantly derepressed. We found that this derepression by cysteine was due to the inhibition of the activity of tyramine oxidase but not that of p-galactosidase. Cysteine and homocysteine were also inhibitory for tyramine oxidase. Ten mM concentration of cysteine inhibited tyramine oxidase activity completely. This inhibition also indicates that the

expression of the atsE operon is regulated by active tyramine oxidase. DISCUSSION

The synthesis of tyramin~ oxidase in enteric bacteria is of interest because it is controlled by carbon, nitrogen and amino compounds. In particular, the enzyme has been implicated in derepression of arylsulfatase synthesis. To sim~

M. YAMASHITA and Y. MUROOKA

1468 TABLE VI.

EFFECT OF cAMP ON THE SYNTHESES OF TYRAMINE OXIDASE AND P-GALACTOSIDASE IN FUSION STRAINSa Enzyme activity (D /mg of protein)

Nitrogen source (0.1 %)

cAMP (10mM)

+ Glutamine

+ Asparagine

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a b

Tyramine oxidase

p-Galactosidase

M37

M63

M10

M14

M37

M63

ND b

ND 50 1 48 83 43

4 21 8 28 23 20

2 6 6 10 28 9

2 164 13 226 403 247

2 200 10 215 446 171

14 ND 17 29 16

Cells were grown in K medium containing 0.5% glucose and 3 mM tyramine. ND, not detected. TABLE VII. ENZYME LEVELS OF TYRAMINE OXIDASE AND P-GALACTOSIDASE IN THE ats: : lac FUSIONS WITH VARIOUS SULFUR.COMPOUNDSa Enzyme activity (D /mg of protein)

Sulfur compound (1 mM)

Tyramine (3mM)

Na2S04

+ Na2S03

+ Na2S203

+ Cysteine

+ + Methionine

+ Taurine

+ a

b C

Tyramine oxidase

p-Galactosidase

M37

M63

M37

M63

ND b

ND 78 ND 51 ND 61 ND 3 NDc ND 73 ND 63

7 549 9 687 6 695 9 52

4 236 3 154 3 227 3 49 7C 4 216 3 203

25 ND 31 ND 38 ND 2 NDc ND 32 ND 22

SC

8 722 4 523

Cells were grown in K medium containing 0.5% succinate, 0.1 % asparagine and various sulfur compounds, and harvested after approximately three doublings. ND, not detected. The cells were grown with 10mM cysteine.

plify the study of the regulation of the ats-tyn system, we used the method of fusing the lacZ gene to the ats and tyn operons and thus brought expression of fJ-galactosidase ·under ats or tyn promoter control. In tyn: : lac fusion strain of E. coli, fJ-galactosidase was induced by tyramine, histamine and tryptamine in addition to catecholamines, such as dopamine and octopamine, but ·not

norepinephrine~

Tyramine oxidase synthesis in

K. aerogenes is strongly induced by tyramine,

dopamine, octopamine and norepinephrine, but· not histamine Of tryptamine,2) whereas in S. typhimurium 16 ) ·and Serratia marcescens1 ,20) the enzyme is not induced by norepinephrine. Differences in the regulation of tyramine oxidase synthesis between enteric bacteria were also seen in the catabolite control of the


ats-Iac and tyn-Iac Fusions

enzyme. Usually, the .degree of catabolite repression varies in different strains of bacteria and with different carbon sources used. This variability is probably due to differences in assimilation rates for carbon and nitrogen compounds in different bacteria. Carbon and nitrogen sources which promote good growth (xylo,se and NH4 + in E. coli) repress the tyn operon, whereas poor carbon and nitrogen sources (xylose and tyramine in K. aerogenes; succinate and asparagine in E. coli) allow the expression of the tyn operon. It is interesting to determine whether the tyn gene is subject to regulation by the nitrogen regulatory genes (ntr), which generally control operons subject to nitrogen regulation in enteric bacteria,1s,21) although we could not find any effect of glutamine synthetase on ammonium repression for tyramine oxidase in K. aerogenes. 2 ,7) For this and further study on the molecular mechanism of the ats-tyn system, in vitro cloning of the ats-tyn region is in progress. The degree of repression of' arysulfatase synthesis by carbon, notrogen and sulfur compounds, especially by methionine and taurine, also varied in different strains of bacteria: Methionine and taurine caused little, if any, repression of arylsulfatase synthesis in K. aerogenes,4) but strongly repressed the enzyme synthesis in S. typhimurium 16 ) and S. marcescens. 20 ) However, this repression is also relieved by addition of tyramine. We did not observe expression of the lac gene in the ats: : lac fusion strains with methionine or taurine as the sole sulfur source in the absence of tyramine, but f3-galactosidase was synthesized under conditions of induction of tyramine oxidase synthesis. This result suggests that the atsE is repressed by methionine and taurine in addition· to inorganic sulfate and cysteine. Synthesis of arylsulfatase in K. aerogenes, in fact, is not subject to catabolite and ammonium repressions, since the non-repressing synthesis of arylsulfatase with methionine or taurine as· the sole source of sulfur is not subject to catabolite control. 7) Comparative studies on the regulation of the syntheses of

1469

tyramine oxidase and arylsulfatase 7 ) and ge~ netic studies on mutant strains of K. aerogenesS ) led us to the conclusion. that the apparent level ofderepressed synthesis ofarylsulfatase by tyramine under conditions .of catabolite and ammonium control is just a reflection of the regulation pattern of tramine oxidase synthesis: that is, repression of arylsulfatase by the sulfate ion is relieved by tyramine oxidase. 3 ,S) We found that the inhibition of tyramine oxidase activity by cysteine resulted in a decrease of the derepressed synthesis of f3galactosidase with the ats : : lac fusion. The ats promoter seems to be more effective than the tyn promoter, since the induction ratio of f3galactosidase by tyramine was higher in the ats: : lac fusions than in the tyn: : lac fusions. Thus,we observed the expression of the latent operon for arylsulfatase in E. coli on lac gene fusion with Mud 1, and the ats£ operon is regulated by the expression of the tyn gene. Acknowledgments. We thank Professor T. Harada, Kobe Women College, and M. Oka, Takeda Co., Ltd., for their valuable discussions.

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17)

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