Mutants of <i>Bacillus stearothermophilus</i> De-repressed for Isocitrate Lyase and Malate Synthase

Chell, R. M.; Sundaram, T. K.

doi:10.1042/bst0030306

Biochemical Society Transactions

1975

DOI: 10.1042/bst0030306

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Mutants of Bacillus stearothermophilus De-repressed for Isocitrate Lyase and Malate Synthase

R. M. Chell

T. K. Sundaram

Abstract: A mutant of Bacillus stearothermophilus deficient in pyruvate carboxylase (EC 6.4.1. l) is inhibited in its growth in a salts-acetate medium on the addition of lactate or glucose to the acetate culture. Secondary mutants isolated for ability to grow in a salts-acetate lactate medium are still deficient in pyruvate carboxylase but arecapable of synthesizing the glyoxylate-cycle enzyme, isocitrate lyase (EC 4.1.3.1), at an elevated rate (Sundaram,

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“…The isocitrate lyase from these two strains is strongly activated by salts, unlike the same enzyme from several mesophilic micro-organisms (Griffiths & Sundaram, 1973; R. M. Chell & T. K. Sundaram, unpublished work), and mutants of the latter thermophile have Vol. 173 been isolated that are de-repressed for the two glyoxylate-cycle enzymes (Sundaram, 1973;Chell & Sundaram, 1975). The salt activation, which appeared to be an interestingly peculiar property of the thermophile isocitrate lyase, prompted the isolation in pure state and a detailed characterization of this enzyme.…”

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confidence: 99%

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp

Chell¹,

Sundaram²,

Wilkinson³

1978

Biochemical Journal

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Isocitrate lyase was isolated in homogeneous state from a thermophilic Bacillus. The enzyme has a mol.wt. of 180000 and a pI of 4.5 and contains threonine as the N-terminal residue. It resembles in size the cognate enzyme from the mesophilic bacterium Pseudomonas indigofera, but is smaller than the enzyme from the eukaryotic fungus Neurospora crassa. All three lyases are tetramers and similar in amino acid composition, but the thermophile enzyme is distinctive from its mesophilic coutnerparts in possessing a lower catalytic-centre activity, greater resistance to chemical and thermal denaturation and fewer thiol groups and in being strongly activated by salts. Salt activation, by 0.4M-KCl, is about 3-fold at 30 degrees C and pH 6.8 and weakens progressively as the temperature or pH is raised. The activation is probably due to a change in the enzyme conformation caused by the electrolyte modifying the interaction between charged groups or between hydrophobic groups in protein. The possible significance of the salt activation, of the relative paucity of thiol groups and of the greater resistance to chemical denaturants is discussed. Besides its effect on the Vmax., KCl produces large increases in the magnitude of several kinetic parameters. A rise in reaction temperature from 30 to 55 degrees C produces a somewhat similar result. In view of these peculiar features, the patterns of inhibition of enzyme activity by compounds such as succinate and phosphoenolpyruvate were examined at 30 and 55 degrees C in the presence and absence of KCl.

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mentioning

confidence: 99%

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp

Chell¹,

Sundaram²,

Wilkinson³

1978

Biochemical Journal

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Monomeric malate synthase from a thermophilic Bacillus

Sundaram¹,

Chell²,

Wilkinson³

1980

Archives of Biochemistry and Biophysics

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The Control of the Synthesis of Isocitrate Lyase in a Thermophilic Bacillus

Griffiths

1979

Journal of General Microbiology

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From a strain of Bacillus stearothermophilus, devoid of active pyruvate carboxylase, a mutant (NG-15) was selected that grew on acetate in the presence of glucose. This mutant differed from its parent organism in possessing high activities of isocitrate lyase when grown on all carbon sources tested except nutrient broth, in possessing unusually low activities of NADPf-dependent isocitrate dehydrogenase and in containing increased amounts of isocitrate. Revertants of mutant NG-15 which regained the ability to synthesize active pyruvate carboxylase also synthesized isocitrate lyase and isocitrate dehydrogenase to the same extent as the wild-type strain. These results suggest that the regulatory mechanism for the synthesis of isocitrate lyase in the thermophile may be different from that in mesophilic bacilli. (Sundaram, 1973). However, one such mutant, designated NG-15, has been found which may not be truly constitutive for the enzyme. The properties of this mutant are described here and the results are discussed in relation to a possible regulatory system for isocitrate lyase synthesis in this thermophilic strain of Bacillus. I N T R O D U C T I O N METHODSIsolation of mutant. A mutant designated PC-2, devoid of active pyruvate carboxylase, was isolated from wild-type cells of a thermophilic strain of Bacillus (Sundaram, 1973). This organism was used as a source of further mutants which were not inhibited by glucose when they were grown on acetate as carbon source. Cultures of strain PC-2 were grown in nutrient broth and were inoculated into flasks containing salts , supplemented with biotin (0.1 pg ml-l) and sodium acetate (50 mM). The cultures were harvested in the mid-exponential phase of growth by centrifuging at 1OOOOg for 10 min at 18 "C. The cells were resuspended to about 0.1 mg dry mass ml-l in the salts solution and samples (0.05 to 0.2 ml) of the suspensions were spread on to plates containing a mixture of sodium acetate (50 m), glucose (10 m), biotin (0.1 pg ml-l) and nutrient broth (160 pg ml-l), solidified with 1.5 % (w/v) agar. A crystal of 5'-methyl-5'-nitrosoguanidine was placed in the middle of each plate and the plates were incubated at 55 "C for *

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Mutants of Bacillus stearothermophilus De-repressed for Isocitrate Lyase and Malate Synthase

Cited by 3 publications

References 1 publication

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp

Monomeric malate synthase from a thermophilic Bacillus

The Control of the Synthesis of Isocitrate Lyase in a Thermophilic Bacillus

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