A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.
Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.
Guilloton
M B
MB
Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth 55812.
Lamblin
A F
AF
Kozliak
E I
EI
Gerami-Nejad
M
M
Tu
C
C
Silverman
D
D
Anderson
P M
PM
Fuchs
J A
JA
D001639:Bicarbonates / Q000378:metabolism; D015139:Blotting, Southern; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003485:Cyanates / Q000378:metabolism*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D015151:Immunoblotting; D007700:Kinetics; D009154:Mutation; D015183:Restriction Mapping
D001639:Bicarbonates; D003485:Cyanates; D002256:Carbonic Anhydrases