Guilloton M B, Lamblin A F, Kozliak E I, Gerami-Nejad M, Tu C, Silverman D, Anderson P M, Fuchs J A
Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth 55812.
J Bacteriol. 1993 Mar;175(5):1443-51. doi: 10.1128/jb.175.5.1443-1451.1993.
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.
氰酸盐可诱导大肠杆菌中cyn操纵子的表达。cyn操纵子包含编码氰酸酶的cynS基因，该酶催化氰酸盐与碳酸氢盐反应生成氨和二氧化碳。最近发现cyn操纵子的第一个基因cynT编码一种碳酸酐酶活性；有人提出，碳酸酐酶可防止在氰酸盐分解过程中由于二氧化碳通过扩散从细胞中损失而导致碳酸氢盐耗尽（M. B. Guilloton、J. J. Korte、A. F. Lamblin、J. A. Fuchs和P. M. Anderson，《生物化学杂志》267:3731 - 3734，1992）。该操纵子第三个基因cynX产物的功能尚不清楚。在本文报道的研究中，通过构建使三个基因中的每一个都失活的染色体突变体来研究cynT和cynX的生理作用。缺失cynT的染色体突变体表达了活性氰酸酶，但没有活性碳酸酐酶。与野生型菌株相比，缺失cynT的菌株的生长受到氰酸盐的抑制，并且该突变菌株无法降解氰酸盐，因此在0.03%（空气）的部分二氧化碳压力（pCO2）下生长时不能将氰酸盐用作唯一氮源。然而，在高pCO2（3%）时，缺失cynT的菌株表现得与野生型菌株一样；它对氰酸盐的毒性作用明显不那么敏感，并且可以降解氰酸盐并将氰酸盐用作生长的唯一氮源。这些结果与碳酸酐酶的推测功能一致。携带cynS::kan的染色体突变体表达了诱导的碳酸酐酶活性，但没有活性氰酸酶。发现cynS::kan突变体在低pCO2时对氰酸盐的敏感性比缺失cynT的突变体低得多，这表明由氰酸酶催化的碳酸氢盐与氰酸盐反应导致的碳酸氢盐耗尽对生长的危害比氰酸盐的直接抑制更大。携带无功能cynX基因的突变体（cynX::kan和缺失cynT cynX::kan）在氰酸盐敏感性、碳酸酐酶和氰酸酶的存在或全细胞对氰酸盐的降解方面与亲本菌株没有差异；cynX产物的生理作用仍然未知。
