The sulfate activation locus of Escherichia coli K12: cloning, genetic, and enzymatic characterization.
The sulfate activation locus of Escherichia coli K12 has been cloned by complementation. The genes and gene products of this locus have been characterized by correlating the enzyme activity, complementation patterns, and polypeptides associated with subclones of the cloned DNA. The enzymes of the sulfate activation pathway, ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (ATP:adenosine-5'-phosphosulfate 3'-phosphotransferase, EC 2.7.1.25) have been overproduced approximately 100-fold. Overproduction of ATP sulfurylase requires the expression of both the cysD gene, encoding a 27-kDa polypeptide, and a previously unidentified gene, denoted cysN, which encodes a 62-kDa polypeptide. Purification of ATP sulfurylase to homogeneity reveals that the enzyme is composed of two types of subunits which are encoded by cysD and cysN. Insertion of a kanamycin resistance gene into plasmid or chromosomal cysN prevents sulfate activation and decreases expression of the downstream cysC gene. cysC appears to be the APS kinase structural gene and encodes a 21-kDa polypeptide. The genes are adjacent and are transcribed counterclockwise on the E. coli chromosome in the order cysDNC. cysN and cysC are within the same operon and cysDNC are not in an operon containing cysHIJ.
Leyh
T S
TS
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Taylor
J C
JC
Markham
G D
GD
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005001:Ethionine / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D010452:Peptide Biosynthesis; D010770:Phosphotransferases / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D013430:Sulfate Adenylyltransferase / Q000096:biosynthesis / Q000235:genetics; D013431:Sulfates / Q000378:metabolism*
D013431:Sulfates; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D013430:Sulfate Adenylyltransferase; D005001:Ethionine