THE ACONITASE OF ESCHERICHIA-COLI - PURIFICATION OF THE ENZYME AND MOLECULAR-CLONING AND MAP LOCATION OF THE GENE (ACN)

被引:31
作者
PRODROMOU, C [1 ]
HAYNES, MJ [1 ]
GUEST, JR [1 ]
机构
[1] UNIV SHEFFIELD,DEPT MOLEC BIOL & BIOTECHNOL,KREBS INST BIOMOLEC RES,POB 594,FIRTH COURT,SHEFFIELD S10 2UH,ENGLAND
来源
JOURNAL OF GENERAL MICROBIOLOGY | 1991年 / 137卷
关键词
D O I
10.1099/00221287-137-11-2505
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The aconitase of Escherichia coli was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of M(r) 95000 or 97500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Bacillus subtilis enzyme (citB product), but the similarity at the DNA level was insufficient to allow detection of the E. coli acn gene using a 456 bp citB probe. Phages containing the acn gene were isolated from a lambda-E. coli gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E. coli chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara et al. (Cell 50, 495-508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (M(r) 120 000) enzymes.
引用
收藏
页码:2505 / 2515
页数:11
相关论文
共 38 条
[1]   CLONING, SEQUENCING, AND MAPPING OF THE BACTERIOFERRITIN GENE (BFR) OF ESCHERICHIA-COLI K-12 [J].
ANDREWS, SC ;
HARRISON, PM ;
GUEST, JR .
JOURNAL OF BACTERIOLOGY, 1989, 171 (07) :3940-3947
[2]  
Arrand J., 1985, NUCLEIC ACID HYBRIDI, P17
[3]   CLONING OF THE UVRD GENE OF ESCHERICHIA-COLI AND IDENTIFICATION OF THE PRODUCT [J].
ARTHUR, HM ;
BRAMHILL, D ;
EASTLAKE, PB ;
EMMERSON, PT .
GENE, 1982, 19 (03) :285-295
[4]   LINKAGE MAP OF ESCHERICHIA-COLI K-12, EDITION-8 [J].
BACHMANN, BJ .
MICROBIOLOGICAL REVIEWS, 1990, 54 (02) :130-197
[5]   NUCLEOTIDE-SEQUENCE OF THE FNR-REGULATED FUMARASE GENE (FUMB) OF ESCHERICHIA-COLI K-12 [J].
BELL, PJ ;
ANDREWS, SC ;
SIVAK, MN ;
GUEST, JR .
JOURNAL OF BACTERIOLOGY, 1989, 171 (06) :3494-3503
[6]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[7]   PURIFICATION OF ACONITASE FROM BACILLUS-SUBTILIS AND CORRELATION OF ITS N-TERMINAL AMINO-ACID-SEQUENCE WITH THE SEQUENCE OF THE CITB GENE [J].
DINGMAN, DW ;
SONENSHEIN, AL .
JOURNAL OF BACTERIOLOGY, 1987, 169 (07) :3062-3067
[8]  
EMPTAGE MH, 1983, J BIOL CHEM, V258, P1106
[9]   COMPLETE NUCLEOTIDE-SEQUENCE OF THE ESCHERICHIA-COLI PTR GENE ENCODING PROTEASE-III [J].
FINCH, PW ;
WILSON, RE ;
BROWN, K ;
HICKSON, ID ;
EMMERSON, PT .
NUCLEIC ACIDS RESEARCH, 1986, 14 (19) :7695-7703
[10]   COMPLETE NUCLEOTIDE-SEQUENCE OF THE ESCHERICHIA-COLI RECC GENE AND OF THE THYA-RECC INTERGENIC REGION [J].
FINCH, PW ;
WILSON, RE ;
BROWN, K ;
HICKSON, ID ;
TOMKINSON, AE ;
EMMERSON, PT .
NUCLEIC ACIDS RESEARCH, 1986, 14 (11) :4437-4451