MOLECULAR-ORGANIZATION OF THE ESCHERICHIA-COLI-GAB CLUSTER - NUCLEOTIDE-SEQUENCE OF THE STRUCTURAL GENES GABD AND GABP AND EXPRESSION OF THE GABA PERMEASE GENE

被引:50
作者
NIEGEMANN, E
SCHULZ, A
BARTSCH, K
机构
[1] HOECHST AG, BIOL FORSCH BEREICH C, H872N, D-65926 FRANKFURT, GERMANY
[2] UNIV FRANKFURT, INST MIKROBIOL, D-60054 FRANKFURT, GERMANY
关键词
SUCCINIC SEMIALDEHYDE DEHYDROGENASE; GABA PERMEASE; GAB CLUSTER; REP ELEMENTS; GABA TRANSPORT; ESCHERICHIA-COLI;
D O I
10.1007/BF00245306
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We have determined the nucleotide sequences of two structural genes of the Escherichia coli gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway: gabD, coding for succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16) and gabP, coding for the 4-aminobutyrate (GABA) transport carrier (GABA permease). We have previously reported the nucleotide sequence of the third structural gene of the cluster, gabT, coding for glutamate: succinic semialdehyde transaminase (EC 2.6.1.19). All three gab genes are transcribed unidirectionally and their orientation within the cluster is 5'-gabD-gabT-gabP-3'. gabT and gabP are separated by an intergenic region of 234-bp, which contains three repetetive extragenic palindromic (REP) sequences. The gabD gene consists of 1,449 nucleotides specifying a protein of 482 amino acids with a molecular mass of 51.7 kDa. The protein shows significant homologies to the NAD+-dependent aldehyde dehydrogenase (EC 1.2.1.3) from Aspergillus nidulans and several mammals, and to the tumor associated NADP+-dependent aldehyde dehydrogenase (EC 1.2.1.4) from rat. The permease gene gabP comprises 1,401 nucleotides coding a highly hydrophobic protein of 466 amino acids with a molecular mass of 51.1 kDa. The GABA permease shows features typical for an integral membrane protein and is highly homologous to the aromatic acid carrier from E. coli, the proline, arginine and histidine permeases from Saccharomyces cerevisiae and the proline transport protein from A. nidulans. Uptake of GABA was increased ca. 5-fold in transformants of E. coli containing gabP plasmids. Strong overexpression of the gabP gene under control of the isopropyl-2-D-thiogalactoside (IPTG) inducible tac promoter, however, resulted in a severe growth inhibition of the transformed strains. The GABA carrier was characterized using moderately overexpressing transformants. The K(m) of GABA uptake was found to be 11.8 muM and the V(max) 0.33 nmol/min . mg cells. Uptake of GABA was stimulated by ammonium sulfate and abolished by 2,4-dinitrophenol. Aspartate competed with GABA for uptake.
引用
收藏
页码:454 / 460
页数:7
相关论文
共 36 条
[1]   STEREOSPECIFIC PRODUCTION OF THE HERBICIDE PHOSPHINOTHRICIN (GLUFOSINATE) BY TRANSAMINATION - CLONING, CHARACTERIZATION, AND OVEREXPRESSION OF THE GENE ENCODING A PHOSPHINOTHRICIN-SPECIFIC TRANSAMINASE FROM ESCHERICHIA-COLI [J].
BARTSCH, K ;
DICHMANN, R ;
SCHMITT, P ;
UHLMANN, E ;
SCHULZ, A .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1990, 56 (01) :7-12
[2]   MOLECULAR ANALYSIS OF 2 GENES OF THE ESCHERICHIA-COLI GAB CLUSTER - NUCLEOTIDE-SEQUENCE OF THE GLUTAMATE SUCCINIC SEMIALDEHYDE TRANSAMINASE GENE (GABT) AND CHARACTERIZATION OF THE SUCCINIC SEMIALDEHYDE DEHYDROGENASE GENE (GABD) [J].
BARTSCH, K ;
VONJOHNNMARTEVILLE, A ;
SCHULZ, A .
JOURNAL OF BACTERIOLOGY, 1990, 172 (12) :7035-7042
[3]  
BENNETZEN JL, 1982, J BIOL CHEM, V257, P3026
[4]   SEQUENCE OF THE LACTOSE PERMEASE GENE [J].
BUCHEL, DE ;
GRONENBORN, B ;
MULLERHILL, B .
NATURE, 1980, 283 (5747) :541-545
[5]   SUPERCOIL SEQUENCING - A FAST AND SIMPLE METHOD FOR SEQUENCING PLASMID DNA [J].
CHEN, EY ;
SEEBURG, PH .
DNA-A JOURNAL OF MOLECULAR & CELLULAR BIOLOGY, 1985, 4 (02) :165-170
[6]   CONTROL OF PATHWAY OF GAMMA-AMINOBUTYRATE BREAKDOWN IN ESCHERICHIA-COLI K-12 [J].
DOVER, S ;
HALPERN, YS .
JOURNAL OF BACTERIOLOGY, 1972, 110 (01) :165-&
[7]  
DOVER S, 1974, J BACTERIOL, V117, P494, DOI 10.1128/JB.117.2.494-501.1974
[8]   UTILIZATION OF GAMMA-AMINOBUTYRIC ACID AS SOLE CARBON AND NITROGEN SOURCE BY ESCHERICHIA-COLI K-12 MUTANTS [J].
DOVER, S ;
HALPERN, YS .
JOURNAL OF BACTERIOLOGY, 1972, 109 (02) :835-&
[9]   OVERPRODUCTION OF TRUNCATED SUBUNIT A OF H+-ATPASE CAUSES GROWTH-INHIBITION OF ESCHERICHIA-COLI [J].
EYA, S ;
MAEDA, M ;
TOMOCHIKA, KI ;
KANEMASA, Y ;
FUTAI, M .
JOURNAL OF BACTERIOLOGY, 1989, 171 (12) :6853-6858
[10]   PRIMARY STRUCTURES OF RAT AND BOVINE LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASES DEDUCED FROM CDNA SEQUENCES [J].
FARRES, J ;
GUAN, KL ;
WEINER, H .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1989, 180 (01) :67-74