The Bacillus stearothermophilus argCJBD operon harbours a strong promoter as evaluated in Escherichia coli cells

• Published in: Gene Vol. 212; no. 2; pp. 167 - 177
• Main Authors: Savchenko, Alexey, Weigel, Pierre, Dimova, Diliana, Lecocq, Michèle, Sakanyan, Vehary
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08-06-1998
• Subjects: Acetyltransferases - genetics; Aldehyde Oxidoreductases; Arginine - genetics; arginine biosynthesis; Bacillus subtilis - genetics; Bacterial Proteins - genetics; Base Sequence; Escherichia coli - genetics; Genes, Bacterial; Geobacillus stearothermophilus - genetics; heterologous expression; Integration Host Factors; Molecular Sequence Data; Operon - genetics; overexpression; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; thermophiles; moaD, gene for molybdopterin converting factor (subunit 1); ArgJ, argJ, N-acetylornithine: l-glutamate- N-acetyltransferase and coding gene; ArgF, argF, ornithine carbamoyl transferase and coding gene; dATP, deoxyadenosine 5′-triphosphate; P lac, promoter of the lac-operon in E. coli; ywiD, gene for hypothetical 18.1 kDa protein (in narK-narG intergenic region of B. subtilis); P trp, promoter of the trp-operon in E. coli; Fnr, fnr, anaerobic regulatory protein and encoding gene; ipi, intracellular proteinase inhibitor BsuPI gene; ArgC, argC, N-acetylglutamate 5-semialdehyde dehydrogenase and coding gene; narK, gene for nitrite extrusion protein; ArgE, argE, N-acetylornithine aminohydrolase and coding gene; overexpression; nprB, extracellular neutral protease gene; ArgR, argR, repressor of arginine biosynthesis and encoding gene; bla, sequence coding for ampicillin; ywiC, gene for hypothetical 27.6 kDa protein (in fnr-narG intergenic region of B. subtilis); BSTM, synthetic medium for B. stearothermophilus; narG, gene for nitrate reductase ( α-chain); ArgA, argA, N-acetylglutamate synthase and coding gene; argCo1, operator site upstream of argC in B. subtilis; AhrC, ahrC, arginine hydroxamate resistance (ArgR) in B. subtilis and coding gene; HEPES, N-(2-hydoxyethyl) piperazine-N′-(2-ethanesulfonic acid); UP-element, sequence upstream of the −35 site of a promoter; kb, kilobase pairs; argCo2, operator site in the coding region of argC in B. subtilis; T7 RNA polymerase, polymerase of the phage T7; UTP, uracyl triphosphate; argCJBD, arginine biosynthetic genes; P L2, bacteriophage λ promoter; arginine biosynthesis; PD-10 columns, desalting columns for cell extracts; tet, sequence coding for tetracycline resistance; SD, Shine–Dalgarno (sequence); moaE, gene for molybdopterin converting factor (subunit 2); Arg box', consensus sequences for interaction with ArgR; cpa, gene for carboxypeptidase A precursor (phospholipase C); P arg, promoter of argCJBD genes; heterologous expression; pb, pair base; ArgD, argD, N-acetylornithine 5-aminotransferase and coding gene; LB, Luria–Bertani medium; P tac, chimeric promoter combining −10 site of P lac with the −35 site or P trp; yitY, gene for hypothetical protein similar to l-gulonolactone oxidase; thermophiles; SDS-PAGE, sodium dodecyl sulphate polyacrylamide-gel electrophoresis; 10, −35 sites, consensus sequences in a promoter; IHF, integrational host factor of E. coli; mobB, gene for molybdopterin-guanine dinucleotide biosynthesis protein B; argCo, operator site of argC in B. stearothermophilus; M9, minimal medium for E. coli; narH, gene for nitrate reductase ( β-chain); NADP/NADP +, nicotinamide-adenine dinucleotide phosphate/reduced form; AT-rich sequence, sequence rich in adenine and thymine; kDa, kilodalton; RBS, ribosome binding site(s); RocR, rocR, activator of arginine catabolism and encoding gene; ArgB, argB, N-acetylglutamate kinase and coding gene; ArgS, argS, arginyl-tRNA synthase and coding gene; IPTG, isopropyl β- d-thiogalactopyranoside
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(98)00174-7

We have shown that the
B. stearothermophilus argCJBD genes form a single operon. In
B. stearothermophilus, a specific repressor governs operon expression by binding to the
argCo operator site overlapping the P
arg promoter sequence (
Dion et al., 1997). Therefore, the enzymatic and transcriptional analyses performed in this work did not reflect the potential strength of P
arg in the native host. For evaluation of the P
arg promoter strength,
E. coli was used as a host since its own ArgR repressor does not interact with the
B. stearothermophilus heterologous operator. P
arg-promoted
argC gene expression dramatically increased, reaching up to 38% of the total protein in
E. coli cells. An AT-rich sequence upstream of a −35 site of P
arg was found to be indispensable for the promoter strength. Plasmids carrying the
B. stearothermophilus argCJBD operon linked with its P
arg/
argCo region were unstable in
E. coli. Stabilization of plasmids was achieved by repression of
B. stearothermophilus arg genes through the action of the
B. subtilis AhrC repressor.