The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor–operator interactions

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis . Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC–lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90°C, whereas B. subtilis AhrC was largely inactivated at 65°C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85°C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus . This pronounced resistance of the repressor–operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.     

Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli

The region required for regulation of a previously characterized arginine-regulatable promoter upstream from the argC gene in the argCAEBD-cpa-argF cluster of Bacillus subtilis was defined by integration of argC-lacZ translational fusions into the chromosome at a site distant from the arginine loci. Some sequence similarity was detected between the argC regulatory region and the well-characterized Escherichia coli arginine operators (ARG boxes). This similarity was shown to be functional in vivo in that the B. subtilis repressor regulated the E. coli arginine genes, but the E. coli repressor, even when encoded by a multicopy plasmid, could not repress the B. subtilis argC promoter. In vitro binding studies using purified repressors on DNA fragments encoding operators from both E. coli and B. subtilis demonstrated interactions by both proteins.     

Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production.

The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33. The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da. The argC gene is linked to argE, as shown by complementation of argE mutants of E. coli. Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S. lividans and in the argC mutant S. lividans 1674. Formation of AGPR is repressed by addition of arginine to the culture medium. The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E. coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp. A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein. Transformation of S. clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production.     

Regulation of arginine biosynthesis in the psychropiezophilic bacterium Moritella profunda: in vivo repressibility and in vitro repressor-operator contact probing

We report the cloning of the arginine repressor gene from the psychropiezophilic Gram-negative bacterium Moritella profunda, the purification of its product (ArgR(Mp)), the identification of the operator in the bipolar argECBFGH(A) operon, in vivo repressibility studies, and an in vitro analysis of the repressor-operator interaction, including binding to mutant and heterologous arginine operators. The ArgR(Mp) subunit shows about 70% amino acid sequence identity with Escherichia coli ArgR (ArgR(Ec)). Binding of purified hexameric ArgR(Mp) to the control region of the divergent operon proved to be arginine-dependent, sequence-specific, and significantly more sensitive to heat than complex formation with ArgR(Ec). ArgR(Mp) binds E.coli arginine operators very efficiently, but hardly recognizes the operator from Bacillus stearothermophilus or Thermotoga maritima. ArgR(Mp) binds to a single site overlapping the -35 element of argC(P), but not argE(P). Therefore, the arrangement of promoter and operator sites in the bipolar argECBFGH(A) operon of M.profunda is very different from the organization of control elements in the bipolar argECBH operon of E.coli, where both promoters overlap the common operator and are equally repressible. We demonstrate that M.profunda argC(P) is about 44-fold repressible, whereas argE(P) is fully constitutive. A high-resolution contact map of the ArgR(Mp)-operator interaction was established by enzymatic and chemical footprinting, missing contact and base-specific premodification binding interference studies. The results indicate that the argC operator consists of two ARG box-like sequences (18bp imperfect palindromes) separated by 3bp. ArgR(Mp) binds to one face of the DNA helix and establishes contacts with two major groove segments and the intervening minor groove of each ARG box, whereas the minor groove segment facing the repressor at the center of the operator remains largely uncontacted. This pattern is reminiscent of complex formation with the repressors of E.coli and B.stearothermophilus, and suggests that each ARG box is contacted by two ArgR subunits belonging to opposite trimers. Moreover, the premodification interference patterns and mutant studies clearly indicate that the inner, center proximal halves of each ARG box in the M.profunda argC operator are more important for complex formation and repression than the outermost halves. A close inspection of sequence conservation and of single base-pair O(c)-type mutations indicate that the same conclusion can be generalized to E.coli operators.     

Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis.

The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.     

Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis

The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.     

Crystallization and preliminary X-ray diffraction studies of nucleoside diphosphate kinase from Dictyostelium discoideum.

Nucleoside diphosphate kinase from the slime mold Dictyostelium discoideum is highly homologous to gene products that are involved in development in Drosophila and in oncogenesis in human cells. The cloned protein expressed in Escherichia coli has been purified and crystallized in a hexagonal space group with a = b = 74.9 A, c = 211.4 A. The asymmetric unit contains either one or two 17,000 Mr subunits of the hexamer.     

Bacillus subtilis DNA gyrase: purification of subunits and reconstitution of supercoiling activity

DNA gyrase from Bacillus subtilis 168 was purified by affinity chromatography on novobiocin-Sepharose and shown to consist of two subunits, A and B, with molecular weights of 100,000 and 85,000, respectively. The B subunits, which contains novobiocin-sensitive. ATPase activity, could complement the gyrA protein of Escherichia coli. No complementation was detected between the A subunit and the E. coli gyrB protein.     

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.     

Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase

Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth. The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation. PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization. Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization. The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA). The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E. coli cells. The PHA synthase expressed in the recombinant E. coli cells was purified to homogeneity. Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases. Two major complexes were identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period. The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E. shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex.     

Crystallization of the arginine-dependent repressor/activator AhrC from Bacillus subtilis

The arginine-dependent repressor/activator AhrC from Bacillus subtilis has been crystallized in space group C222(1), with unit cell dimensions a = 229.8 A, b = 72.8 A, c = 137.7 A and one aporepressor hexamer per asymmetric unit. Preliminary X-ray photographs show measurable intensities beyond 3.0 A.     

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis.

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.     

Secretory production of human granulocyte colony-stimulating factor in Escherichia coli.

Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein, consisting of 174 amino acids, which plays an important role in hematopoietic cell proliferation, differentiation of hemopoietic precursor cells, and activation of mature neutrophilic granulocytes. In this study, secretory production of hG-CSF in the periplasmic space of Escherichia coli using the Bacillus sp. endoxylanase signal peptide was examined. For the efficient expression of hG-CSF gene, the first five codons at the N-terminal were altered based on the E. coli high-frequency codon database. The hG-CSF gene fused to the endoxylanase signal sequence was expressed using an inducible trc promoter. However, recombinant E. coli cells were completely lysed after induction with 1 mM isopropyl-beta-D-thiogalactopyranoside. Insertion of a small oligopeptide (13 amino acids) containing the histidine hexamer and factor Xa cleavage site between the signal peptide and the mature hG-CSF protein allowed successful secretion of hG-CSF into the periplasm without cell lysis. Among the several E. coli strains examined, E. coli BL21(DE3) and E. coli MC4100 allowed production of hG-CSF to the highest levels (20-22% of total proteins) with the secretion efficiencies greater than 98%. The circular dichroism spectra showed that the conformation of purified hG-CSF is almost identical to native hG-CSF.     

Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis.

Various streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Strepotmyces coelicolor A3 (2)] acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli. The expression level in these hosts was two to three times lower than in S. reticuli, indicating the absence of positive regulatory elements. Like S. reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part. The cel-1 gene with its original upstream region was not expressed within Escherichia coli. When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E. coli. However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms. As the cellulase (Avicelase) synthesized by S. reticuli is not cleaved by the E. coli proteases, its posttranslational modification is proposed. With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter.