Uncoupled release of protein and lipid in a protein-secreting bacterium, Bacillus brevis 47.

Bacillus brevis 47 was found to release approx. 10% of the total cellular lipids into the medium, and the protein secretion process in B. brevis 47 was studied to determine whether any relationship exists with lipid synthesis or alteration in the lipid composition. B. brevis 47 contained the usual phospholipids such as phosphatidylethanolamine, phosphatidylglycerol and cardiolipin as well as diglycerides as major neutral lipids. The extracellular lipid consisted of the same constituents as the cellular lipid but with a significantly altered composition. Divalent cations such as Ca2+, which specifically inhibit protein secretion, had no effect on the lipid release. A nonprotein-secreting mutant released lipid to the same extent as wild-type cells. Based on these results, we conclude that protein secretion occurs independently of lipid release.

Structurally different rat liver medium-chain acyl CoA dehydrogenases directed by complementary DNAs differing in their 5'-region.

Different forms of rat liver medium-chain acyl CoA dehydrogenase (MCAD) (EC 1.3.99.3) were produced in Escherichia coli carrying expression plasmids (pRMCADm-1 approximately 9) differing at the 5'-region of the cDNA. The proteins expressed could be readily extracted from the cells. The protein (approximately 44 kDa) directed by pRMCADm-3 showed the highest activity and was readily purified to homogeneity. The purified enzyme contained non-covalently bound FAD and was similar to rat liver mitochondrial enzyme in all respects examined. The purified protein (approximately 45 kDa) directed by pRMCADm-1 did not contain FAD and showed no enzymatic activity. Therefore, the leader peptide disturbs the binding of FAD to the apoprotein. The purified protein (approximately 40 kDa) directed by pRMCADm-6 did not contain FAD. Thus, the deletion of the NH2-terminal portion of the apoprotein to some extent results in its inability to combine with FAD.

Hexagonal surface array in a protein-secreting bacterium, Bacillus brevis 47.

Bacillus brevis 47, a protein-secreting bacterium, contained two major proteins with approximate molecular weights of 150 000 and 130 000 in the cell wall. The cell surface was covered with a hexagonally arranged array of six structural units about 4 nm in diameter with a lattice constant of 14.5 nm. The regular array structure as well as the chemical composition of cell envelopes remained the same regardless of the growth conditions. A mutant, strain 47-57, which was isolated as a phage resistant colony, contained only the 150 000 protein as a major cell wall protein. Although the mutant had hexagonally arranged arrays with the same lattice constant as that of wild-type cells, the distribution of mass in the unit cell differed considerably from that of the wild-type cells. The number of structural units in the unit cell of the mutant was reduced from six to three. Taking these results together with filtered images of the wild-type and mutant envelopes, two possible models for the surface array of B. brevis 47 are discussed.

Isolation of a cDNA encoding Aspergillus oryzae Taka-amylase A: evidence for multiple related genes.

Complementary and genomic DNAs encoding Aspergillus oryzae Taka-amylase A (Taa) were cloned and sequenced. The coding sequence of the cDNA comprised the signal peptide [21 amino acids (aa)] and mature Taa (478 aa). The deduced aa sequence agrees well with the published aa sequence, except for one insertion, one deletion and ten aa substitutions. These differences might be due to the difference in the strains used. Sequence comparison of the cDNA and genomic DNA indicates the presence of eight introns ranging in size from 55 to 86 bp. Southern-blot analysis showed the presence of at least two Taa genes, and the second gene (Taa-G2) was isolated. All the intron/exon junctions follow the 'GT-AG' rule, except for intron I of the first gene (Taa-G1). The 5'-noncoding region was well conserved among the genomic genes and contained sequences similar to 'CAAT' and 'TATA' boxes at nucleotides -121 and -31, counted from the transcription start point, respectively. The 3'-noncoding regions, however, differed significantly from each other. Taa-G2 contains a sequence identical to that of several independent cDNA clones, suggesting that it may be the major transcribed gene in A. oryzae.

Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein.

A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.

Human interleukin-1 receptor antagonist: large-scale expression in Bacillus brevis 47-5Q.

Interleukin-1 receptor antagonist (IL-1RA) has been used as a tool to study the biologic activity of IL-1 and as a possible therapeutic substance for inflammatory disease. To perform in vivo study, however, large quantities of IL-1RA are required. Bacillus brevis strains secrete large amounts of protein but little protease into the medium. Using B. brevis 47-5Q, we developed a large-scale expression system of human IL-1RA (HuIL-1RA). The bacteria secreted HuIL-1RA into the culture medium at very high levels, approximately 200 mg/L. The protein was isolated in one-step purification with monoclonal antibody (mAb) against HuIL-1RA. The IL-1RA molecule was determined to be functionally active by the inhibiting assay of HuIL-1-induced cell proliferation in a mouse T cell line, D10N4M.

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites.

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.

Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification.

Sphingomyelinase (sphingomyelin cholinephosphohydrolase) [EC 3.1.4.12] of Bacillus cereus was overproduced in a protein-hyperproducing strain, B. brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene. From 1 liter of culture, about 10 mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B. cereus IAM 1208. The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B. cereus was processed properly in B. brevis 47.

Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences.

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.

Major proteins released by a protein-producing bacterium, Bacillus brevis 47, are derived from cell wall protein.

B. brevis 47 secretes a vast amount of protein consisting mainly of two kinds with approximate molecular weights of 130,000 and 150,000. The two major extracellular proteins were indistinguishable from those of cell wall protein by SDS-polyacrylamide gel electrophoresis. Based on the results of analysis of amino acid composition, limited proteolysis followed by electrophoresis, and the cross-reactivity of antisera, we conclude that the 130K and 150K extracellular proteins are derived from the respective cell wall proteins. Furthermore, the NH2-terminal amino acid analysis suggests that the two major extracellular proteins are released from the cell wall without any modification of the NH2-terminal portion.

The efficient production of human epidermal growth factor by Bacillus brevis.

A system has been developed for the efficient production of heterologous proteins using Bacillus brevis as a host that secretes large amounts of cell wall protein into the medium. The promoter region and signal peptide-encoding region of the cell wall protein gene were used to construct an expression-secretion vector. Bacterial proteins such as amylases can be produced in large amounts by this system (1 g/l or more), but mammalian proteins such as human alpha-amylase are produced at a low level (one or two orders of magnitude less than for bacterial proteins). The highly efficient secretion of human epidermal growth factor (h-EGF, more than 1 g/l) was obtained with B. brevis HPD31 as the host and plasmid pHY481, derived from B. brevis 481, as the vector. Recombinant hEGF was purified easily from the culture supernatant by two steps. Purified hEGF had the identical NH2-terminal amino acid sequence and COOH-terminal amino acid sequence with those of the authentic hEGF, and it was fully active in biological assays. This recombinant hEGF has been shown to be successful for biological wool harvesting (CSIRO, Australia). These results, in combination with previous results, indicate that foreign proteins of diverse origins can be produced efficiently as functional proteins in B. brevis.

Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis.

We have constructed a very efficient synthesis and secretion system for cholera toxin B subunit (CTB) of Vibrio cholerae 569B using Bacillus-brevis. The constructed expression-secretion vector has the multiple promoters and the signal peptide coding region of the mwp gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, directly followed by the gene encoding the mature CTB. A large amount of mature CTB (1.4 g per liter of culture) was secreted into the medium. It had the same amino terminal amino acid sequence as that of authentic CTB and was fully active in GM1 ganglioside binding assay.

Production of human protein disulfide isomerase by Bacillus brevis.

Human protein disulfide isomerase (PDI; EC 5.3.4.1) was expressed and secreted into the culture medium using Bacillus brevis as host and pNU200 which codes the promoter and signal sequence of major cell wall protein of B. brevis as vector. The accumulation of recombinant human PDI (rhPDI) reached about 5 mg l-1 in the late exponential phase of the bacterial growth. The purified rhPDI was found to be exactly processed at the carboxyl terminus of the signal sequence. It was as active as natural PDI derived from human placenta as determined by its ability to reactivate scrambled ribonuclease that was a fully oxidized mixture containing randomly formed disulfide bonds. The activity was significantly accelerated in the presence of dithiothreitol or a mixture of reduced and oxidized glutathione. These indicate that the characteristics of rhPDI are similar to those reported for mammalian PDI and that it can be used for refolding inactive proteins having incorrect disulfide bonds.

Formation of mutagens by sorbic acid-nitrite reaction: effects of reaction conditions on biological activities.

Conditions of the reaction between sorbic acid and sodium nitrite generating mutagenic principles were examined. In the rec-assay and the Ames reversion assay, the maximal mutagenic activity was obtained in a pH range of 3.5-4.2. Mutagenic and growth-inhibitor activities of five C-nitro and C-nitroso compounds were studied. The product Y, 2-methyl-1,4-dinitropyrrole, was the strongest mutagen among them.

Antimutagenic effects of N-methyl-valyl-amiclenomycin (BA-2) isolated from the metabolites of Streptomyces sp.

A novel antimutagenic factor, BA-2, active against UV-induced mutagenesis in Escherichia coli WP2 was isolated from the metabolites of Streptomyces sp. strain AJ9455. BA-2 also suppressed mutations induced by 4-nitroquinoline N-oxide (4-NQO) and furylfuramide (AF-2) in E. coli WP2s (uvrA) without any decrease of cellular viability. BA-2 strongly inhibited the UV induction of SOS repair functions when it was monitored by beta-galactosidase activity expressed from the sulA::lacZ fusion gene of strain PQ37. It is assumed that the antimutagenic effect of BA-2 on mutagenesis induced by UV, 4-NQO or AF-2 was the result of inhibition of induction of the inducible error-prone SOS repair. The structure of BA-2 was considered to be N-methyl-valyl-amiclenomycin, and the structural unit of 4-amino-2,5-cyclohexadiene must be essential for the antimutagenic activity, since deamination by heating results in the loss of antimutagenic activity of BA-2.

A new test system for screening macromolecular antitumor antibiotics and its application to culture fluids of actinomycetes.

In searching for macromolecular antitumor antibiotics, a new screening method was developed that consisted of 1) a macromolecular antibiotic detecting system employing macro-molecule permeable mutants of Escherichia coli, 2) a system to detect DNA-affecting antibiotics using DNA repair mutants, and 3) a mutagenicity detecting system, employing a valine resistance test. This new test system was applied to about 2,900 kinds of culture fluids of Actinomycetes and consequently 15 samples were found which contained macromolecular antibiotics with DNA affecting properties.

Screening and some properties of new macromolecular peptide antibiotics.

In searching for macromolecular antitumor antibiotics of microbial origin, 2,875 kinds of Actinomycetes culture fluids were applied to a newly developed test system which consisted of antimicrobial assay using a macromolecule permeable mutant, DNA damage assay and mutagenicity test. As a result, 78 macromolecular antibiotics were found. Among them, 15 antibiotics precipitable with ammonium sulfate were macromolecular peptide antibiotics (protein antibiotics), of which molecular weight ranged from 10,000 to 14,000. Macromolecular peptide antibiotics AN-1, -5 and -15, termed type I antibiotics, showed stronger growth inhibitory effect on the uvrA and recA mutants, as compared to the effect on their parent, MP2. They also had mutagenic activity. AN-7, -9, -16, -18, -20, -22, -23, -25, and -26, termed type II, exhibited an increased inhibitory activity to a recA mutant but did not to an uvrA mutant. They all showed mutagenicity. AN-3, -11 and -13, type III antibiotics, gave similar influence on the DNA repair mutants, and on their parent, MP2. They had no mutagenic activity. Except for AN-11 and -13 of type III antibiotics, all antibiotics were inhibitory to the cell growth of a cancer cell, L1210.

The fermentation, isolation and characterization of a macromolecular peptide antibiotic: AN-3.

A new macromolecular peptide antibiotic, named AN-3, was isolated from the culture broth of Streptomyces albulus. From 19 liters of culture broth containing AN-3 with 90 units/ml activity, a 400 mg sample with a specific activity of 109 units/mg was obtained. Purified AN-3 gave a single band on polyacrylamide gel electrophoresis. AN-3 was a basic polypeptide with a molecular weight of 12,000 approximately 12,500 and an isoelectric point of pH 7.6. It showed a peak of absorption at 280 nm and seemed to have no nonprotein chromophoric component. It was soluble in water but insoluble in ethanol, butanol and acetone, and was stable at pH 4 approximately 9 but unstable at pH2. AN-3 had no antibacterial activity against Gram-positive and Gram-negative bacteria so far as tested. But, it showed a strong inhibitory effect on a macromolecule permeable mutant of Escherichia coli. It was not mutagenic. It appeared to inhibit synthesis of DNA and RNA without affecting DNA itself. It also inhibited the in vitro cell growth of L1210 and its ED50 was 5 micrograms/ml. AN-3 had antitumor activity against Lewis lung carcinoma in mouse in vivo.

The fermentation, isolation and characterization of macromolecular peptide antibiotics: AN-7A, -7B and -7D.

A group of new macromolecular peptide antibiotics, named AN-7, was isolated from the culture broth of Streptomyces griseoincarnatus AJ9424. AN-7 was fractionated into three different components, A, B and D. From 18 liters of culture broth (78 units/ml), 10 mg of AN-7A with a specific activity of 2,053 units/mg, 9 mg of AN-7B (1,167 units/mg) and 11 mg of AN-7D (6,225 units/mg) were obtained. All of these samples gave single bands on polyacrylamide gel electrophoresis. They are acidic polypeptides with molecular weights ranging from 12,400 to 13,000. Their UV absorption spectra showed maxima peaks at 280 nm and shoulders at 290 nm. Each AN-7 component has a nonprotein chromophoric component. AN-7A, -7B and -7D have no antibacterial activity against the Gram-negative bacteria tested but strongly inhibit the growth of Gram-positive bacteria and Escherichia coli MP2, a macromolecule permeable mutant strain. The AN-7 components are mutagenic. These antibiotics inhibit the in vitro growth of L1210 cells (ED50 0.13 approximately 0.18 micrograms/ml). AN-7A, -7B and -7D also inhibit the growth of L1210 cells in mice.

The fermentation, isolation and characterization of a macromolecular peptide antibiotic: AN-1.

A new macromolecular peptide antibiotic, named AN-1, was isolated from the culture broth of Streptomyces albus AJ9003. From 18 liters of culture broth (110 units/ml activity) a 300 mg sample of AN-1 was obtained with a specific activity of 1,160 units/mg was obtained. AN-1 is a basic polypeptide with a molecular weight of 12,000, isoelectric point of pH 8.3, and gives a single band on SDS polyacrylamide gel electrophoresis. It is soluble in water but insoluble in ethanol, butanol and acetone. It was stable at pH 6 approximately 9 but very unstable at pH 2. The UV absorption spectrum shows a maximum at 280 nm. AN-1 had no antibacterial activity against the Gram-positive and Gram-negative bacteria tested, but shows strong inhibitory activity toward Escherichia coli MP2, a macromolecule permeable mutant. In addition to being highly mutagenic, AN-1 inhibits the in vitro cell growth of L1210 (ED50 0.41 micrograms/ml). However, AN-1 had no antitumor activity against mouse leukemia L1210 or Lewis lung carcinoma in mouse.