Reassembly in vitro of hexagonal surface arrays in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 had two protein layers (the outer and middle walls) and a peptidoglycan layer (the inner wall) and contained two major proteins with approximate molecular weights of 130,000 and 150,000 in the cell wall. Both the total and Triton-insoluble envelopes revealed a hexagonal lattice array with a lattice constant of 14.5 nm. The proteins of 130,000 and 150,000 molecular weight isolated from the Triton-insoluble envelopes were serologically different from each other and assembled in vitro on the peptidoglycan layer. A mixture of 130,000- and 150,000-molecular-weight proteins led to the formation of a five-layered cell wall structure, two layers on each side of the peptidoglycan layer, which resembled closely the Triton-insoluble envelopes. A three-layered cell wall structure, one layer on each side of the peptidoglycan layer, was reconstituted when only the 150,000-molecular-weight protein was used. Both five- and three-layered cell walls reconstituted in vitro also contained hexagonally arranged arrays with the same lattice constant as that of the total and Triton-insoluble envelopes. A mutant, strain 47-57, which was isolated as a phage-resistant colony, had a two-layered cell wall consisting of the middle and inner wall layers and contained only 150,000-molecular-weight protein as the major cell wall protein. The cell envelopes of the mutant revealed the hexagonal arrays with the same lattice constant as that of the wild-type cell envelopes. We conclude that the outer and middle wall layers consist of proteins with approximate molecular weights of 130,000 and 150,000, respectively. Furthermore, the 150,000-molecular-weight protein formed the hexagonal arrays in the middle wall layer.     

Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 secreted vast amounts of protein into the medium and had a characteristic three-layered cell wall. The three layers are designated, from the outermost to the innermost layer, as the outer wall (4.2 nm), the middle wall(8.5 nm), and the inner wall (2.1-3.7 nm). The inner wall might be a peptidoglycan layer. The fine cell wall structure was morphologically altered to various extents, depending on the growth period. At the early stationary phase of growth, cells began to shed the outer two layers of a limited area of the surface. This shedding was complete after further cell growth. The morphological alterations in the cell wall occurred concomitantly with a prominent increase in protein excretion. When protein secretion was severely inhibited by growing cells with Mg2+, morphological alterations in the cell wall were not observed, even at the late stationary phase of growth. This was also the case with a nonprotein-producing mutant, strain 47-5-25. When cells were incubated in buffers, the outer two layers of the cell wall were specifically removed, leaving cells surrounded only by the inner wall layer. The layers removed by incubation were recovered by high-speed centrifugation. This fraction consisted of two layers resembling the outer and middle wall layers. Protein secreted by B. brevis 47-5 consisted mainly of two proteins with approximate molecular weights of 150,000 and 130,000. Proteins released by incubating cells in buffers and proteins in the outer- and middle-wall-enriched fraction were also composed mainly of two proteins with the same molecular weights as those secreted into the medium. Therefore, we conclude that B. brevis 47 secretes proteins derived from the outer two layers of cell wall and these components are synthesized even after the shedding of the outer two layers.     

A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium.

A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.     

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.     

A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium

A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.     

Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA.

A method has been developed for introducing plasmid DNA into Bacillus brevis 47, a protein-secreting bacterium. Treatment of B. brevis 47 cells with 50 mM Tris-hydrochloride buffer of alkaline pH was effective for inducing DNA uptake competence. In the presence of polyethylene glycol, the Tris-treated cells incorporated plasmid DNA with a frequency of 10(-4) (transformants per viable cell) when 1 microgram of plasmid DNA was added to 10(9) Tris-treated cells. The pH of Tris-hydrochloride buffer as well as the concentration and molecular weight of the polyethylene glycol affected the transformation frequency. The growth phase of B. brevis 47 cells strongly influenced the frequency. Two plasmids, pHW1 and pUB110, have been introduced into B. brevis 47 by this method. The mechanism of induction of competence for DNA uptake in connection with removal of the outer two protein layers of the cell wall by treatment of B. brevis 47 cells with Tris-hydrochloride buffer is discussed.     

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.     

Molecular cloning of a major cell wall protein gene from protein-producing Bacillus brevis 47 and its expression in Escherichia coli and Bacillus subtilis

Bacillus brevis 47 contains two major cell wall proteins. Each protein forms a hexagonal array in the cell wall. A 4.8-kilobase HindIII fragment of B. brevis 47 DNA cloned into Escherichia coli with pBR322 as a vector directed the synthesis of polypeptides cross-reactive with antibody to the middle wall protein. A 700-base-pair BamHI-HpaI fragment was shown to be the essential region for the synthesis of immunoreactive polypeptides. Furthermore, this fragment appeared to contain the promoter activity. The 3.5-kilobase BamHI fragment covering the essential region as well as its downstream sequence was subcloned into the corresponding restriction site of pUB110 by using Bacillus subtilis as the cloning host. Both E. coli and B. subtilis carrying the cloned DNA synthesized several immunoreactive polypeptides which were mainly found in the cytoplasm. B. subtilis secreted polypeptides cross-reactive with antibody to the middle wall protein. These extracellular polypeptides were degraded upon prolonged culture.     

Comparative study on the cell wall structure of protein-producing Bacillus brevis

Abstract Among eight strains of protein-producing Bacillus brevis , three morphological groups have been identified according to the structure of the cell walls.

• (I)

  Cell wall consisting of a peptidoglycanlayer
• (II)

  Two-layered cell wall consisting of a peptidoglycan-layer and an S-layer
• (III)

  Three-layered cell wall consisting of a peptidoglycan-layer and two S-layers

Group I and group II cell walls have not been described yet for protein-producing bacteria. The S-layers observed in this study all had hexagonal symmetry and lattice constants of approximately 18 nm. The immunological relation between the S-layer proteins of the newly isolated B. brevis strains and those of B. brevis 47 has been examined using antisera against both S-layer-proteins of B. brevis 47. S-layers from protein-producing B. brevis strains, which were adjacent to the peptidoglycan-layer, were similar to each other, whether they were the outermost cell wall layer (group II) or not (group III). However, no similarity was found between these layers and the outermost S-layer of B. brevis 47 (group III).     

Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene.

Bacillus brevis 47 contains two surface (S)-layer proteins, termed the outer wall protein (OWP) and the middle wall protein (MWP), which form a hexagonal array in the cell wall. The MWP and OWP genes are contained in the 9-kilobase-pair (kbp) BclI fragment and constitute an operon under coordinate control of their expression. The nucleotide sequence of a 3.8-kbp EcoRI-SacI fragment containing the entire MWP gene has been determined in this study. Together with the DNA sequence of the promoter region for the MWP-OWP gene operon (H. Yamagata, T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 169:1239-1245, 1987) and that of the OWP gene (A. Tsuboi, R. Uchihi, R. Tabata, Y. Takahashi, H. Hashiba, T. Sasaki, H. Yamagata, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 168:365-373, 1986), the complete nucleotide sequence of the MWP-OWP gene operon has been determined. The MWP gene encodes a secretory precursor of the MWP, consisting of a total of 1,053 amino acid residues with a signal peptide of 23 amino acid residues at its amino-terminal end. Bacillus subtilis harboring the MWP gene synthesized an immunoreactive polypeptide with almost the same molecular weight as the authentic MWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compositions deduced from the MWP and OWP genes were similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic amino acids compared with basic amino acids and in the very low content of sulfur-containing amino acids. The acidic nature of the MWP and OWP was confirmed by isoelectric focusing on polyacrylamide gels. In addition, circular dichroism spectra indicated that the S-layer proteins in B. brevis 47 were composed of approximately 30% beta-sheet and 5% alpha-helical structures, with the remainder of the polypeptide backbone being aperiodic in nature.     

Production of swine pepsinogen by protein-producing Bacillus brevis carrying swine pepsinogen cDNA

Summary An expression-secretion vector, pNU100, was constructed, utilizing the promoter and coding sequences for the signal peptide and nine amino-terminal amino acids of the middle wall protein, to produce foreign proteins by protein-producing Bacillus brevis. Expression of swine pepsinogen cDNA in B. brevis was examined with pNU100 as a vector. The recombinant swine pepsinogen synthesized by B. brevis was found to accumulate extracellularly in the form of a soluble protein and to have acid protease activity. The acid protease activity was completely inhibited by pepstatin. Furthermore, the recombinant pepsinogen was converted autocatalytically to pepsin under acidic conditions. This indicates that B. brevis produces a pepsinogen with the same conformation as authentic pepsinogen. Efficient production of the enzyme (11 mg/l) was achieved by regulating the pH of the medium. The enzyme produced by B. brevis remained stable on cultivation for a long period, up to 40 h. This is suggested to be due to a unique property of protein-producing B. brevis, i. e. a deficiency in extracellular protease production.     

Transformation of Bacillus brevis 47 by electroporation

Summary Bacillus brevis 47 was successfully and reproducibly transformed with pUB110 plasmid DNA by electroporation with an efficiency of 10⁴ transformants per g of DNA. This represents a 10-fold improvement over the chemical transformation method previously used for this organism.     

Stimulation of extracellular protein production in Bacillus brevis 47

Summary Bacillus brevis 47 was cultivated in 2-1 fermentors to study the effect of medium supplementation on extracellular protein production. Additional polypeptone, when supplied initially or at 12 h (late exponential phase), had little stimulatory effect on extracellular protein levels, which reached 6–7 g/l after 48h. A large increase in protein production was observed, however, when polypeptone was added at 21 h (stationary phase). This addition resulted in the accumulation in the medium of 14 g/l protein after 48 h, and a total of 16 g/l when cell-bound protein was included. In all cases, glucose was consumed only very slowly.     

Protein Production in Chemically Defined Medium by Bacillus brevis No. 47

A simple chemically defined medium was devised for exoprotein production by Bacillus brevis No. 47. About 2 mg/ml of proteins was produced in the synthetic medium containing 4% glucose and 1% ammonium sulfate. An essential component of fermentation medium was Ca salt which is required by this organism for assimilating glucose.

Studies on the effects of various medium components on protein production revealed that the conditions appropriate for growth are also suitable for protein accumulation. Some compounds, especially inhibitors of cell wall synthesis and certain detergents, were found to enhance protein production.     

Effects of Phosphate in Medium on Protein Secretion in a Protein-Producing Bacterium, Bacillus brevis 47

Bacillus brevis 47 secreted up to 1 mg of protein per ml in a chemically defined medium, depending on phosphate concentration. The composition of exoproteins was altered quantitatively by the concentration of external phosphate. Morphologically, B. brevis 47 showed a distinct three-layered cell wall structure and shed the outer two layers during growth.