Major Proteins Released by a Protein-Producing Bacterium, Bacillus brevis 47, Are Derived from Cell Wall Protein1

Ohmizu, Hiroshi; Sasaki, Takuji; Tsukagoshi, Norihiro; Udaka, Shigezo; Kaneda, Norio; Yagi, Kunio

doi:10.1093/oxfordjournals.jbchem.a134450

Cited by 20 publications

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“…The results are averages of three time points, 24, 48, and 72 h. The analysis results for the three time points were quite stable and consistent with the absence of large amounts of carbohydrate in the purified sample. The content of 47% nonpolar residues was similar to the values 44 to 50% determined for other Bacillus S-layer proteins analyzed to date (25,31). The contents of 15% hydroxyl and 15% basic residues were also within published ranges of 11 to 18% for hydroxyl and basic residues in Bacillus S-layer proteins (25,31).…”

Section: Fig 3 Sds-page Of Sds Cell Extracts and Culture Supernatansupporting

confidence: 81%

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1

et al. 1995

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Many prokaryotic organisms possess surface layer (S-layer) proteins that are components of the outermost cell envelope. With immunogold labeling, it was demonstrated that the protein extractable antigen 1 (EA1) was localized on the outer surface and specifically to cell wall fragments from Bacillus anthracis which retained the S layer. When grown in rich medium under aerobic conditions, the avirulent strain Delta Sterne-1 released large amounts of EA1 into the medium. This EA1 had no higher-order structure initially but formed twodimensional crystals under defined conditions. The released EA1 was purified in aqueous buffers with a three-step procedure and found to have a mass of 95 kDa when subjected to denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence data revealed exact identity to the first eight residues of the S-layer protein from B. thuringiensis 4045. Gel permeation chromatography of the purified EA1 under nondenaturing conditions revealed a single peak corresponding to a mass of approximately 400 kDa, suggesting that a tetramer or dimer of dimers was the primary species in solution. SDS-PAGE of EA1 purified in the absence of protease inhibitors revealed specific proteolytic processing to an 80-kDa form, which immunoreacted with polyclonal anti-EA1 antibodies. This proteolytic cleavage of EA1 to 80 kDa was duplicated with purified EA1 and the protease trypsin or pronase.The gram-positive organism Bacillus anthracis is the etiological agent of anthrax and the only member of the genus Bacillus capable of causing invasive disease in humans and other mammals. Fully virulent strains of B. anthracis harbor two endogenous plasmids, pXO1 (22, 33) and pXO2 (10, 34), which code for the major known virulence factors of this organism, a poly-D-glutamic acid capsule and two bipartite exotoxins. The genes for capsule synthesis are located on the 95-kb plasmid pXO2 (10,19,20), while the genes for toxin synthesis are located on the 184-kb plasmid pXO1 (24,28,29,35).It was discovered during the late 1800s and early 1900s that cultures of virulent B. anthracis could be attenuated by growth at 42 to 43ЊC. The attenuation observed with such Pasteur-type vaccine strains resulted from curing the cells of the plasmid pXO1 (22). Fully virulent pXO2 ϩ pXO1 ϩ strains were thus attenuated by conversion to the pXO1 Ϫ pXO2 ϩ genotype. Other attenuated strains, such as the Sterne strain, spontaneously lost pXO2 while retaining pXO1. Culturing the Sterne strain at 42ЊC resulted in the loss of pXO1 and produced the avirulent pXO1 Ϫ pXO2 Ϫ strain referred to as Delta Sterne-1 (15).Immunological analysis of animals vaccinated with the current veterinary attenuated live spore vaccine revealed that serum antibody strongly recognizes a vegetative cell-associated protein termed extractable antigen 1 (EA1) (6). Two B. anthracis vegetative cell-associated proteins, EA1 and EA2, were initially identified from these studies (6). These highly antigenic proteins are extractable from vegetative cell...

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Section: Fig 3 Sds-page Of Sds Cell Extracts and Culture Supernatansupporting

confidence: 81%

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1

et al. 1995

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“…The cell wall layers (surface layers [S layers]) observed in these strains have a hexagonal array with lattice constants of 14.5 to 18 nm (11,42,48). The S-layer proteins of strains 47 and HPD31 are composed of proteins with approximate molecular weights of 130 x 103 (outer wall protein) and 150 x lo3 (MWP) (strain 47) (24,43) and 135 x lo3 (HWP) (strain HPD31) (5,11). The deduced amino acid sequence obtained from the nucleotide sequence of the HWP gene shows a high level of homology (78%) with the sequence of the MWP gene Therefore, an interesting question is how the taxonomic properties of protein-secreting bacteria correlate with their cell wall morphology.…”

Section: Discussionmentioning

confidence: 99%

“…Western blot (immunoblot) analysis of the extracellular proteins was performed as described by Towbin et al (41). Rabbit antisera to the middle cell wall protein (protein MWP) of strain 47 and to the cell wall protein (protein HWP) of strain HPD31 were prepared as reported previously (24,43).…”

Section: Quinone Composition Cells Cultivated Overnight In 72mentioning

confidence: 99%

Characterization of Bacillus brevis with Descriptions of Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov.

Takagi¹,

Sato²,

Kadowaki³

et al. 1993

International Journal of Systematic Bacteriology

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Thirty-five Bacillus brevis strains obtained from culture collections, including protein-producing isolates, were taxonomically studied by using numerical analysis, DNA base composition, and DNA-DNA hybridization. Six DNA relatedness groups were represented, and these groups correlated well with clusters based on the numerical analysis. The B. brevis strains were separated into B. brevis sensu stricto, four new species, and an unidentified species of the genus Bacillus. Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov, are proposed.The heterogeneity of Bacillus brevis has been indicated by the wide range of maximum growth temperatures (10) and the results of numerical classification (13,26,27) and classification based on API test data (17). The protein producers identified as B. brevis (45) had guanine-plus-cytosine (G+C) contents ranging from 47 to 55 mol%. These observations suggest that this taxon is genetically heterogeneous. Recently, Nakamura (23) reported that the phenotypic heterogeneity of B. brevis was due to variability introduced by the presence of genetically unrelated strains. Previously, Udaka (44) and Takagi et al. (37) reported the bacterial production of large amounts of proteins in culture fluids; many of the bacteria involved appeared phenotypically to be B. brevis (37,44). These strains showed either weak or no detectable proteolytic activity in the culture supernatant (37). B. brevis 47 and HPD31 have been employed successfully as host bacteria for extracellular production of foreign proteins, such as bacterial thermophilic a-amylase (38) and human epidermal growth factor (49). Additional unique characteristics of B. brevis that have been reported are gramicidin production (9) and molluscicidal activity (31).In order to clarify the taxonomic positions of 14 B. brevis strains from culture collections and of 21 protein-producing isolates, we examined their phenotypic characteristics and processed the data obtained by using standard numerical analysis. In addition, the relationships between clusters resulting from the numerical analysis were evaluated by DNA-DNA hybridization.As a result, the above-mentioned 35 strains assigned to B. brevis were divided into six independent species, four of which are proposed below as the new species Bacillus migulanus, Bacillus choshinensis, Bacillus parabrevis, and Bacillus gala c toph ilus. MATERIALS AND METHODSBacterial strains. B. brevis strains and reference strains obtained from culture collections and other sources and the protein-producing strains described previously (37, 44) were * Corresponding author. used in this study. The sources and histories of these strains are listed in Table 1. Working stocks were cultivated on T2 agar (44) for 24 h at 30 or 37°C and were stored at a room temperature.Characterization, Unless indicated otherwise, the methods described by Gordon et al. (10) were used for characterization of organisms. Acid production from carbohydrates was determin...

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“…Crude cell envelopes were centrifuged (48,000 x g, 30 min, 4°C) after removal of unbroken cells by centrifugation, washed three times by suspension in the same buffer, and then treated with Triton X-100 to prepare Triton-insoluble envelopes according to the method of Tsuboi et al (34). The cell wall proteins were solubilized SDS as described by Ohmizu et al (17). The (23) after subcloning of appropriate restriction fragments into derivatives of M13 phages or pUC plasmids.…”

Section: Methodsmentioning

confidence: 99%

Conserved structures of cell wall protein genes among protein-producing Bacillus brevis strains

et al. 1990

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Bacillus brevis HPD31 contains a surface (S)-layer protein, termed the HWP, which forms a hexagonal array in the cell wall. The 5' region of the HWP gene was isolated from a DNA library constructed in bacteriophage vector EMBL3 from a partial BamHI digest of the chromosomal DNA. The 3' region contained in a 2.7-kilobase BgEl fragment of the DNA was cloned into Escherichia coli, using pUC118 as a vector. On the basis of the chemically determined N-terminal amino acid sequence, the HWP gene was found to encode a polypeptide consisting of 1,087 amino acid residues with a signal peptide of 53 or 23 amino acid residues. The deduced amino acid composition was similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic relative to basic amino acids and in the very low content of sulfur-containing amino acids. The deduced amino acid sequence showed high homology (78%) with that of the middle wall protein of B. brevis 47. Furthermore, the multiple 5' ends of the HWP gene transcripts detected on S1 nuclease analysis closely resembled those of the middle wall protein gene transcripts. This complex structure was also conserved (greater than 85%) in the regulatory regions of two other cell wall protein genes isolated from B. brevis HPD52 and HP033, suggesting that the synthesis of the cell wall proteins is intricately regulated through a similar mechanism in protein-producing B. brevis.A number of gram-positive and gram-negative bacteria possess a regular surface layer, the so-called S layer, which is now defined as a two-dimensional crystalline array of proteinaceous subunits forming a surface layer on procaryotic cells (28,29). The morphological properties of S layers have been extensively characterized for a wide range of microorganisms (25-27), whereas the genes for S-layerforming proteins have been isolated from only a few microorganisms, such as Bacillus brevis 47 (35, 37, 39, 41), Halobacterium halobium (11), Deinococcus radiodurans (18, 19), Caulobactor crescentus (30), and Aeromonas salmonicida (4). Too little is known at present about the regulation of these genes to elucidate the mechanisms involved in the biosynthesis, transport, and assembly of Slayer proteins. Isolation and characterization of the genes encoding S-layer-forming proteins from diverse origins are essential.Recently, we newly isolated many protein-producing B. brevis strains from soil from diverse origins (32) for comparison with B. brevis 47, the S layer of which has been extensively characterized from various aspects: morphology (40), chemical and immunological properties of the S-layerforming proteins, and characterization of genes for those proteins (35, 37). Protein-producing B. brevis strains all have S layers showing hexagonal symmetry, with a lattice constant of approximately 18 nm (8).The genes for two S-layer proteins, termed the outer wall protein and middle wall protein (MWP), of B. brevis 47 constitute a cotranscriptional unit (cwp operon) and are transcribed from several tandem promoters lo...

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Major Proteins Released by a Protein-Producing Bacterium, Bacillus brevis 47, Are Derived from Cell Wall Protein1

Cited by 20 publications

References 0 publications

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1

Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1

Characterization of Bacillus brevis with Descriptions of Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov.

Conserved structures of cell wall protein genes among protein-producing Bacillus brevis strains

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