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1.  Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis. 
Journal of Bacteriology  1978;135(3):952-960.
Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.
PMCID: PMC222469  PMID: 99441
2.  Comparison of Two Serologically Distinct Ribonucleic Acid Bacteriophages II. Properties of the Nucleic Acids and Coat Proteins 
Journal of Bacteriology  1966;92(3):739-745.
Overby, L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman. Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins. J. Bacteriol. 92:739–745. 1966.—The ribonucleic acid (RNA) molecules and coat proteins of two RNA coliphages, MS-2 and Qβ, have been characterized. MS-2 RNA shows an S20,w of 25.8 and a molecular weight by light scattering of 106. The corresponding parameters for Qβ-RNA were 28.9 and 0.9 × 106. A difference in base composition was reflected in the adenine-uracil ratio, which was 0.95 for MS-2 and 0.75 for Qβ. The two RNA preparations are readily separated by chromatography on columns of methylated albumin. Both gave identical bouyant densities in cesium sulfate of 1.64 g/ml. The coat protein subunits were of similar molecular weights: 15,500 (Qβ) and 14,000 (MS-2). They differed, however, in that the Qβ-protein lacked tryptophan and histidine, whereas the MS-2 protein lacked only histidine.
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PMCID: PMC276317  PMID: 5922545
3.  Comparison of Two Serologically Distinct Ribonucleic Acid Bacteriophages I. Properties of the Viral Particles 
Journal of Bacteriology  1966;91(1):442-448.
Overby, L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman. Comparison of two serologically distinct ribonucleic acid bacteriophages. I. Properties of the viral particle. J. Bacteriol. 91:442–448. 1966.—Two ribonucleic acid (RNA) coliphages, MS-2 and Qβ, have been characterized physically and serologically. MS-2 has an S20, w value of 79, a molecular weight of 3.6 × 106, a density of 1.422, and pH 3.9 as its isoelectric point. Qβ has an S20, w of 84, a molecular weight of 4.2 × 106, a density of 1.439, and an isoelectric point at pH 5.3. One host (Escherichia coli A-19) permits a distinction between the two on the basis of a marked difference in plaque size. They are distinct immunochemically, no serological cross-reaction being detectable.
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PMCID: PMC315966  PMID: 5903109
4.  Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion. 
Journal of Bacteriology  1985;164(2):550-555.
Translational coupling was demonstrated in a gene fusion in which the promoter and the N-terminal region of the Bacillus subtilis subtilisin (aprA) gene were fused to a promoterless Tn9-derived chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. Expression of this gene fusion results in the production of a native-sized CAT product, whereas the Tn9-derived CAT gene is usually not translated from its own ribosome binding site in B. subtilis (D. S. Goldfarb, R. L. Rodriguez, and R. H. Doi, Proc. Natl. Acad. Sci. USA 79:5886-5890, 1982). A 178-base-pair deletion, which removed part of the signal peptide and the propeptide of the aprA gene and created a translational stop codon 230 base pairs upstream of the CAT gene ribosome binding site, reduced expression of the CAT gene. A BamHI 10-mer linker insertion into this deletion site, which restored the reading frame and simultaneously removed the translation stop codon, restored CAT gene expression. The data indicate that expression of the CAT gene was dependent on translation of the truncated aprA gene into the ribosome binding site of the CAT gene.
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PMCID: PMC214287  PMID: 2997117
5.  Bacterial Sporulation and Regulation of Dihydrodipicolinate Synthase in Ribonucleic Acid Polymerase Mutants of Bacillus subtilis 
Journal of Bacteriology  1975;124(3):1628-1629.
The activity of dihydrodipicolinate synthase increased late in sporulation in Bacillus subtilis. Mutants blocked at several stages of sporulation due to having an altered ribonucleic acid polymerase failed to exhibit this increase.
PMCID: PMC236086  PMID: 811651
6.  rpoD operon promoter used by sigma H-RNA polymerase in Bacillus subtilis. 
Journal of Bacteriology  1988;170(4):1617-1621.
Three promoters direct transcription of the sigA (rpoD) operon in Bacillus subtilis. Promoters P1 and P2 are used during the exponential growth phase, whereas P3 is used only during the stationary phase. We examined the use of these promoters in promoter-probe plasmids and found that expression from P3 was prevented by a mutation in spoOH, which encodes the secondary RNA polymerase sigma factor sigma H. Moreover, we found that sigma H-containing RNA polymerase efficiently and accurately used the P3 promoter in vitro. Evidently, this operon, which is essential for exponential growth, is transcribed during the early phase of sporulation by this secondary form of RNA polymerase. Comparison of the nucleotide sequences of the P3 promoter and the spoVG promoter, which also is used by sigma H-RNA polymerase, revealed sequences at the -10 and -35 regions of these promoters that may signal recognition of promoters by sigma H-RNA polymerase.
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PMCID: PMC211009  PMID: 3127379
7.  Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation. 
Journal of Bacteriology  1977;129(1):422-432.
The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation. The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma). Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000. Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2. The delta2 factor had a molecular weight of around 20,000. The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme. The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.
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PMCID: PMC234942  PMID: 401498
11.  A mutation in P23, the first gene in the RNA polymerase sigma A (sigma 43) operon, affects sporulation in Bacillus subtilis. 
Journal of Bacteriology  1990;172(4):2175-2177.
Mutations within P23, the first gene of the Bacillus subtilis sigma A operon, were not detrimental to vegetative growth or sporulation. One deletion of P23 resulted in a strain that sporulated earlier than the wild type. This aberrant phenotype may be due to the simultaneous deletion of a sigma H promoter from the sigma A operon.
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PMCID: PMC208719  PMID: 2108133
12.  Bacillus subtilis ribonucleic acid polymerase mutants conditionally temperature sensitive at various stages of sporulation. 
Journal of Bacteriology  1977;129(1):433-444.
Rifampin-resistant mutants of Bacillus subtilis that are conditionally temperature sensitive during sporulation have been isolated and characterized. The mutants can grow at the same rate as the wild type at the nonpermissive temperature but cannot sporulate. Depending on the mutation, they are blocked at either stage 0 to I, II, II to III, or IV of sporulation. The mutants showed an altered pattern of RNA synthesis after the stage at which they were blocked. The effect of rifampin on the activity of enzymes from mutant vegetative cells and sporulating cells was significantly different, suggesting that the RNA polymerase from sporulating cells was different from the RNA polymerase of vegetative cells. These results suggest that the conformation of the RNA polymerase core plays an important role in determining correct transcription during sporulation.
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PMCID: PMC234943  PMID: 401499
13.  Mutation analysis of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. 
Journal of Bacteriology  1994;176(23):7328-7334.
Cellulose-binding protein A (CbpA) has been previously shown to mediate the interaction between crystalline cellulose substrates and the cellulase enzyme complex of Clostridium cellulovorans. CbpA contains a family III cellulose-binding domain (CBD) which, when expressed independently, binds specifically to crystalline cellulose. A series of N- and C-terminal deletions and a series of small internal deletions of the CBD were created to determine whether the entire region previously described as a CBD is required for the cellulose-binding function. The N- and C-terminal deletions reduced binding affinity by 10- to 100-fold. Small internal deletions of the CBD resulted in substantial reduction of CBD function. Some, but not all, point mutations throughout the sequence had significant disruptive effects on the binding ability of the CBD. Thus, mutations in any region of the CBD had effects on the binding of the fragment to cellulose. The results indicate that the entire 163-amino-acid region of the CBD is required for maximal binding to crystalline cellulose.
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PMCID: PMC197122  PMID: 7961505
14.  Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes. 
Journal of Bacteriology  1994;176(22):6952-6956.
Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources. Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity. Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose. Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity. Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose. The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA. These data strongly indicate that crystalline cellulose promotes cellulosome assembly.
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PMCID: PMC197066  PMID: 7961457
15.  The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases. 
Journal of Bacteriology  1993;175(21):7119-7122.
We overexpressed one of the hydrophobic repeated domains (HBDs) (110 amino acid residues) of the cellulose-binding protein (CbpA) from Clostridium cellulovorans by making a hybrid protein with the Escherichia coli maltose-binding protein (MalE). The HBD was purified to homogeneity, and interactions between the HBD and endoglucanases were analyzed by a novel interaction Western blotting (immunoblotting) method. The HBD had specific interactions with endoglucanases (EngB and EngD) from C. cellulovorans. These results indicated that the HBD was an endoglucanase binding site of CbpA.
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PMCID: PMC206844  PMID: 8226657
16.  Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. 
Journal of Bacteriology  1993;175(18):5762-5768.
Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.
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PMCID: PMC206653  PMID: 8376323
17.  Temperature-sensitive sporulation caused by a mutation in the Bacillus subtilis secY gene. 
Journal of Bacteriology  1993;175(11):3656-3660.
A thermosensitive sporulation mutant of Bacillus subtilis containing a mutation in the secY gene was isolated and characterized. No asymmetric septum specific to the sporulation was detected by electron microscopy at the nonpermissive temperature, indicating that the block occurred at a very early stage of sporulation. Furthermore, competence development in the mutant cell was affected even at the sporulation-proficient temperature. It is assumed that the SecY protein of B. subtilis has multiple roles both in the regulation of spore formation and in stationary-phase-associated phenomena.
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PMCID: PMC204768  PMID: 8501070
18.  Effects of amino acid substitutions in the promoter -10 binding region of the sigma A factor on growth of Bacillus subtilis. 
Journal of Bacteriology  1993;175(8):2470-2474.
On the bases of structural and functional information about the Bacillus subtilis sigma A protein and the techniques of site-directed mutagenesis, we constructed a B. subtilis sigA mutant (DB1005) which grows normally at 37 degrees C but is sensitive to higher temperatures. DNA sequencing analyses revealed that DB1005 has Ile-198-->Ala and Ile-202-->Ala amino acid substitutions in the alpha-helix of the 2.4 region of the sigma A protein. Western blotting (immunoblotting) revealed that this mutant sigma A protein is stable at both 37 and 49 degrees C. These results suggest that Ile-198 and Ile-202 separately or in combination are critical for the sigma A protein to be functional at the restrictive temperature.
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PMCID: PMC204541  PMID: 8468306
19.  Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli. 
Journal of Bacteriology  1992;174(4):1403-1409.
By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium cellulovorans were hyperexpressed and purified from Escherichia coli. The two enzymes demonstrated both endoglucanase and xylanase activities. The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl beta-1,4-cellobiosidase activity. The two proteins were very homologous (80%) up to the Pro-Thr-Thr region which divided the protein into -NH2- and -COOH-terminals. The -COOH- region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocellum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel). However, the -COOH- region of EngD, which had homology to the cellulose-binding domains of Cellulomonas fimi exo- and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB. By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C. cellulovorans. Many proteins homologous to EngB and EngD were also present on the complex.
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PMCID: PMC206438  PMID: 1735727
20.  Localization of a new promoter, P5, in the sigA operon of Bacillus subtilis and its regulation in some spo mutant strains. 
Journal of Bacteriology  1991;173(21):7050-7054.
The sigA operon of Bacillus subtilis is transcribed from at least two SigA and two SigH promoters. Primer extension and promoter probe analyses have localized a fifth promoter, P5, that is active only at later sporulation stages (T3 to T5). Mutations in the genes for the sigma factors SigG, SigK, SigH, and SigE do not block transcription from P5. The expression from P5 is blocked or severely reduced in spo0A, spo0B, spo0E, and spo0K mutants.
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PMCID: PMC209066  PMID: 1840586
21.  Localization of a second SigH promoter in the Bacillus subtilis sigA operon and regulation of dnaE expression by the promoter. 
Journal of Bacteriology  1990;172(10):5631-5636.
The presence of a second SigH promoter in the sigA operon of Bacillus subtilis was demonstrated by use of a promoter probe plasmid, a sigH deletion mutant, primer extension studies, and in vitro transcription with E sigma H holoenzyme. Both SigH promoters were expressed at low levels even during the growth phase but were expressed at higher levels during the early stationary phase. Expression from the upstream SigH promoter allowed the expression of both dnaE and sigA genes; however, expression from the downstream SigH promoter, which was located in the ribosome-binding site of the dnaE gene, resulted only in the expression of the sigA gene, since the truncated dnaE ribosome-binding site could not be used for initiating translation. Thus, promoter switching during the early stationary phase resulted not only in expression from SigH promoters but also in differential expression of the genes in the sigA operon.
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PMCID: PMC526875  PMID: 1698762
22.  Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor. 
Journal of Bacteriology  1990;172(6):3257-3263.
By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.
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PMCID: PMC209133  PMID: 2111806
23.  Complex character of senS, a novel gene regulating expression of extracellular-protein genes of Bacillus subtilis. 
Journal of Bacteriology  1990;172(4):1939-1947.
The senS gene of Bacillus subtilis, which in high copy number stimulates the expression of several extracellular-protein genes, has been cloned, genetically mapped, and sequenced. The gene codes for a highly charged basic protein containing 65 amino acid residues. The gene is characterized by the presence of a transcription terminator (attenuator) located between the promoter and open reading frame, a strong ribosome-binding site, and a strong transcription terminator at the 3' end of this monocistronic gene. The amino acid sequence of SenS showed partial homology with the N-terminal core binding domain region of bacterial RNA polymerase sigma factors and a helix-turn-helix motif found in DNA-binding proteins. The gene can be deleted without any effect on growth or sporulation.
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PMCID: PMC208689  PMID: 2108127
24.  Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A (sigma 43) promoter in vitro and in vivo. 
Journal of Bacteriology  1989;171(5):2657-2665.
In vitro studies demonstrated that the Bacillus subtilis subtilisin gene (aprE) could be transcribed by RNA polymerase holoenzyme reconstituted from core and sigma A factor obtained from vegetative cells. Upstream deletions (from -45) reduced the amount of transcription from the promoter. A deletion downstream of the promoter that overlapped a putative downstream minor promoter did not affect transcription from the sigma A promoter, which indicated that the putative downstream promoter is not utilized in vivo. S1 nuclease mapping studies showed that there was a low level of transcription from the subtilisin promoter during the growth phase and that the site of transcription initiation was the same during log and stationary phases. We conclude from these findings that there is only one promoter for the subtilisin gene and that it can be transcribed by the sigma A form of RNA polymerase in vitro.
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PMCID: PMC209949  PMID: 2496113
25.  Developmental expression of three proteins from the first gene of the RNA polymerase sigma 43 operon of Bacillus subtilis. 
Journal of Bacteriology  1987;169(9):4190-4195.
The first gene of the Bacillus subtilis RNA polymerase sigma 43 operon, P23, has a protein-coding capacity of 23,000 daltons. Sequence analysis revealed three potential translational initiation sites within the same reading frame, which could encode proteins of 23,000 (P23), 19,000 (P19), and 9,000 (P9) daltons, respectively. An internal promoter (P3), which is expressed only during the sporulation stage, is located between the second and the third translational start sites. By protein fusion to the Escherichia coli beta-galactosidase gene, we showed that all three translational initiation sites of the P23 gene are used in vivo in both E. coli and B. subtilis, and regulation for differential expression of the three proteins during the development of B. subtilis is coupled to the transcriptional promoter switching mechanism. The physiological function of these multiple gene products is unknown and is currently under investigation.
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PMCID: PMC213728  PMID: 3040682

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