scholarly journals Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

2003 ◽  
Vol 185 (4) ◽  
pp. 1443-1454 ◽  
Author(s):  
Erh-Min Lai ◽  
Nikhil D. Phadke ◽  
Maureen T. Kachman ◽  
Rebecca Giorno ◽  
Santiago Vazquez ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Anthracis ◽  
Proteomic Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Detection Methods ◽  
Coat Proteins ◽  
Bona Fide ◽  
Spore Proteins ◽  
Spore Coat Proteins

ABSTRACT The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.

Comparison of spore proteins among species of the cellular slime moulds Dictyostelium and Polysphondylium as examined by immunological cross-reactivity

1988 ◽  
Vol 34 (7) ◽  
pp. 891-896 ◽  
Author(s):  
Yohko Yamada ◽  
Koji Okamoto ◽  
Ikuo Takeuchi
Keyword(s):  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Developmental Changes ◽  
Coat Proteins ◽  
Slime Moulds ◽  
Cellular Slime ◽  
Spore Proteins ◽  
Spore Coat Proteins ◽  
Cellular Slime Moulds

Spore proteins of six cellular slime mould species, Dictyostelium discoideum, D. mucoroides, D. purpureum, D. lacteum, Polysphondylium violaceum, and P. pallidum were studied. The spore proteins were cross-reacted with four different polyclonal antibodies produced against D. mucoroides spores and D. discoideum major spore coat proteins SP96, SP70, and SP60 by SDS polyacrylamide gel electrophoresis and immunoblotting. The spore proteins of D. discoideum and D. mucoroides showed the strongest cross-reactivity with all the antisera and also produced many common protein bands, thus reflecting their morphological similarities. Polysphondylium violaceum, which is quite distinct in morphology from D. discoideum and D. mucoroides, produced the second strongest cross-reactivity. In contrast, the proteins of D. purpureum showed little cross-reactivity, although morphologically it closely resembles D. mucoroides. The developmental changes of these spore proteins were investigated by cross-reacting the antisera against vegetative and slug cell proteins. In the cases of D. discoideum and D. mucoroides, the band patterns of slug proteins coincided with those of spores, which suggested that most of the spore proteins had already accumulated at the slug stage. However, this was not the case with P. violaceum, which suggested that spore proteins of this species cross-reactive with the antisera were synthesized or modified at the culmination stage.

scholarly journals Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers

2008 ◽  
Vol 190 (20) ◽  
pp. 6741-6748 ◽  
Author(s):  
Sonali Ghosh ◽  
Barbara Setlow ◽  
Paul G. Wahome ◽  
Ann E. Cowan ◽  
Marco Plomp ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Dipicolinic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Great Majority ◽  
Coat Proteins ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Wet Heat ◽  
Coat Material

ABSTRACT Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca2+ and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.

Identification of proteins in the exosporium of Bacillus anthracis

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 355-363 ◽  
Author(s):  
Caroline Redmond ◽  
Leslie W. J. Baillie ◽  
Stephen Hibbs ◽  
Arthur J. G. Moir ◽  
Anne Moir
Keyword(s):  
Bacillus Anthracis ◽  
Amino Acid Sequences ◽  
Coat Proteins ◽  
Wild Type ◽  
Sds Page ◽  
Adsorbed Proteins ◽  
Spore Coat Proteins ◽  
Ames Strain ◽  
A Site ◽  
Novel Protein

Spores of Bacillus anthracis, the causative agent of anthrax, possess an exosporium. As the outer surface layer of these mature spores, the exosporium represents the primary contact surface between the spore and environment/host and is a site of spore antigens. The exosporium was isolated from the endospores of the B. anthracis wild-type Ames strain, from a derivative of the Ames strain cured of plasmid pXO2−, and from a previously isolated pXO1−, pXO2− doubly cured strain, B. anthracis UM23Cl2. The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis. A number of loosely adsorbed proteins were identified from amino acid sequences determined by either nanospray-MS/MS or N-terminal sequencing. Salt and detergent washing of the exosporium fragments removed these and revealed proteins that are likely to represent structural/integral exosporium proteins. Seven proteins were identified in washed exosporium: alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, named ExsK. CotY, ExsY and CotB are homologues of Bacillus subtilis outer spore coat proteins, but ExsF and ExsK are specific to B. anthracis and other members of the Bacillus cereus group.

Synthesis of acid-soluble spore proteins by Bacillus subtilis

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss
Keyword(s):  
Bacillus Subtilis ◽  
Maximum Rate ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Spore Proteins ◽  
Major Acid

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

scholarly journals Glycerol Monolaurate Inhibits Virulence Factor Production in Bacillus anthracis

2005 ◽  
Vol 49 (4) ◽  
pp. 1302-1305 ◽  
Author(s):  
Sara M. Vetter ◽  
Patrick M. Schlievert
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
The United States ◽  
Human Populations ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Glycerol Monolaurate

ABSTRACT Anthrax, caused by Bacillus anthracis, has been brought to the public's attention because of the 2001 bioterrorism attacks. However, anthrax is a disease that poses agricultural threats in the United States as well as human populations in Europe, China, Africa, and Australia. Glycerol monolaurate (GML) is a compound that has been shown to inhibit exotoxin production by Staphylococcus aureus and other gram-positive bacteria. Here, we study the effects of GML on growth and toxin production in B. anthracis. The Sterne strain of B. anthracis was grown to post-exponential phase with 0-, 10-, 15-, or 20-μg/ml concentrations of GML and then assayed quantitatively for protective antigen (PA) and lethal factor (LF). After 8 h, GML at concentrations greater than 20 μg/ml was bacteriostatic to growth of the organism. However, a 10-μg/ml concentration of GML was not growth inhibitory, but amounts of PA and LF made were greatly reduced. This effect was not global for all proteins when total secreted protein from culture fluids was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Through quantitative reverse transcription-PCR assays, this toxin-inhibitory effect was shown to occur at the transcriptional level, since amounts of mRNA for pagA (PA), lef (LF), and cya (edema factor) were reduced. Surprisingly, mRNA levels of atxA, a regulator of exotoxin gene expression, rose in the presence of GML. These data will be useful in developing therapeutic tools to treat anthrax disease, whether in animals or humans. These results also suggest that mechanisms of virulence regulation exist independent of atxA.

scholarly journals The purification of a novel amylase from Bacillus subtilis and its inhibition by wheat proteins

1983 ◽  
Vol 209 (2) ◽  
pp. 561-564 ◽  
Author(s):  
A R Orlando ◽  
P Ade ◽  
D Di Maggio ◽  
C Fanelli ◽  
L Vittozzi
Keyword(s):  
Bacillus Subtilis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alpha Amylase ◽  
Wheat Protein ◽  
Wheat Proteins ◽  
Maximal Inhibition ◽  
Molecular Weights ◽  
Amylase Inhibitors

A new alpha-amylase (EC 3.2.1.1) from Bacillus subtilis was purified by affinity chromatography. The molecular weight of the purified enzyme, estimated from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 93000, which is very different from the molecular weights of two well-characterized amylases from B. subtilis. Electrofocusing showed an isoelectric point of 5. Amylase shows a broad maximum of activity between pH 6 and 7; maximal inhibition of enzyme by wheat-protein alpha-amylase inhibitors is displayed at pH 7.

scholarly journals Identification of a Second Collagen-Like Glycoprotein Produced by Bacillus anthracis and Demonstration of Associated Spore-Specific Sugars

2005 ◽  
Vol 187 (13) ◽  
pp. 4592-4597 ◽  
Author(s):  
Lashanda N. Waller ◽  
Michael J. Stump ◽  
Karen F. Fox ◽  
William M. Harley ◽  
Alvin Fox ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bacillus Anthracis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Carbohydrate Components ◽  
Protein Moiety

ABSTRACT Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these spore-specific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.

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