scholarly journals The ExsY Protein Is Required for Complete Formation of the Exosporium of Bacillus anthracis

2006 ◽  
Vol 188 (21) ◽  
pp. 7440-7448 ◽  
Author(s):  
Jeremy A. Boydston ◽  
Ling Yue ◽  
John F. Kearney ◽  
Charles L. Turnbough
Keyword(s):  
Bacillus Anthracis ◽  
Solid Medium ◽  
Basal Layer ◽  
Wild Type ◽  
Nucleoside Hydrolase ◽  
Spore Resistance ◽  
Normal Hair ◽  
Spore Outgrowth ◽  
Microscopic Inspection ◽  
Complete Formation

ABSTRACT The outermost layer of the Bacillus anthracis spore is the exosporium, which is composed of a paracrystalline basal layer and an external hair-like nap. The filaments of the nap are formed by a collagen-like glycoprotein called BclA, while the basal layer contains several different proteins. One of the putative basal layer proteins is ExsY. In this study, we constructed a ΔexsY mutant of B. anthracis, which is devoid of ExsY, and examined the assembly of the exosporium on spores produced by this strain. Our results show that exosporium assembly on ΔexsY spores is aberrant, with assembly arrested after the formation of a cap-like fragment that covers one end of the forespore—always the end near the middle of the mother cell. The cap contains a normal hair-like nap but an irregular basal layer. The cap is retained on spores prepared on solid medium, even after spore purification, but it is lost from spores prepared in liquid medium. Microscopic inspection of ΔexsY spores prepared on solid medium revealed a fragile sac-like sublayer of the exosporium basal layer, to which caps were attached. Examination of purified ΔexsY spores devoid of exosporium showed that they lacked detectable levels of BclA and the basal layer proteins BxpB, BxpC, CotY, and inosine-uridine-preferring nucleoside hydrolase; however, these spores retained half the amount of alanine racemase presumed to be associated with the exosporium of wild-type spores. The ΔexsY mutation did not affect spore production and germination efficiencies or spore resistance but did influence the course of spore outgrowth.

scholarly journals Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in Bacillus anthracis

2009 ◽  
Vol 191 (7) ◽  
pp. 2237-2247 ◽  
Author(s):  
Jared D. Heffron ◽  
Benjamin Orsburn ◽  
David L. Popham
Keyword(s):  
Bacillus Anthracis ◽  
High Performance ◽  
Structural Characteristics ◽  
Wild Type ◽  
Lytic Enzymes ◽  
Spectroscopy Analysis ◽  
Lytic Transglycosylase ◽  
Spore Resistance ◽  
Sequence Similarities ◽  
Mass Spectroscopy Analysis

ABSTRACT The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

scholarly journals The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

2007 ◽  
Vol 189 (18) ◽  
pp. 6704-6713 ◽  
Author(s):  
Brian M. Thompson ◽  
Lashanda N. Waller ◽  
Karen F. Fox ◽  
Alvin Fox ◽  
George C. Stewart
Keyword(s):  
Bacillus Anthracis ◽  
Mutant Strain ◽  
Basal Layer ◽  
Structural Defects ◽  
Bacterial Cells ◽  
Wild Type ◽  
Fatal Disease ◽  
Specific Location ◽  
Immunofluorescence Studies ◽  
Kinetics Of

ABSTRACT Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis ΔSterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.

scholarly journals Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination

2009 ◽  
Vol 191 (24) ◽  
pp. 7587-7596 ◽  
Author(s):  
Kari M. Severson ◽  
Michael Mallozzi ◽  
Joel Bozue ◽  
Susan L. Welkos ◽  
Christopher K. Cote ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Bacillus Anthracis ◽  
Molecular Mass ◽  
Causative Agent ◽  
Basal Layer ◽  
Surface Protein ◽  
High Molecular Mass ◽  
Spore Surface ◽  
Wild Type ◽  
Active Investigation

ABSTRACT The Bacillus anthracis spore is the causative agent of the disease anthrax. The outermost structure of the B. anthracis spore, the exosporium, is a shell composed of approximately 20 proteins. The function of the exosporium remains poorly understood and is an area of active investigation. In this study, we analyzed the previously identified but uncharacterized exosporium protein ExsK. We found that, in contrast to other exosporium proteins, ExsK is present in at least two distinct locations, i.e., the spore surface as well as a more interior location underneath the exosporium. In spores that lack the exosporium basal layer protein ExsFA/BxpB, ExsK fails to encircle the spore and instead is present at only one spore pole, indicating that ExsK assembly to the spore is partially dependent on ExsFA/BxpB. In spores lacking the exosporium surface protein BclA, ExsK fails to mature into high-molecular-mass species observed in wild-type spores. These data suggest that the assembly and maturation of ExsK within the exosporium are dependent on ExsFA/BxpB and BclA. We also found that ExsK is not required for virulence in murine and guinea pig models but that it does inhibit germination. Based on these data, we propose a revised model of exosporium maturation and assembly and suggest a novel role for the exosporium in germination.

scholarly journals Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1133-1145 ◽  
Author(s):  
Rebecca Giorno ◽  
Michael Mallozzi ◽  
Joel Bozue ◽  
Krishna-Sulayman Moody ◽  
Alex Slack ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Mother Cell ◽  
Complex Structure ◽  
Basal Layer ◽  
Subcellular Location ◽  
Bacterial Spores ◽  
Cell Cytoplasm ◽  
Nucleoside Hydrolase ◽  
Gfp Fluorescence ◽  
Protective Structures

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine–uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB–GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB–GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB–GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH–GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB–GFP and IunH–GFP resides in the region between the coat and the exosporium, called the interspace.

scholarly journals Characterization of the Exosporium Basal Layer Protein BxpB of Bacillus anthracis

2005 ◽  
Vol 187 (17) ◽  
pp. 5868-5876 ◽  
Author(s):  
Christopher T. Steichen ◽  
John F. Kearney ◽  
Charles L. Turnbough
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Bacillus Anthracis ◽  
Basal Layer ◽  
Immunogold Electron Microscopy ◽  
Stable Complex ◽  
Wild Type

ABSTRACT Bacillus anthracis spores, the cause of anthrax, are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA, whereas several different proteins form or are tightly associated with the basal layer. In this study, we used immunogold electron microscopy to demonstrate that BxpB (also called ExsF) is a component of the exosporium basal layer. Binding to the basal layer by an anti-BxpB monoclonal antibody was greatly increased by the loss of BclA. We found that BxpB and BclA are part of a stable complex that appears to include the putative basal layer protein ExsY and possibly other proteins. Previous results suggested that BxpB was glycosylated; however, our results indicate that it is not a glycoprotein. We showed that ΔbxpB spores, which lack BxpB, contain an exosporium devoid of hair-like nap even though the ΔbxpB strain produces normal levels of BclA. These results indicated that BxpB is required for the attachment of BclA to the exosporium. Finally, we found that the efficiency of production of ΔbxpB spores and their resistance properties were similar to those of wild-type spores. However, ΔbxpB spores germinate faster than wild-type spores, indicating that BxpB suppresses germination. This effect did not appear to be related to the absence from ΔbxpB spores of a hair-like nap or of enzymes that degrade germinants.

scholarly journals A Novel Spore Protein, ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape

2010 ◽  
Vol 192 (15) ◽  
pp. 4012-4021 ◽  
Author(s):  
Monica M. Fazzini ◽  
Raymond Schuch ◽  
Vincent A. Fischetti
Keyword(s):  
Electron Microscopy ◽  
Bacillus Cereus ◽  
Bacillus Anthracis ◽  
Insertional Mutagenesis ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Basal Layer ◽  
Wild Type ◽  
Wide Range ◽  
Green Fluorescent

ABSTRACT Bacillus cereus spores are assembled with a series of concentric layers that protect them from a wide range of environmental stresses. The outermost layer, or exosporium, is a bag-like structure that interacts with the environment and is composed of more than 20 proteins and glycoproteins. Here, we identified a new spore protein, ExsM, from a β-mercaptoethanol extract of B. cereus ATCC 4342 spores. Subcellular localization of an ExsM-green fluorescent protein (GFP) protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutant spores were smaller and rounder than wild-type spores, which had an extended exosporium (spore length for the wt, 2.40 ± 0.56 μm, versus that for the exsM mutant, 1.66 ± 0.38 μm [P < 0.001]). Thin-section electron microscopy revealed that exsM mutant spores were encased by a double-layer exosporium, both layers of which were composed of a basal layer and a hair-like nap. Mutant exsM spores were more resistant to lysozyme treatment and germinated with higher efficiency than wild-type spores, and they had a delay in outgrowth. Insertional mutagenesis of exsM in Bacillus anthracis ΔSterne resulted in a partial second exosporium and in smaller spores. In all, these findings suggest that ExsM plays a critical role in the formation of the exosporium.

scholarly journals Role of DNA Repair by Nonhomologous-End Joining in Bacillus subtilis Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV, and Ionizing Radiation

2007 ◽  
Vol 189 (8) ◽  
pp. 3306-3311 ◽  
Author(s):  
Ralf Moeller ◽  
Erko Stackebrandt ◽  
Günther Reitz ◽  
Thomas Berger ◽  
Petra Rettberg ◽  
...  
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Ionizing Radiation ◽  
Ultrahigh Vacuum ◽  
Nonhomologous End Joining ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Spore Resistance

ABSTRACT The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation, and ultrahigh vacuum was studied in wild-type and DNA repair mutants (recA, splB, ykoU, ykoV, and ykoU ykoV mutants) of Bacillus subtilis. NHEJ-defective spores with mutations in ykoU, ykoV, and ykoU ykoV were significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.

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.

Effect of Small, Acid-Soluble Proteins on Spore Resistance and Germination under a Combination of Pressure and Heat Treatment

2007 ◽  
Vol 70 (9) ◽  
pp. 2168-2171
Author(s):  
JONG-KYUNG LEE ◽  
SARA MOVAHEDI ◽  
STEPHEN E. HARDING ◽  
BERNARD M. MACKEY ◽  
WILLIAM M. WAITES
Keyword(s):  
Heat Treatment ◽  
High Pressure ◽  
High Temperature ◽  
Spore Germination ◽  
Soluble Proteins ◽  
Wild Type ◽  
Spore Resistance ◽  
Pressure Resistance ◽  
The One

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of α/β-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65°C and 100 to 500 MPa) and applied to wild-type and SASP-α−/β− Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65°C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65°C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65°C inactivated wild-type SASP-α+/β+ spores more than mutant SASP-α−/β− spores, and this was attributed to less pressure-induced germination in SASP-α−/β− spores than in wild-type SASP-α+/β+ spores. However, there was no difference in the pressure resistance between SASP-α+/β+ and SASP-α−/β− spores at 100 MPa and ambient temperature (approximately 22°C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that α/β-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.

scholarly journals Light Gradient-Based Screening of Arabidopsis thaliana on a 384-Well Type Plant Array Chip

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 191
Author(s):  
Youn-Hee Park ◽  
Je-Kyun Park
Keyword(s):  
Arabidopsis Thaliana ◽  
White Light ◽  
Solid Medium ◽  
Red Light ◽  
Phytochrome B ◽  
Wild Type ◽  
Light Gradient ◽  
Gradient Based ◽  
Light Effects ◽  
Germination And Growth

Arabidopsis thaliana (Arabidopsis), as a model for plant research, is widely used for various aspects of plant science. To provide a more sophisticated and microscopic environment for the germination and growth of Arabidopsis, we report a 384-well type plant array chip in which each Arabidopsis seed is independently seeded in a solid medium. The plant array chip is made of a poly(methyl methacrylate) (PMMA) acrylic material and is assembled with a home-made light gradient module to investigate the light effects that significantly affect the germination and growth of Arabidopsis. The light gradient module was used to observe the growth pattern of seedlings according to the intensity of the white light and to efficiently screen for the influence of the white light. To investigate the response to red light (600 nm), which stimulates seed germination, the light gradient module was also applied to the germination test. As a result, the germination results showed that the plant array chip can be used to simultaneously screen wild type seeds and phytochrome B mutant seeds on a single array chip according to the eight red light intensities.

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