scholarly journals The Spore-Specific Alanine Racemase of Bacillus anthracis and Its Role in Suppressing Germination during Spore Development

2008 ◽  
Vol 191 (4) ◽  
pp. 1303-1310 ◽  
Author(s):  
Olga N. Chesnokova ◽  
Sylvia A. McPherson ◽  
Christopher T. Steichen ◽  
Charles L. Turnbough
Keyword(s):  
Bacillus Anthracis ◽  
Mother Cell ◽  
Basal Layer ◽  
Structural Proteins ◽  
Alanine Racemase ◽  
Spore Development ◽  
Germination Inhibitor ◽  
Atypical Localization ◽  
Late Stages ◽  
Premature Germination

ABSTRACT Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinant l-alanine to the germination inhibitor d-alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of the alr gene, which encodes Alr, is restricted to sporulating cells and that the bulk of alr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped two alr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Δalr mutant of B. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Δalr spores germinate more efficiently in the presence of l-alanine, presumably because of their inability to convert exogenous l-alanine to d-alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Δalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produce d-alanine during the late stages of sporulation to suppress premature germination of the developing spore.

scholarly journals Identification of the UDP-N-Acetylglucosamine 4-Epimerase Involved in Exosporium Protein Glycosylation in Bacillus anthracis

2009 ◽  
Vol 191 (22) ◽  
pp. 7094-7101 ◽  
Author(s):  
Shengli Dong ◽  
Olga N. Chesnokova ◽  
Charles L. Turnbough ◽  
David G. Pritchard
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Basal Layer ◽  
Amino Sugar ◽  
Protein Glycosylation ◽  
Side Chains ◽  
Protein Backbone ◽  
Gene Encoding ◽  
Multiple Copies ◽  
Late Stages

ABSTRACT Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation.

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 Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

BMB Reports ◽  
2009 ◽  
Vol 42 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Shivani Kanodia ◽  
Shivangi Agarwal ◽  
Priyanka Singh ◽  
Shivani Agarwal ◽  
Preeti Singh ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Biochemical Characterization ◽  
Alanine Racemase

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 Critical Role for Arginine Methylation in Adenovirus-Infected Cells

2007 ◽  
Vol 81 (23) ◽  
pp. 13209-13217 ◽  
Author(s):  
Demetris C. Iacovides ◽  
Clodagh C. O'Shea ◽  
Juan Oses-Prieto ◽  
Alma Burlingame ◽  
Frank McCormick
Keyword(s):  
Amino Acids ◽  
Critical Role ◽  
Arginine Methylation ◽  
Structural Proteins ◽  
Adenovirus Infection ◽  
The Third ◽  
C Terminus ◽  
Arginine Residues ◽  
Infected Cells ◽  
Late Stages

ABSTRACT During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection.

scholarly journals Characterization of the Enzymes Encoded by the Anthrose Biosynthetic Operon of Bacillus anthracis

2010 ◽  
Vol 192 (19) ◽  
pp. 5053-5062 ◽  
Author(s):  
Shengli Dong ◽  
Sylvia A. McPherson ◽  
Yun Wang ◽  
Mei Li ◽  
Pengfei Wang ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Biosynthetic Pathway ◽  
Basal Layer ◽  
Side Chain ◽  
Side Chains ◽  
Aminotransferase Activity ◽  
Acyltransferase Activity ◽  
Hydratase Activity ◽  
Etiological Agents

ABSTRACT Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-β-l-rhamnose α-1,3-l-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit β-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.

scholarly journals Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

2009 ◽  
Vol 9 (1) ◽  
pp. 53 ◽  
Author(s):  
Rafael M Couñago ◽  
Milya Davlieva ◽  
Ulrich Strych ◽  
Ryan E Hill ◽  
Kurt L Krause
Keyword(s):  
Bacillus Anthracis ◽  
Structural Characterization ◽  
Alanine Racemase

scholarly journals Identification of the Immunodominant Protein and Other Proteins of the Bacillus anthracis Exosporium

2003 ◽  
Vol 185 (6) ◽  
pp. 1903-1910 ◽  
Author(s):  
Christopher Steichen ◽  
Ping Chen ◽  
John F. Kearney ◽  
Charles L. Turnbough,
Keyword(s):  
Bacillus Anthracis ◽  
Tandem Repeats ◽  
Genomic Island ◽  
Alanine Racemase ◽  
Permeability Barrier ◽  
Immunodominant Antigen ◽  
Large Panel ◽  
Specific Variation ◽  
And Function ◽  
Molecular Structure And Function

ABSTRACT Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose-fitting, balloon-like layer called the exosporium. Although the exosporium serves as the source of surface antigens and a primary permeability barrier of the spore, its molecular structure and function are not well characterized. In this study, we identified five major proteins in purified B. anthracis (Sterne strain) exosporia. One protein was the recently identified collagen-like glycoprotein BclA, which appears to be a structural component of the exosporium hair-like nap. Using a large panel of unique antispore monoclonal antibodies, we demonstrated that BclA is the immunodominant antigen on the B. anthracis spore surface. We also showed that the BclA protein and not a carbohydrate constituent directs the dominant immune response. In addition, the length of the central (GXX)n repeat region of BclA appears to be strain specific. Two other unique proteins, BxpA and BxpB, were identified. BxpA is unusually rich in Gln and Pro residues and contains several different tandem repeats, which also exhibit strain-specific variation. In addition, BxpA was found to be cleaved approximately in half. BxpB appears to be glycosylated or associated with glycosylated material and is encoded by a gene that (along with bclA) may be part of an exosporium genomic island. The other two proteins identified were alanine racemase and superoxide dismutase, both of which were reported to be associated with the surface of other Bacillus spores. Possible functions of the newly identified proteins are discussed.

scholarly journals Orientation within the Exosporium and Structural Stability of the Collagen-Like Glycoprotein BclA of Bacillus anthracis

2005 ◽  
Vol 187 (15) ◽  
pp. 5310-5317 ◽  
Author(s):  
Jeremy A. Boydston ◽  
Ping Chen ◽  
Christopher T. Steichen ◽  
Charles L. Turnbough
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Triple Helix ◽  
Thermal Denaturation ◽  
Sodium Dodecyl ◽  
Basal Layer ◽  
Structural Data ◽  
Immunogold Electron Microscopy

ABSTRACT Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T m ) of 37°C, like mammalian collagen. However, rBclA trimers exhibited T m values of 84°C and 95°C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T m values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.

scholarly journals FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

PLoS Biology ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. e3001314
Author(s):  
Ane Landajuela ◽  
Martha Braun ◽  
Christopher D. A. Rodrigues ◽  
Alejandro Martínez-Calvo ◽  
Thierry Doan ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Mother Cell ◽  
Negative Curvature ◽  
Lipid Binding ◽  
Membrane Curvature ◽  
Membrane Topology ◽  
Cell Cytoplasm ◽  
Curvature Sensing ◽  
Membrane Fission ◽  
Late Stages

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

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