Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention. Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention. Bacillus anthracis is a Gram-positive, rod-shaped, aerobic soil bacterium that causes anthrax in humans and other mammals (1Mock M. Fouet A. Annu. Rev. Microbiol. 2001; 55: 647-671Google Scholar). Like other Bacillus species, B. anthracis forms endospores (or spores) when vegetative cells are deprived of an essential nutrient (2Priest F.G. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Biology. American Society for Microbiology, Washington, D. C.1993: 3-16Google Scholar). The mature spore is dormant and highly resistant to extreme temperatures, radiation, harsh chemicals, desiccation, and physical damage (3Nicholson W.L. Munakata N. Horneck G. Melosh H.J. Setlow P. Microbiol. Mol. Biol. Rev. 2000; 64: 548-572Google Scholar). These properties allow the spore to persist in the soil for many years until encountering a signal to germinate (4Paidhungat M. Setlow P. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Its Closest Relatives: From Genes to Cells. American Society for Microbiology, Washington, D. C.2002: 537-548Google Scholar). Anthrax is typically contracted by contact with spores (1Mock M. Fouet A. Annu. Rev. Microbiol. 2001; 55: 647-671Google Scholar). Because of their ability to cause a potentially fatal disease and to withstand harsh conditions, spores of B. anthracis have been developed into weapons of mass destruction by numerous countries and terrorist groups (5Webb G.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4355-4356Google Scholar). The effectiveness of B. anthracis spores as a biological weapon was demonstrated when letters laden with spores were mailed in the United States in the fall of 2001. In response to the threat of future releases of lethal spores, new research has been undertaken to enhance our knowledge of B. anthracis biology and pathogenesis. A major goal of such studies is to identify components of the B. anthracis spore that can serve either as molecular targets of spore inactivation or as unique markers that allow rapid and accurate spore detection (6Williams D.D. Benedek O. Turnbough Jr., C.L. Appl. Environ. Microbiol. 2003; 69: 6288-6293Google Scholar). Sporulation in the genus Bacillus begins in the starved vegetative cell with an asymmetric septation that produces large and small genome-containing compartments called the mother cell and forespore, respectively (7Errington J. Microbiol. Rev. 1993; 57: 1-33Google Scholar). The mother cell then engulfs the forespore and surrounds it with a layer of modified peptidoglycan called the cortex and a more external proteinaceous layer called the coat. The spore coat, composed of three sublayers and many different proteins, forms the outermost detectable layer for spores of many species (e.g. B. subtilis) (8Lai E.M. Phadke N.D. Kachman M.T. Giorno R. Vazquez S. Vazquez J.A. Maddock J.R. Driks A. J. Bacteriol. 2003; 185: 1443-1454Google Scholar, 9Henriques A.O. Moran Jr., C.P. Methods. 2000; 20: 95-110Google Scholar). For other Bacillus species, such as B. anthracis, the spore coat is surrounded by another prominent layer called the exosporium, which is synthesized by the mother cell concurrently with the cortex and coat (10Gerhardt P. Fed. Proc. 1967; 26: 1504-1517Google Scholar). After a final stage of maturation, during which covalent modifications occur in the outer layers of the spore, the mother cell lyses and releases the spore (9Henriques A.O. Moran Jr., C.P. Methods. 2000; 20: 95-110Google Scholar, 11Roels S. Losick R. J. Bacteriol. 1995; 177: 6263-6275Google Scholar). Of particular interest in current studies of the B. anthracis spore is the exosporium, which is the primary permeability barrier of the spore and the source of spore surface antigens (10Gerhardt P. Fed. Proc. 1967; 26: 1504-1517Google Scholar, 12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). As the outermost surface of the spore, the exosporium interacts with the soil environment, detection devices, spore-binding cells in the mammalian host, and host defenses. Thus, it is likely that the exosporium plays an important role in spore survival and/or pathogenesis (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). To demonstrate such a role, it is necessary to characterize individual exosporium components. Early studies revealed that spores of B. anthracis and closely related species (e.g. Bacillus cereus and Bacillus thuringiensis) possess an exosporium composed of a paracrystalline basal layer and an external hairlike nap, which exhibits a strain-specific length up to 600 Å (10Gerhardt P. Fed. Proc. 1967; 26: 1504-1517Google Scholar, 13Gerhardt P. Ribi E. J. Bacteriol. 1964; 88: 1774-1789Google Scholar, 14Hachisuka Y. Kojima K. Sato T. J. Bacteriol. 1966; 91: 2382-2384Google Scholar, 15Beaman T.C. Pankratz H.S. Gerhardt P. J. Bacteriol. 1971; 107: 320-324Google Scholar, 16Kramer M.J. Roth I.L. Can. J. Microbiol. 1968; 14: 1297-1299Google Scholar). The exosporium constitutes about 2% of the mass of the spore and contains approximately 50% protein, 20% lipid, 20% carbohydrate, and 10% other components (17Matz L.L. Beaman T.C. Gerhardt P. J. Bacteriol. 1970; 101: 196-201Google Scholar). A recent proteomic analysis of the exosporium suggested that it contains at least 137 different proteins (18Liu H. Bergman N.H. Thomason B. Shallom S. Hazen A. Crossno J. Rasko D.A. Ravel J. Read T.D. Peterson S.N. Yates J.I. Hanna P.C. J. Bacteriol. 2004; 186: 164-178Google Scholar). However, this analysis was performed with an exosporium fraction prepared from spores that were not purified sufficiently to remove contaminating proteins released into the growth medium by lysed cells (44Williams D.D. Turnbough Jr., C.L. J. Bacteriol. 2004; 186: 566-569Google Scholar). Analyses of the exosporium prepared from highly purified spores indicates that about 20 different protein species are present in or tightly associated with the exosporium (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar, 19Todd S.J. Moir A.J.G. Johnson M.J. Moir A. J. Bacteriol. 2003; 185: 3373-3378Google Scholar, 44Williams D.D. Turnbough Jr., C.L. J. Bacteriol. 2004; 186: 566-569Google Scholar). The first B. anthracis exosporium protein identified, and one of the most interesting, was a glycoprotein called BclA (for Bacillus collagen-like protein of anthracis) (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar, 20Sylvestre P. Couture-Tosi E. Mock M. Mol. Microbiol. 2002; 45: 169-178Google Scholar). BclA is the structural component of the hairlike nap and contains multiple, collagen-like Xaa-Yaa-Gly (or XXG) repeats in its central region (20Sylvestre P. Couture-Tosi E. Mock M. Mol. Microbiol. 2002; 45: 169-178Google Scholar). The number of XXG repeats in BclA varies among strains (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar, 21Sylvestre P. Couture-Tosi E. Mock M. J. Bacteriol. 2003; 185: 1555-1563Google Scholar). This variation is responsible for the different lengths of the hairlike nap found on spores of different B. anthracis strains (21Sylvestre P. Couture-Tosi E. Mock M. J. Bacteriol. 2003; 185: 1555-1563Google Scholar). BclA has also been shown to be the immunodominant protein on the B. anthracis spore surface, because most antibodies raised against spores react with this protein (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). Finally, most of the XXG repeats in the collagen-like region of BclA have the sequence PTG, and nearly all of the XXG repeats contain a threonine residue, which may be a site of attachment of an O-linked oligosaccharide (22Schmidt M.A. Riley L.W. Benz I. Trends Microbiol. 2003; 11: 554-561Google Scholar, 23Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Google Scholar). In this report, we describe two O-linked oligosaccharides that are attached to BclA: a 715-Da tetrasaccharide and a 324-Da disaccharide. We show that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. The attachment of each oligosaccharide to BclA may occur through a GalNAc linker, which is lost during oligosaccharide release. Using several analytical techniques, we determine the complete structure of the tetrasaccharide. It contains a unique sugar residue that may be useful for species-specific detection of B. anthracis spores or even serve as a new target for preventing anthrax. Bacterial Strains—The Sterne veterinary vaccine strain of B. anthracis along with B. cereus T and B. thuringiensis ssp. kurstaki were obtained from John Ezzell (U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD). B. subtilis (trpC2) 1A700 (originally designated 168) was obtained from the Bacillus Genetic Stock Center (Ohio State University, Columbus, OH). The Sterne strain of B. anthracis is not a human pathogen because it lacks plasmid pXO2, which carries the genes necessary to produce the protective poly-γ-d-glutamic acid capsule of the vegetative cell. Spores of the Sterne strain appear to be essentially identical to spores of virulent strains of B. anthracis (24Read T.D. Peterson S.N. Tourasse N. Baillie L.W. Paulsen I.T. Nelson K.E. Tettelin H. Fouts D.E. Eisen J.A. Gill S.R. Holtzapple E.K. Okstad O.A. Helgason E. Rilstone J. Wu M. Kolonay J.F. Beanan M.J. Dodson R.J. Brinkac L.M. Gwinn M. DeBoy R.T. Madpu R. Daugherty S.C. Durkin A.S. Haft D.H. Nelson W.C. Peterson J.D. Pop M. Khouri H.M. Radune D. Benton J.L. Mahamoud Y. Jiang L. Hance I.R. Weidman J.F. Berry K.J. Plaut R.D. Wolf A.M. Watkins K.L. Nierman W.C. Hazen A. Cline R. Redmond C. Thwaite J.E. White O. Salzberg S.L. Thomason B. Friedlander A.M. Koehler T.M. Hanna P.C. Kolsto A.B. Fraser C.M. Nature. 2003; 423: 81-86Google Scholar). Plasmid and Strain Constructions—Recombinant DNA techniques, preparation of plasmid DNA from Escherichia coli, and transformation of E. coli were carried out by standard procedures (25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1989Google Scholar). Electroporation of B. anthracis was performed using unmethylated plasmid DNA isolated from E. coli strain GM1684 (dam-4) (26Koehler T.M. Dai Z. Kaufman-Yarbray M. J. Bacteriol. 1994; 176: 586-595Google Scholar). Mutants of the B. anthracis Sterne strain were constructed by allelic exchange between the chromosome and a mutant locus carried by the shuttle vector pUTE29 as previously described (27Saile E. Koehler T.M. J. Bacteriol. 2002; 184: 370-380Google Scholar, 28Dai Z.J.-C. Sirard M. Mock M. Koehler T.M. Mol. Microbiol. 1995; 16: 1171-1181Google Scholar). Without selection with tetracycline, plasmid pUTE29 is not maintained in B. anthracis (26Koehler T.M. Dai Z. Kaufman-Yarbray M. J. Bacteriol. 1994; 176: 586-595Google Scholar). Sitedirected mutagenesis was performed with the QuikChange™ kit from Stratagene. To construct a ΔbclA deletion strain, a DNA segment containing the bclA gene and about 1 kb of flanking sequence on each side was PCR-amplified using genomic B. anthracis Sterne DNA as the template. The PCR product was cloned into plasmid pCR-Blunt II-TOPO (Invitrogen), and a unique BglII restriction site (used below) was introduced 56 bp after the bclA stop codon, in an apparent intergenic region (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar), by site-directed mutagenesis. The unique ApaI restriction site in this plasmid (from the TOPO vector) was also removed by site-directed mutagenesis, and the resultant plasmid was designated pCLT1159. Using this plasmid as the template, outward PCR was used to create a new plasmid in which most of the bclA gene (i.e. codons 27–382 of 400) was removed and replaced with an ApaI site. The region containing the bclA deletion and flanking B. anthracis DNA was then excised and inserted into the multiple cloning site (between PstI and KpnI) of plasmid pUTE29. A kanamycin resistance cassette from plasmid pUC18::Ωkm-2 (29Perez-Casal J. Caparon M.G. Scott J.R. J. Bacteriol. 1991; 173: 2617-2624Google Scholar) was inserted into the unique BglII site of the plasmid. After passage through E. coli strain GM1684 (dam-4), the plasmid was electroporated into B. anthracis. The transformant was grown under conditions that allowed allelic exchange to replace the wild-type bclA locus with the ΔbclA mutation and adjacent kanamycin resistance cassette, while permitting the loss of the recombinant pUTE29 (27Saile E. Koehler T.M. J. Bacteriol. 2002; 184: 370-380Google Scholar, 28Dai Z.J.-C. Sirard M. Mock M. Koehler T.M. Mol. Microbiol. 1995; 16: 1171-1181Google Scholar). The mutant locus was confirmed by PCR amplification of the bclA region and sequencing the DNA product, and the mutant strain was designated CLT292. To construct mutant strains carrying a reduced number of XXG repeats in the bclA gene, we first performed outward PCR with primers 5′-ACTGGGCCCACTGGTGCTACCGGACTG and 5′-AGTGGGCCCAGTTGGTCCAGTAGTACC and plasmid pCLT1159 (bclA+) as the template. One primer hybridizes to a unique site, whereas the other hybridizes to multiple sites within the central region of bclA. The resulting PCR products were digested with ApaI and circularized to produce plasmids carrying a partial deletion (not all the same) of the bclA repeat region. After cloning and propagating the plasmids in E. coli, the deletions were defined by sequence analysis. For each deletion plasmid, a fragment containing the mutant bclA gene and flanking sequences was excised and inserted into plasmid pUTE29 as above. A spectinomycin-resistance cassette from plasmid pJRS312 (Ω-sp) (27Saile E. Koehler T.M. J. Bacteriol. 2002; 184: 370-380Google Scholar) was inserted into the unique BglII site downstream of bclA. The resulting plasmid was used for allelic replacement of the bclA locus using strain CLT292 (ΔbclA kan) as the recipient. We confirmed that each mutant strain was kanamycin-sensitive and spectinomycin-resistant, and carried the expected deletion. To construct a strain unable to synthesize l-rhamnose, we generated a derivative of plasmid pUTE29 that contained a spectinomycin-resistance cassette (i.e. Ω-sp) flanked by sequences upstream and downstream of the rmlD gene of B. anthracis. This plasmid was used for allelic replacement of the rmlD locus of the Sterne strain as described above. The mutant strain, designated CLT274, contains a deletion that removes codons 11–264 of the 284 rmlD codons, and the deleted codons are replaced by the Ω-sp cassette. Preparation of Spores and Purified Exosporium—Spores were prepared from cultures grown at 37 °C for 48–72 h on liquid or solid Difco sporulation medium, extensively washed in cold (4 °C) distilled water, sedimented through 50% Renografin to remove vegetative cells and debris, and washed again in cold water (30Knurr J. Benedek O. Heslop J. Vinson R.B. Boydston J.A. McAndrew J. Kearney J.F. Turnbough Jr., C.L. Appl. Environ. Microbiol. 2003; 69: 6841-6847Google Scholar, 31Nicholson W.L. Setlow P. Harwood C.R. Cutting S.M. Molecular Biological Methods for Bacillus. John Wiley & Sons, Ltd., West Sussex, UK1990: 391-450Google Scholar). Spores were stored in water at 4 °C (protected from light) and quantitated microscopically using a Petroff-Hausser counting chamber. The exosporium was removed from spores by passage through a French press and then highly purified by differential centrifugation as previously described (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). Monosaccharide Analysis by Gas Chromatography—The monosaccharide compositions of spores, exosporium, and other samples were determined by gas chromatographic analysis of the trimethylsilyl derivatives of the sugar methyl glycosides. Samples were dried in a vacuum centrifuge, resuspended in 400 μl of 1.45 n methanolic HCl, and heated at 80 °C overnight. The methanolic HCl was removed by vacuum centrifugation, and the sample was resuspended in 200 μl of methanol, followed by the addition of 20 μl of acetic anhydride and 20 μl of pyridine. This mixture was allowed to react for 30 min at room temperature and then evaporated to dryness. The samples were then trimethylsilylated using 50 μl of Tri-Sil (Pierce), and the vials were sealed under argon. The trimethylsilylated glycosides were separated and quantitated on an HP 5890 gas chromatograph equipped with a 30-m HP-1 wide bore fused silica column coated with a 0.88-μm layer of cross-linked methyl silicone gum. Samples were applied to the column with an automatic injector, and sugars were detected by flame ionization. Determination of the Absolute Configuration of Rhamnose Residues—To distinguish between the d- and l-forms of rhamnose residues, the (+)-2-butyl glycosides were prepared and analyzed by gas chromatography as previously described (32Leontein K. Lindberg B. Lonngren J. Carbohydr. Res. 1978; 62: 359-362Google Scholar). However, HCl rather than trifluoroacetic acid was used as the catalyst, and rather than using acetate derivatives, as in the original procedure, trimethylsilyl derivatives were prepared as described above. The retention time of the uncommon d-rhamnose, for which a standard was not available, was determined by chromatography of the (–)-2-butyl glycoside of l-rhamnose. Hydrazinolysis of Glycoproteins—Selective hydrazinolysis was used to release O-linked oligosaccharides from spore glycoproteins. B. anthracis spores (1010) or exosporium samples were dried in a vacuum centrifuge and desiccated overnight over P2O5 under vacuum. Anhydrous hydrazine (1 ml) was added to each sample in a glass ampoule, which was flushed with argon and flame-sealed. The samples were heated at 60 °C for 5 h to specifically release O-linked oligosaccharides (33Patel T. Bruce J. Merry A. Bigge C. Wormald M. Jaques A. Parekh R. Biochemistry. 1993; 32: 679-693Google Scholar). The hydrazine was evaporated under vacuum and the residue was resuspended in 3 ml of water. The mixture was centrifuged at 14,000 × g for 10 min, and the supernatant containing oligosaccharides was collected. Gel Filtration Chromatography and Assay of Rhamnose-containing Oligosaccharides—The supernatant obtained from the hydrazinolysis procedure was loaded onto a 170 × 2.2 cm Bio-Gel P4 (fine; Bio-Rad) column, and the oligosaccharides were eluted with 0.1 m acetic acid. Three-ml fractions were collected, and a 250-μl sample of each fraction was assayed for 6-deoxy sugars (e.g. rhamnose) using the Dische-Shettles protocol (34Dische Z. Shettles L.B. J. Biol. Chem. 1948; 175: 595-603Google Scholar). Mass Spectrometry—Mass spectrometry was performed with a Micromass Q-TOF 2 mass spectrometer. Samples were introduced by flow injection into a stream of 50% acetonitrile containing 0.1% formic acid delivered by a Harvard model 22 syringe pump and were ionized by the electrospray mode. NMR Spectroscopy—Approximately 750 μg of the purified tetrasaccharide was lyophilized, dissolved in 450 μl of Me2SO-d6 (99.99% deuterium), and transferred to a 5-mm NMR tube. NMR data were collected on a Bruker DRX-500 NMR spectrometer using a 5 mm TXI probe equipped with x, y, z gradients at a probe temperature of 25 °C. A few measurements were repeated at 600 MHz on an Avance DRX-600 NMR spectrometer. NMR experiments were performed on samples stored for 3–4 days at 4 °C. Standard Bruker pulse sequences were used, except for the 13C-coupled HSQC, where the program was modified to remove the 13C decoupling during acquisition. Proton and carbon chemical shifts were referenced to an internal Me2SO peak (2.490 ppm for proton and 39.5 ppm for 13C). In addition to standard 1H and 13C one-dimensional NMR spectra, a series of homo- and heteronuclear two-dimensional NMR data sets were obtained. DQF-COSY 1The abbreviations used are: DQF-COSY, double quantum filtered-correlated spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; HMBC, heteronuclear multiple bond correlation; ESI-Q-TOF, electrospray ionization-quadrupole-time of flight; Ant, anthrose. was collected with 4096 data points and 0.409 s acquisition time in the F2 dimension with 800 increments in the indirect dimension. The data matrix was zero-filled in the F1 dimension to give a matrix of 4096 × 2048 points. The two-dimensional TOCSY experiments were performed with various spin lock times of 20, 40, 50, and 70 ms. The two-dimensional NOESY experiments were performed using 400- and 800-ms mixing times. The heteronuclear two-dimensional experiments HSQC, HSQC-TOCSY, and HMBC (with and without bilinear rotation decoupling filter) were performed using pulse field gradient programs. Data processing and plotting were performed using the Bruker Xwinplot program. Analysis of the Exosporium Monosaccharide Composition—As the first step in the identification of BclA oligosaccharides, we analyzed the monosaccharide composition of exosporium preparations purified from spores of wild-type and ΔbclA strains of B. anthracis (Sterne). Equal amounts of each exosporium sample were subjected to methanolysis, and trimethylsilyl derivatives of the resulting methyl glycosides were separated by gas chromatography. Methanolysis gives rise to several isomeric methyl glycosides in defined ratios for a particular sugar (35Bhatti T. Chambers R.E. Clamp J.R. Biochim. Biophys. Acta. 1970; 222: 339-347Google Scholar). The results showed that the wild-type exosporium contained at least four major monosaccharides that were absent in the ΔbclA exosporium (Fig. 1). Based on the retention times and isomeric ratios of sugar standards, we identified the most abundant monosaccharide residue as rhamnose and a second residue as GalNAc. Because bacteria are known to make both d- and l-rhamnose, we determined the absolute configuration of the rhamnose in the exosporium. The chiral 2-butyl glycosides of rhamnose were prepared, and the trimethylsilyl derivatives were analyzed by gas chromatography (32Leontein K. Lindberg B. Lonngren J. Carbohydr. Res. 1978; 62: 359-362Google Scholar). Only l-rhamnose was present (data not shown). The other two major monosaccharide residues that appear to be associated with BclA are labeled A and B in Fig. 1A. Component A is a unique monosaccharide described in detail below, and component B is most likely 3-O-methyl l-rhamnose based on a preliminary characterization (data not shown). The latter assignment is supported by the previous identification of 3-O-methyl rhamnose as a component of B. anthracis spores (36Fox A. Black G.E. Fox K. Rostovtseva S. J. Clin. Microbiol. 1993; 31: 887-894Google Scholar). The gas chromatogram of the ΔbclA exosporium contained a minor peak of a sugar with a retention time similar to that of the major methyl glycoside isomer of rhamnose (Fig. 1B). However, this sugar does not appear to be rhamnose. The same minor peak was present in a chromatogram of a ΔrmlD derivative of the Sterne strain that is unable to synthesize l-rhamnose (data not shown). Based on retention times and isomeric ratios of sugar standards, the minor sugar was tentatively identified as ribose, which was previously reported to be present in low levels in the exosporium of B. cereus T (17Matz L.L. Beaman T.C. Gerhardt P. J. Bacteriol. 1970; 101: 196-201Google Scholar) and in spores of B. anthracis (36Fox A. Black G.E. Fox K. Rostovtseva S. J. Clin. Microbiol. 1993; 31: 887-894Google Scholar). The only BclA-associated peak present in the chromatogram of the ΔrmlD strain was that of GalNAc. Although the four BclA-associated monosaccharides described above were major components of the exosporium carbohydrate, they were relatively minor components of the carbohydrate present in intact spores (data not shown). Thus, these monosaccharides appear to be components of glycoconjugates that are primarily or uniquely found in the exosporium. Isolation of Rhamnose-containing Oligosaccharides Associated with BclA—The numerous threonine residues in the collagen-like region of BclA and the monosaccharide composition of the exosporium indicated that one or more rhamnose-containing oligosaccharides were O-linked to BclA. To isolate these oligosaccharides, 1010 purified spores of the Sterne strain of B. anthracis were treated with anhydrous hydrazine under conditions that released only O-linked oligosaccharides. The free oligosaccharides were separated on a Bio-Gel P4 column (2.2 × 170 cm), and column fractions were assayed for rhamnose. Two oligosaccharide peaks were detected (Fig. 2). Based on the elution times of oligosaccharide standards, the larger peak corresponded to a tetrasaccharide, whereas the smaller peak corresponded to a disaccharide. Analysis of individual column fractions by ESI-Q-TOF mass spectrometry indicated that each oligosaccharide peak was essentially homogeneous and that the tetrasaccharide and disaccharide had masses of 715 and 324 Da, respectively. Both oligosaccharides were also isolated from purified exosporium of the Sterne strain following hydrazinolysis and gel filtration chromatography as described above. In addition, two minor peaks corresponding to a pentasaccharide and a trisaccharide were observed with masses of 918 and 527 Da, respectively (data not shown). These masses are equal to those of the tetrasaccharide and disaccharide, respectively, with the addition of a GalNAc residue. The significance of the minor oligosaccharides detected in the exosporium is discussed below. To determine whether the two major oligosaccharides were present in spores lacking BclA, we subjected 1010 spores of a ΔbclA derivative of the Sterne strain to hydrazinolysis and assayed for oligosaccharides as described above. No oligosaccharides were detected in the column fractions (Fig. 2). This result indicated that the tetrasaccharide and disaccharide found in wild-type spores were attached to or at least associated with BclA. Each purified oligosaccharide from wild-type sp
Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.
Several mycoplasma species have been shown to form biofilms that confer resistance to antimicrobials and which may affect the host immune system, thus making treatment and eradication of the pathogens difficult. The present study shows that the biofilms formed by two strains of the human pathogen Mycoplasma pneumoniae differ quantitatively and qualitatively. Compared with strain UAB PO1, strain M129 grows well but forms biofilms that are less robust, with towers that are less smooth at the margins. A polysaccharide containing N-acetylglucosamine is secreted by M129 into the culture medium but found in tight association with the cells of UAB PO1. The polysaccharide may have a role in biofilm formation, contributing to differences in virulence, chronicity and treatment outcome between strains of M. pneumoniae. The UAB PO1 genome was found to be that of a type 2 strain of M. pneumoniae, whereas M129 is type 1. Examination of other M. pneumoniae isolates suggests that the robustness of the biofilm correlates with the strain type.
Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high-resolution mass spectrometry. O-linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O-linked modification, and glucose was inferred by (13) C-labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.
Although they lack a cell wall, mycoplasmas do possess a glycocalyx. The interactions between the glycocalyx, mycoplasmal surface proteins and host complement were explored using the murine pathogen Mycoplasma pulmonis as a model. It was previously shown that the length of the tandem repeat region of the surface lipoprotein Vsa is associated with susceptibility to complement-mediated killing. Cells producing a long Vsa containing about 40 repeats are resistant to complement, whereas strains that produce a short Vsa of five or fewer repeats are susceptible. We show here that the length of the Vsa protein modulates the affinity of the M. pulmonis EPS-I polysaccharide for the mycoplasma cell surface, with more EPS-I being associated with mycoplasmas producing a short Vsa protein. An examination of mutants that lack EPS-I revealed that planktonic mycoplasmas were highly susceptible to complement killing even when the Vsa protein was long, demonstrating that both EPS-I and Vsa length contribute to resistance. In contrast, the mycoplasmas were resistant to complement even in the absence of EPS-I when the cells were encased in a biofilm.
Few mycoplasmal polysaccharides have been described and little is known about their role in pathogenesis. The infection of mice with Mycoplasma pulmonis has been utilized in many in vivo and in vitro studies to gain a better understanding of host-pathogen interactions during chronic respiratory infection. Although alveolar macrophages have a primary role in host defence, M. pulmonis is killed inefficiently in vitro. One antiphagocytic factor produced by the mycoplasma is the family of phase- and size-variable Vsa lipoproteins. However, bacteria generally employ multiple strategies for combating host defences, with capsular polysaccharide often having a key role. We show here that mutants lacking the EPS-I polysaccharide of M. pulmonis exhibit increased susceptibility to binding and subsequent killing by alveolar macrophages. These results give further insight into how mycoplasmas are able to avoid the host immune system and sustain a chronic infection.
Many proteins that have a primary function as a cytoplasmic protein are known to have the ability to moonlight on the surface of nearly all organisms. An example is the glycolytic enzyme enolase, which can be found on the surface of many types of cells from bacteria to human. Surface enolase is not enzymatic because it is monomeric and oligomerization is required for glycolytic activity. It can bind various molecules and activate plasminogen. Enolase lacks a signal peptide and the mechanism by which it attaches to the surface is unknown. We found that treatment of whole cells of the murine pathogen Mycoplasma pulmonis with phospholipase D released enolase and other common moonlighting proteins. Glycostaining suggested that the released proteins were glycosylated. Cytoplasmic and membrane-bound enolase was isolated by immunoprecipitation. No post-translational modification was detected on cytoplasmic enolase, but membrane enolase was associated with lipid, phosphate and rhamnose. Treatment with phospholipase released the lipid and phosphate from enolase but not the rhamnose. The site of rhamnosylation was identified as a glutamine residue near the C-terminus of the protein. Rhamnose has been found in all species of mycoplasma examined but its function was previously unknown. Mycoplasmas are small bacteria with have no peptidoglycan, and rhamnose in these organisms is also not associated with polysaccharide. We suggest that rhamnose has a central role in anchoring proteins to the membrane by linkage to phospholipid, which may be a general mechanism for the membrane association of moonlighting proteins in mycoplasmas and perhaps other bacteria.
The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates.Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology.The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins.Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage.In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine.Protein glycosylation occurred at numerous sites on surfaceexposed lipoproteins with no apparent amino acid sequence specificity.The lipoproteins of Mycoplasma pneumoniae also are glycosylated.Glycosylation was dependent on the glycosidic linkages from host oligosaccharides.As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related.The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.
Mycoplasma genitalium is an important etiologic agent of non-gonococcal urethritis (NGU), known for chronicity and multidrug resistance, in which biofilms may play an integral role. In some bacterial species capable of forming biofilms, extracellular polymeric substances (EPS) composed of poly-N-acetylglucosamine (PNAG) are a crucial component of the matrix. Monosaccharide analysis of M. genitalium strains revealed high abundance of GlcNAc, suggesting a biofilm-specific EPS. Chromatograms also showed high concentrations of galactose and glucose as observed in other mycoplasma species. Fluorescence microscopy of M. genitalium biofilms utilizing fluor-coupled lectins revealed differential staining of biofilm structures. Scanning electron microscopy (SEM) showed increasing maturation over time of bacterial "towers" seen in biofilm development. As seen with Mycoplasma pneumoniae, organisms within fully mature M. genitalium biofilms exhibited loss of cell polarization. Bacteria associated with disrupted biofilms exhibited decreased dose-dependent viability after treatment with antibiotics compared to bacteria with intact biofilms. In addition, growth index analysis demonstrated decreases in metabolism in cultures with disrupted biofilms with antibiotic treatment. Taken together, these data suggest that M. genitalium biofilms are a contributing factor in antibiotic resistance.
The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome-scale model for M. florum and will guide future genome engineering endeavors in this simple organism.
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