Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.     

Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis

Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.     

Plant Cell Wall Matrix Polysaccharide Biosynthesis

The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemicellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysaccharides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrils, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like （Csl） family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incrementally unravel the mechanisms of Golgi polysaccharide biosynthesis.     

Yeast Cell Wall Bound Proteolytic Enzymes

Nearly all the amino group-producing activity of the autolysate of cells of Saccharomyces sake was recovered in the cell wall fraction obtained from the autolysis residue. The activity of the cell wall fraction was not lost even after repeated use.

The proteolytic activity of the fraction was not solubilized by incubation with detergents, disruption with cell mill or by freezing and thawing method, but was solubilized to some extent by incubation with a commercial yeast cell-lytic enzyme preparation.

The cell wall fraction hydrolysed casein to about 50%. When casein was previously treated with certain proteinases, more than 60% was digested. The activity of the fraction was significantly increased by the addition of Zn²⁺ while it was decreased by several proteolytic enzyme inhibitors. The interesting fact was that in the presence of EDTA the cell wall fraction showed only carboxypeptidase-like activity, and attacked the oxidized insulin B-chain to release two amino acids from the carboxyl terminal in known order.     

Cell Wall Assembly in Saccharomyces cerevisiae

An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.     

Contributions of Four Cortex Lytic Enzymes to Germination of Bacillus anthracis Spores

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca²⁺-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore''s integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca²⁺-DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.The Gram-positive bacterium Bacillus anthracis is the etiologic agent of cutaneous, gastrointestinal, and inhalational anthrax (24). An anthrax infection begins when the host is infected with highly resistant, quiescent B. anthracis spores (1, 24). Within the host, the spore''s sensory mechanism recognizes chemical signals, known as germinants, and triggers germination, which leads to the resumption of metabolism (36). Spores that have differentiated into vegetative cells produce a protective capsule and deadly toxins. These virulence factors allow the bacteria to evade the host''s immune system and establish an infection resulting in septicemia, toxemia, and frequently death (24). Although vegetative cells produce virulence factors that are potentially fatal, these cells cannot initiate infections and are much more susceptible to antimicrobial treatments than spores (24). Therefore, efficient triggering of spore germination may enhance current decontamination methods.Spores are highly resistant to many environmental insults because the spore core (cytoplasm) is dehydrated, dormant, and surrounded by multiple protective layers, including a modified layer of peptidoglycan (PG) known as the cortex (36). The cortex functions to maintain dormancy and heat resistance by preventing core rehydration (9). It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugars (Fig. ​(Fig.1).1). Peptide side chains on the NAM residues are either involved in interstrand cross-linking, cleaved to single l-alanine side chains, or fully removed with accompanying formation of muramic-δ-lactam (2, 31, 38). After germination is initiated by either nutrient or nonnutrient germinants, the cortex is depolymerized, resulting in complete core rehydration, resumption of metabolic activity, and outgrowth (33, 36).Open in a separate windowFIG. 1.Spore PG structure and hydrolysis. The central structure shows a representative spore PG strand with alternating NAG and NAM or muramic-δ-lactam (MδL) residues and with tetrapeptide or l-Ala side chains on the NAM residues. Forked arrows originate at sites of hydrolysis by the indicated enzymes and point to muropeptide products. The indicated “aG” muropeptide names are as previously published (7, 11). SleB lytic transglycosylase activity produces muropeptides terminating in anhydro-NAM. Cleavage at adjacent NAM residues produces the tetrasaccharide aG7a or aG7b, while cleavage further apart can produce octasaccharides or larger fragments. These can be further cleaved by muramidase treatment, resulting in the production of tetrasaccharide N, which terminates in NAM. The N-acetylglucosaminidase activity of SleL produces tetrasaccharides terminating in NAG, which can be further cleaved by muramidase to trisaccharides terminating in NAM.Cortex hydrolysis is driven by autolysins called germination-specific cortex lytic enzymes (GSLEs) that recognize the cortex-specific muramic-δ-lactam residues (2, 4, 21, 32). GSLEs fall into two classes: spore cortex lytic enzymes (SCLEs), which are thought to depolymerize intact cortical PG, and cortical fragment lytic enzymes (CFLEs), which further degrade partially hydrolyzed cortex (21). Both SCLEs and CFLEs have been identified in a variety of spore-forming species, including B. anthracis (11, 18, 19), Bacillus cereus (4, 20, 26), Bacillus megaterium (8, 34), Bacillus subtilis (13, 16, 25), Bacillus thuringiensis (12), and Clostridium perfringens (5, 23). Of the four GSLEs identified in B. anthracis, CwlJ1, CwlJ2, and SleB are predicted to be SCLEs (11), whereas SleL is thought to be a CFLE (18).Recently, independent studies showed that CwlJ1 and the lytic transglycosylase SleB (Fig. ​(Fig.1)1) play partially redundant roles and that either is sufficient for spore germination and outgrowth (10, 11). However, these same studies report conflicting results concerning the role of CwlJ2 during germination. Heffron et al. found no effect of CwlJ2 on the biochemistry of cortex hydrolysis or on colony-forming efficiency of spores (11). Giebel et al. reported that loss of CwlJ2 caused a minor defect in germination kinetics and that in the absence of SleB and CwlJ1, further loss of CwlJ2 had a major effect on colony forming efficiency (10). SleL in Bacillus anthracis is proposed to be an N-acetylglucosaminidase (Fig. ​(Fig.1)1) whose role is to further degrade cortex fragments resulting from SCLE hydrolysis (18). SleL is not essential for the completion of germination but does promote the release of small muropeptides to the spore''s surrounding environment (18).This study reports the effects of multiple deletion mutations affecting GSLEs on spore germination efficiency and kinetics of cortex hydrolysis. The data confirm the dominant roles played by CwlJ1 and SleB in the initiation of cortex hydrolysis and the major role of SleL in release of small cortex fragments. A minor role of CwlJ2 in nutrient-triggered germination and the contributions of CwlJ1 and CwlJ2 to Ca²⁺-dipicolinic acid (DPA)-triggered germination were revealed.     

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

Effect of Auxin on Cell Wall Degrading Enzymes

The effect of auxin on the activities of amylase, cellulase, β-1, 3- and/or β-l, 6-glucanase and hemieellulase were observed using etiolated barley coleoptile and pea epicotyl internode segments. The activities of β-1, 3- and/or β-l, 6-glueanase and hemicellulase of barley were increased by indole-3-acetic acid in a 3 hours' treatment. Amylase activity was not influenced by the auxin. Cellulase activity was not detected under the experimental conditions. 2, 4-Dichlorophenoxyacetic acid increased hemicellulase activity, but not cellulase and amylase activities, in pea epicotyl segments in 3 hours. Fungal β-1, 3-glucanase exogenously applied induced the elongation of barley coleoptile segments. The elongation induced by the enzyme was as high as that induced by indole-3-acetic acid at least for the first 1 to 3 hours.     

Cell Wall Protein in Bacillus subtilis

The cell wall of Bacillus subtilis 168 contains protein that is refractory to removal by salts, detergents, and denaturants.     

Cell wall anchor structure of BcpA pili in Bacillus anthracis

Assembly of pili in Gram-positive bacteria and their attachment to the cell wall envelope are mediated by sortases. In Bacillus cereus and its close relative Bacillus anthracis, the major pilin protein BcpA is cleaved between the threonine and the glycine of its C-terminal LPXTG motif sorting signal by the pilin-specific sortase D. The resulting acyl enzyme intermediate is relieved by the nucleophilic attack of the side-chain amino group of lysine within the YPKN motif of another BcpA subunit. Cell wall anchoring of assembled BcpA pili requires sortase A, which also cleaves the LPXTG sorting signal of BcpA between its threonine and glycine residues. We show here that sortases A and D require only the C-terminal sorting signal of BcpA for substrate cleavage. Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the side-chain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. These results represent the first demonstration of a cell wall anchor structure for pili, which are deposited by sortase A into the envelope of many different microbes.     

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

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.Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, rod-shaped soil bacterium that forms spores when deprived of essential nutrients (15). Spore formation begins with an asymmetric septation that divides the developing cell into a forespore compartment and a larger mother cell compartment, each of which contains a copy of the genome. The mother cell then engulfs the forespore and surrounds it with three protective layers: a cortex composed of peptidoglycan, a closely apposed proteinaceous coat, and a loosely fitting exosporium (10). Mother cell lysis releases the mature spore, which is dormant and capable of surviving in harsh environments for many years (17). When spores encounter an aqueous environment containing nutrients, they can germinate and grow as vegetative cells (21).Recently, interest in B. anthracis spores has intensified in response to their use as agents of bioterrorism. Of particular interest has been the outermost layer of the spore, the exosporium, which serves as a semipermeable barrier to potentially harmful macromolecules (8, 25) and as the vital first point of contact with the immune system of an infected host (11, 18, 30). The exosporium of B. anthracis and of closely related species, such as Bacillus cereus and Bacillus thuringiensis, is comprised of a paracrystalline basal layer and an external hairlike nap (1). The basal layer contains approximately 20 different proteins (20, 23), while the filaments of the nap are formed by trimers of a single collagenlike glycoprotein called BclA (2, 26). The central region of BclA contains a large number of GXX repeats, and the region varies in length in naturally occurring strains of B. anthracis, resulting in hairlike naps of differing lengths (22, 27). Most of the GXX repeats are GPT, and many of the threonine residues are glycosylated. Two major oligosaccharide side chains are present, a pentasaccharide and a trisaccharide, and both are linked to the protein through reducing terminal N-acetylgalactosamine (GalNAc) residues (3). Several studies have demonstrated that the oligosaccharides are antigenic and are exposed on the surface of Bacillus anthracis spores (14, 29). This makes them prime targets for both detection devices and immunoprophylaxis.We previously reported our use of hydrazinolysis to release BclA oligosaccharides from exosporium preparations (3). The primary product was a tetrasaccharide that formed as a result of the undesirable loss of the reducing terminal GalNAc residue of the pentasaccharide, a process called “peeling.” We determined that the oligosaccharide consisted of a linear chain of three rhamnose residues with a novel deoxyamino sugar at its nonreducing terminus. This unusual sugar, 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose, was given the trivial name anthrose.Rhamnose is the major sugar present in both the trisaccharide and the pentasaccharide, and a four-gene rhamnose biosynthetic operon was previously identified (22). Previously, we proposed a pathway for anthrose biosynthesis (Fig. ​(Fig.1)1) and identified a four-gene operon (Fig. ​(Fig.2)2) that is essential for its biosynthesis (5). An in-frame deletion of the first gene of the operon reduced the amount of anthrose by approximately 50%, whereas the deletion of any one of the other three genes totally abolished anthrose synthesis. Here, we describe the characterization of the altered oligosaccharide side chains of the four deletion mutants. We also cloned several genes that we predicted are involved in anthrose biosynthesis and demonstrated that the gene products possessed the expected biochemical activities.Open in a separate windowFIG. 1.Proposed biosynthetic pathway of anthrose. The pathway utilizes dTDP-4-keto-6-deoxy-α-d-glucose, an intermediate in rhamnose biosynthesis, and methylcrotonyl-CoA, derived from leucine catabolism. (Modified from reference 5.)Open in a separate windowFIG. 2.Anthrose operon and flanking genes. The four genes of the anthrose operon are antA (BAS3322), antB (BAS3321), antC (BAS3320), and antD (BAS3319). The operon is flanked by genes that encode a putative collagenase (BAS3323) and a putative methyltransferase (BAS3318). (Modified from reference 5.)     

Phase Separation of Plant Cell Wall Polysaccharides and Its Implications for Cell Wall Assembly

Concentrated binary mixtures of polymers in solution commonly exhibit immiscibility, resolving into two separate phases each of which is enriched in one polymer. The plant cell wall is a concentrated polymer assembly, and phase separation of the constituent polymers could make an important contribution to its structural organization and functional properties. However, to our knowledge, there have been no published reports of the phase behavior of cell wall polymers, and this phenomenon is not included in current cell wall models. We fractionated cell walls purified from the pericarp of unripe tomatoes (Lycopersicon esculentum) by extraction with cyclohexane diamine tetraacetic acid (CDTA), Na2CO3, and KOH and examined the behavior of concentrated mixtures. Several different combinations of fractions exhibited phase separation. Analysis of coexisting phases demonstrated the immiscibility of the esterified, relatively unbranched pectic polysaccharide extracted by CDTA and a highly branched, de-esterified pectic polysaccharide present in the 0.5 N KOH extract. Some evidence for phase separation of the CDTA extract and hemicellulosic polymers was also found. We believe that phase separation is likely to be a factor in the assembly of pectic polysaccharides in the cell wall and could, for example, provide the basis for explaining the formation of the middle lamella.     

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides × P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofiuorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubule-associated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.     

Studies on Cell Wall of Alkalophilic Bacillus

Cell walls of alkalophilic Bacillus No. C-125 and No. A-59 which grew in different pH conditions were prepared and analyzed. In the walls from cells grown at pH 10.3 (pH 10.3-cell wall) and the walls from cells grown at pH 7.5 (pH 7.5-cell wall) of the alkalophilic bacilli, the contents of neutral sugar and phosphorus were low as compared with those of Bacillus subtilis 6160, while uronic acid and amino acids were abundant. The uronic acid content of the pH 10.3-cell walls was higher than that of the pH 7.5-cell walls in both strains. The insoluble fraction (peptidoglycan) of cell walls of Bacillus No. C-125 consisted of muramic acid, glutamic acid, alanine, diaminopimelic acid and glucosamine as in neutrophilic bacilli. In the TCA soluble fraction of pH 10.3-cell walls of Bacillus No. C-125, uronic acid was a polymer of glucuronic acid containing a small amount of hexosamine, and 2/3 of the ninhydrin positive material was glutamic acid which was derived mainly from poly γ-L-glutamic acid.     

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides x P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of devel...     

Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research

Characterization of candidate surrogate spores prior to experimental use is critical to confirm that the surrogate characteristics are as closely similar as possible to those of the pathogenic agent of interest. This review compares the physical properties inherent to spores of Bacillus anthracis (Ba) and Bacillus thuringiensis (Bt) that impact their movement in air and interaction with surfaces, including size, shape, density, surface morphology, structure and hydrophobicity. Also evaluated is the impact of irradiation on the physical properties of both Bacillus species. Many physical features of Bt and Ba have been found to be similar and, while Bt is considered typically non-pathogenic, it is in the B. cereus group, as is Ba. When cultured and sporulated under similar conditions, both microorganisms share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 μm (in the respirable size range), have an exosporium with a hairy nap, and have higher relative hydrophobicities than other Bacillus species. While spore size, morphology, and other physical properties can vary among strains of the same species, the variations can be due to growth/sporulation conditions and may, therefore, be controlled. Growth and sporulation conditions are likely among the most important factors that influence the representativeness of one species, or preparation, to another. All Bt spores may, therefore, not be representative of all Ba spores. Irradiated spores do not appear to be a good surrogate to predict the behavior of non-irradiated spores due to structural damage caused by the irradiation. While the use of Bt as a surrogate for Ba in aerosol testing appears to be well supported, this review does not attempt to narrow selection between Bt strains. Comparative studies should be performed to test the hypothesis that viable Ba and Bt spores will behave similarly when suspended in the air (as an aerosol) and to compare the known microscale characteristics versus the macroscale response.     

Lysis of Yeast Cell Wall by Enzymes from Streptomycetes

This paper deals with yeast cell-wall lytic enzymes formed by Streptomyces with regard to the connection with the cell-wall structure.

In the first place, 29 organisms of β-glucanase-producing Streptomycetes were selected among 777 strains belonging to genus Streptomyces by means of a cylinder-plate method employing the yeast glucan as a substrate. As for these organisms, the depolymerizing activity against the yeast glucan was considered to be mainly due to β-1,3-glucanase activity. Against the heat-treated cell of bakers’ yeast, the crude enzymes merely showed poor lytic activities, however, in the combined employment with some protease preparations, especially with an alkaline protease from St. satsumaensis nov. sp., a remarkable increase of the lytic activities was demonstrated. On the other hand, the intact cell wall of bakers’ yeast, or both the heat-treated and the intact cells of Sacch. cerevisiae 18.29 strain were dissolved very easily by a sole action of β-glucanase or of protease, respectively. In consequence, it seemed that the lysis occurred with different mechanisms in response to differences of substrates. On this subject, the results of investigations and discussions were described in special measure. In addition, the possibility, that some other enzymes than β-glucanase or protease might concern to the lysis of the cell wall, was also investigated and discussed.     

The Sortase A Enzyme That Attaches Proteins to the Cell Wall of Bacillus anthracis Contains an Unusual Active Site Architecture

The pathogen Bacillus anthracis uses the Sortase A (SrtA) enzyme to anchor proteins to its cell wall envelope during vegetative growth. To gain insight into the mechanism of protein attachment to the cell wall in B. anthracis we investigated the structure, backbone dynamics, and function of SrtA. The NMR structure of SrtA has been determined with a backbone coordinate precision of 0.40 ± 0.07 Å. SrtA possesses several novel features not previously observed in sortase enzymes including the presence of a structurally ordered amino terminus positioned within the active site and in contact with catalytically essential histidine residue (His¹²⁶). We propose that this appendage, in combination with a unique flexible active site loop, mediates the recognition of lipid II, the second substrate to which proteins are attached during the anchoring reaction. pK_a measurements indicate that His¹²⁶ is uncharged at physiological pH compatible with the enzyme operating through a “reverse protonation” mechanism. Interestingly, NMR relaxation measurements and the results of a model building study suggest that SrtA recognizes the LPXTG sorting signal through a lock-in-key mechanism in contrast to the prototypical SrtA enzyme from Staphylococcus aureus.