Two Kdo-Heptose Regions Identified in Hafnia alvei 32 Lipopolysaccharide: the Complete Core Structure and Serological Screening of Different Hafnia O Serotypes

Hafnia alvei, a gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. Various 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-containing fragments different from known structures of core oligosaccharides were previously found among fractions obtained by mild acid hydrolysis of some H. alvei lipopolysaccharides (LPSs). However, the positions of these segments in the LPS structure were not known. Analysis of de-N,O-acylated LPS by nuclear magnetic resonance spectroscopy and mass spectrometry allowed the determination of the location of a Kdo-containing trisaccharide in the structure of H. alvei PCM 32 LPS. It was established that the trisaccharide {l-α-d-Hepp-(1→4)-[α-d-Galp6OAc-(1→7)]-α-Kdop-(2→} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between →4,7)-α-Kdop and →2)-Glcp in the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes of H. alvei LPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides of H. alvei 2, 32, 600, 1192, 1206, and 1211 LPSs. The weaker cross-reactions with LPSs of strains 974, 1188, 1198, 1204, and 1214 suggest the presence of similar structures in these LPSs, as well. Thus, we have identified new examples of endotoxins among those elucidated so far. This type of core oligosaccharide deviates from the classical scheme by the presence of the structural Kdo-containing motif in the outer-core region.Lipopolysaccharide (LPS) (endotoxin) is the main surface antigen and an important virulence factor of most of the gram-negative bacteria that are pathogenic for humans and animals (46). LPS contributes greatly to the structural integrity of bacteria and constitutes a “pathogen-associated molecular pattern” for host infection (46). As one of the most potent natural activators of the innate immune system, LPS is recognized by different classes of receptors present on macrophages, monocytes, B and T cells, neutrophils, endothelial cells, and epithelial cells (46). Endotoxins stimulate these cells to produce multiple inflammatory mediators responsible for immunotoxicity (e.g., tumor necrosis factor alpha, interleukin 1 [IL-1], IL-6, IL-8, gamma/alpha interferon, NO, platelet-activating factor, and endorphins). Interaction of LPS with the CD14/Toll-like receptor 4/MD-2 receptor complex constitutes a major mechanism responsible for the innate immune response to infection by gram-negative bacteria (1, 46). A large amount of LPS released into the bloodstream triggers the excessive inflammatory response of the innate immune system, leading to sepsis and septic shock (6). High levels of inflammatory mediators have profound effects on the cardiovascular system, kidneys, lungs, liver, central nervous system, and coagulation system. Following their action, renal failure, myocardial dysfunction, acute respiratory distress syndrome, hepatic failure, and disseminated intravascular coagulation can occur, which may result in death (6). Despite intense research on the etiology and treatment of sepsis, its severe form still carries a high mortality rate (6, 46).Hafnia alvei has been reported to be an opportunistic human pathogen. This gram-negative bacterium and its LPS are among the identified causative agents of bacteremia and septicemia in humans and animals (19). For the years 2001 to 2003, up to 42 cases of H. alvei bacteremia were reported annually in the United Kingdom. Most of them were monomicrobic infections, and in ∼33% of the cases, H. alvei was isolated, not only from the blood, but also from hepatic abscesses, pancreatic pseudocyst fluid, sputum, feces, and central venous catheters (19). Besides bacteremia and sepsis, which seem to be the most common syndromes reported, H. alvei is also associated with respiratory diseases and mixed hospital infections in humans. Since the gastrointestinal and respiratory tracts represent very common habitats for hafniae, most cases of H. alvei bacteremia originate there. H. alvei sepsis is also a serious clinical problem in the animal production industry, as infections of H. alvei can be severe, causing septicemia in commercial laying hens, pullets, and rainbow trout (19).Our knowledge of the pathogenicity of H. alvei is limited. LPS is the major virulence factor in cases of H. alvei septicemia and bacteremia (19). Studies of other virulence factors of H. alvei have reported only on the iron-scavenging mechanism, mannose-sensitive/mannose-resistant hemagglutinins, and plasmids encoding bacteriocins and alveicins (19).Most of the elucidated structures of H. alvei LPS are smooth-type molecules built up of O-specific polysaccharide, core oligosaccharide (OS), and lipid A. The O antigens of H. alvei are subdivided into 40 O serotypes (2, 28, 42). The structures of the O-specific polysaccharides from 30 serologically different H. alvei strains have been elucidated (15, 24, 26, 28, 42).So far, four types of core OS have been identified for H. alvei LPSs (9, 17, 25, 27, 30, 43). The most common core OS, isolated by mild acidic hydrolysis from LPSs of smooth H. alvei strains, is a hexasaccharide composed of two d-Glc, three l,d-Hep, and one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues. Two l,d-Hep residues are substituted by phosphoethanolamine (PEtn) and phosphoryl (P) groups (9, 17, 25). In LPSs isolated from H. alvei PCM 1185 and 1204, core OSs are terminated with d-Galp instead of d-Glcp (27). LPSs of H. alvei containing nontypical core OSs, identical with those found in LPSs of Escherichia coli R4 (strains 23 and 1222) and Salmonella enterica Ra (strain 39), were also identified (43).The chemical structures of H. alvei O-specific chains and core OSs were elucidated using fractions obtained by mild acid hydrolysis of LPS. The procedure was optimized for the delipidation of LPS, exploiting the susceptibility of a ketosidic linkage between the inner core and lipid A to acid. However, other acid-labile linkages within the LPS could also be affected, leading to partial degradation of the isolated molecules.The presence of Kdo-containing OSs among fractions obtained by mild acid hydrolysis of LPSs, other than previously identified core OSs, makes the structural analysis of entire H. alvei LPSs difficult. Two types of trisaccharides were previously identified: (i) l-α-d-Hepp-(1→4)-[α-d-Galp-(1→7)]-α-Kdop (l-α-d-Hep is α-l-glycero-d-manno-heptose) for strains 2, 1211, 32, and 1192 (16, 23) and (ii) α-d-Galp-(1→2)-l-α-d-Hepp-(1→4)-α-Kdop for strains 1188 and 1196 (22). These Kdo-containing motifs were never located in any of the LPS segments. Thus, it is of interest to complete the structure of H. alvei LPSs with the locations of such acid-labile motifs in the structures of LPSs isolated from these bacteria.We now report on structural studies of de-N,O-acylated LPS of H. alvei 32 containing a carbohydrate backbone of lipid A, core OS, an additional trisaccharide in the outer region of the core OS, and all of the acid-labile substituents. Additionally, data obtained previously for a trisaccharide isolated from H. alvei 32 LPS (16) was complemented with detailed ¹H and ¹³C nuclear magnetic resonance (NMR) analyses and the assignment of all proton and carbon signals. Screening for the presence of these acid-labile trisaccharides, identical with those found in the strain 32 LPS, was performed on 37 different O serotypes of H. alvei LPSs with antibodies against the conjugate of the de-N,O-acylated H. alvei 32 endotoxin fragment with bovine serum albumin (BSA), specific for the isolated trisaccharide.(Part of this work was presented at the 21st International Carbohydrate Symposium, Cairns, Australia, 7 to 12 July 2002, and the 3rd German-Polish-Russian Meeting on Bacterial Carbohydrates, Wroclaw, Poland, 6 to 9 October 2004.)     

Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.Amino acids are the building blocks of proteins and play an important role in the regulation of the metabolism of living organisms. Among two enantiomers of naturally occurring amino acids, l-amino acids are predominant in living organisms, while d-amino acids are found in both free and bound states in various organisms like bacteria (36), yeasts (35), plants (47), insects (11), mammals (17), bivalves (39), and fish (28). The d-amino acids are mostly endogenous and produced by racemization from their counterparts by the action of a racemase. Thus, the amino acid racemases are involved in d-amino acid metabolism (29, 46). Since the discovery of alanine racemase in 1951 (42), several racemases toward amino acids, such as those for glutamate, threonine, serine, aspartate, methionine, proline, arginine, and phenylalanine, have been reported in bacteria, archaea, and eukaryotes, including mammals (1, 2, 15, 30, 31, 44). They are classified into two groups: pyridoxal 5′-phosphate (PLP)-dependent and PLP-independent enzymes (9, 36).Lysine racemase (Lyr, EC 5.1.1.5) was first reported in Proteus vulgaris ATCC 4669 (19) and proposed to be involved in the lysine degradation of bacterial cells (5, 19). Catabolism of lysine occurs via two parallel pathways. In one of the pathways, δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate (AMA) are the key metabolites (5). d-Lysine catabolism proceeds through a series of cyclized intermediates which are necessary to regenerate an α-amino acid and comprise the following metabolites (AMA pathway): d-lysine→α-keto-ɛ-amino caproate→Δ¹-piperideine-2-carboxylate→pipecolate→Δ¹-piperideine-6-carboxylate→α-amino-δ-formylcaproate→α-AMA→α-ketoadipate (6, 7, 12, 27). The final product is converted to α-ketoglutarate via a series of coenzyme A derivatives and subsequently participates as an intermediate in the Krebs cycle. This pathway suggests that the biological function of d-lysine in the bacteria is that of a carbon or nitrogen source. Racemization of added l-lysine to d-lysine by whole cells of Proteus spp. and Escherichia spp. (19) and by the cell extract of Pseudomonas putida ATCC 15070 (5, 20) has been found. However, the enzyme has not been purified to homogeneity, and thus, its molecular and catalytic characteristics, including its gene structure, have not been elucidated. In this study, we explored a metagenomic library constructed from a garden soil to isolate a novel Lyr enzyme. After expression in Escherichia coli, the purified enzyme was characterized in terms of optimal pH and temperature, thermal stability, and racemization activity.     

Novel Rhamnosyltransferase Involved in Biosynthesis of Serovar 4-Specific Glycopeptidolipid from Mycobacterium avium Complex

Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.The genus Mycobacterium has a unique feature in the cell envelope that contains a multilayered structure consisting of peptidoglycan, mycolyl-arabinogalactan complex, and surface glycolipids (8, 12). It is known that these components play a role in protection from environmental stresses, such as antimicrobial agents and host immune responses (8, 12). Some of them are recognized as pathogenic factors related to mycobacterial diseases, such as tuberculosis and leprosy (8, 12). In case of nontuberculous mycobacteria that are widely distributed in the natural environment as opportunistic pathogens, glycopeptidolipids (GPLs) are abundantly present on the cell envelope as surface glycolipids (34). GPLs have a core structure in which a fatty acyl-tetrapeptide is glycosylated with 6-deoxy-talose (6-d-Tal) and O-methyl-rhamnose (O-Me-Rha) (2, 5, 13). This structure is common to all types of GPLs, and GPLs with this structure that have not undergone further glycosylation are termed non-serovar-specific GPLs (nsGPLs) (2, 5, 13). Structural diversity generated by further glycosylations, such as rhamnosylation, fucosylation, and glucosylation, is observed for the oligosaccharide portion linked to the 6-d-Tal residue of nsGPLs from Mycobacterium avium complex (MAC), a member of the nontuberculous mycobacteria consisting of two species, M. avium and M. intracellulare (2, 5, 34). Consequently, these nsGPLs with varied oligosaccharides lead to the formation of the serovar-specific GPLs (ssGPLs) that define around 30 types of MAC serovars (10).The properties of MAC serovars are known to be notably different from each other and also to be closely associated with the pathogenicity of MAC (3, 6, 18, 30, 31, 32). Various epidemiological studies indicate that serovar 4 is the most prevalent type and is also one of the serovars frequently isolated from AIDS patients (1, 20, 33, 36). Additionally, pulmonary MAC disease caused by serovar 4 is shown to exhibit a poorer prognosis than that caused by other serovars (23). With respect to host immune responses to MAC infection, serovar 4-specific GPL is reported to have characteristic features that are in contrast to those of other ssGPLs (21, 30). Structurally, serovar 4-specific GPL contains a unique oligosaccharide in which the oligosaccharide of serovar 2-specific GPL is further glycosylated with 4-O-methyl-rhamnose (4-O-Me-Rha) residue through a 1→4 linkage (Table ​(Table1)1) (24). Therefore, it is thought that the presence of 4-O-Me-Rha and its linkage position are important in exhibiting the specificity of biological activities. The biosynthesis of the oligosaccharide portion in several ssGPLs is currently being clarified (15, 16, 17, 25, 26), while that of serovar 4-specific GPL is still unresolved. In this study, we have focused on the genomic region predicted to be associated with GPL biosynthesis in the serovar 4 strain and explored the key genes responsible for the formation of 4-O-Me-Rha that might determine the specific properties of MAC serovar 4.

TABLE 1.

Oligosaccharide structures of serovar 2- and 4-specific GPLs

Species-Specific Cell Wall Binding Affinity of the S-Layer Proteins of Mosquitocidal Bacterium Bacillus sphaericus C3-41

The binding affinities and specificities of six truncated S-layer homology domain (SLH) polypeptides of mosquitocidal Bacillus sphaericus strain C3-41 with the purified cell wall sacculi have been assayed. The results indicated that the SLH polypeptide comprised of amino acids 31 to 210 was responsible for anchoring the S-layer subunits to the rigid cell wall layer via a distinct type of secondary cell wall polymer and that a motif of the recombinant SLH polypeptide comprising amino acids 152 to 210 (rSLH_152-210) was essential for the stable binding of the S-layer with the bacterial cell walls. The quantitative assays revealed that the K_D (equilibrium dissociation constant) values of rSLH_152-210 and rSLH_31-210 with purified cell wall sacculi were 1.11 × 10⁻⁶ M and 1.40 × 10⁻⁶ M, respectively. The qualitative assays demonstrated that the SLH domain of strain C3-41 could bind only to the cell walls or the cells treated with 5 M guanidinium hydrochloride of both toxic and nontoxic B. sphaericus strains but not to those from other bacteria, indicating the species-specific binding of the SLH polypeptide of B. sphaericus with bacterial cell walls.Crystalline bacterial cell surface layers (S-layers) cover the cell surfaces of many bacteria and archaea during all stages of growth and division. S-layers are composed of identical protein or glycoprotein subunits, which can assemble into two-dimensional crystalline arrays and exhibit oblique, square, or hexagonal symmetry (27, 28, 30). S-layers play key roles in the interaction between bacterial cells and environment as protective coats, molecular sieves, ion traps, cell adhesion mediators, and attachment structures (4, 21, 26, 29). Many S-layer proteins possess an N-terminal region with highly conserved amino acid sequences, which is called an S-layer homology (SLH) domain. An SLH domain contains one, two, or three repeating SLH motifs (6, 16). Each SLH motif is composed of about 55 amino acids containing 10 to 15 conserved residues (6, 17). It is suggested that the SLH domain of S-layer proteins is responsible for the binding of the S-layer subunits to the rigid cell wall layer (6, 15, 17, 19, 25), while the middle and C-terminal parts include the domains which are involved in the self-assembly process (27). In the case of Bacillaceae, secondary cell wall polymers (SCWP) are responsible for binding with SLH domains (13, 18, 19), but the SLH domains of some other bacteria have an affinity for peptidoglycan (33).Bacillus sphaericus is a gram-positive soil bacterium that represents a strictly aerobic group of mesophilic endospore-forming bacteria. Due to its specific toxicity to target mosquito larvae and the limited environment impact, some strains of this bacterium have been successfully used worldwide in integrated mosquito control programs. Previous studies revealed that some nontoxic strains of B. sphaericus contained S-layer proteins, and the S-layer proteins of B. sphaericus NCTC 9602, JG-A12, P1, and CCM 2177 have been studied in detail elsewhere (3, 7-9, 12, 22).B. sphaericus C3-41, a highly active strain isolated from a mosquito-breeding site in China in 1987, has different levels of toxicity against Culex spp., Anopheles spp., and Aedes spp. This strain belongs to the flagella serotype H5a5b, like strains 2362 and 1593 (32), and it has been developed as a commercial larvicide (JianBao) for mosquito larva control in China during the last decade (31). The genomic analysis of strain C3-41 revealed that an S-layer protein gene (slpC) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF535606","term_id":"145975949","term_text":"EF535606"}}EF535606) exists on the chromosomal genome and its sequence is identical to the S-layer protein of B. sphaericus 2362 (1, 10), composed of 3,531 bp encoding a protein of 1,176 amino acids with a molecular size of 125 kDa. Although the binding function of S-layers has been identified in some nontoxic B. sphaericus strains (6, 11), it is not well documented in mosquitocidal B. sphaericus strains, and there are few reports on the binding function of each SLH motif and the binding specificity.In this study, the binding affinities and specificities of each SLH motif of S-layer protein from mosquitocidal B. sphaericus C3-41 alone and in combination with the different cell wall preparations have been investigated, and the species-specific binding of SLH polypeptide with bacterial cell walls has been demonstrated.     

Exposure to Bioaerosols during the Growth Season of Tomatoes in an Organic Greenhouse Using Supresivit (Trichoderma harzianum) and Mycostop (Streptomyces griseoviridis)

In working environments, especially in confined spaces like greenhouses, elevated concentrations of airborne microorganisms may become a problem for workers'' health. Additionally, the use of microbial pest control agents (MPCAs) may increase exposure to microorganisms. The aim of this study was to investigate tomato growers'' exposure to naturally occurring bioaerosol components [dust, bacteria, fungi, actinomycetes, (1→3)-β-d-glucans, and endotoxin] and MPCAs applied by drip irrigation. Airborne dust was collected with filter samplers and analyzed for microorganisms by plate counts and total counts using a microscope. Analysis of (1→3)-β-d-glucan and endotoxin content was performed by kinetic, chromatic Limulus amoebocyte lysate tests. The fungal strain (Trichoderma harzianum) from the biocontrol product Supresivit was identified by PCR analysis. Measurements were performed on the day of drip irrigation and 1 week, 1 month, and 3 months after the irrigation. T. harzianum from Supresivit could be detected only on the day of treatment. Streptomyces griseoviridis, an applied MPCA, was not detected in the air during this investigation. We found that bioaerosol exposure increases during the growth season and that exposure to fungi, bacteria, and endotoxin can reach levels during the harvest period that may cause respiratory symptoms in growers. The collected data indicate that MPCAs applied by drip irrigation do not become airborne later in the season.In greenhouses, the growth conditions for plants are optimized to increase crop yield. However, the confined environment can increase growers'' exposure to bioaerosols (fungal spores, bacteria, pollen, etc.) and agents for plant protection (25). It is well documented that workers in agriculture are exposed to high concentrations of airborne microorganisms due to the handling of organic materials (31, 52), but less is known about greenhouse workers'' exposure.Inhalation of microorganisms can cause respiratory symptoms and lung function impairment (2, 4, 10, 11, 29, 56). The components (1→3)-β-d-glucan (β-glucan) and endotoxin are found to be associated with respiratory symptoms as well (8, 40, 41). Endotoxin is found predominantly on the outer surface of Gram-negative bacteria, and β-glucans are a part of the cell wall in fungi, as well as in some plants. There are strong indications that inhalation of propagules from the actinomycete Streptomyces may evoke an immunological response leading to respiratory diseases (16). Occupational asthma and rhinitis have been reported for greenhouse workers sensitized to workplace flowers or molds (42, 43, 45, 46). Another health concern is that some fungal species, e.g., the thermophilic Aspergillus fumigatus, can infect immunosuppressed people (30).Microbial pest control agents (MPCAs) are species of microorganisms that can suppress plant pests and pathogens (5). Application of biocontrol products that contain MPCAs may increase growers'' exposure to the microbial agent (9, 19, 26, 27). The biocontrol product Supresivit contains the ascomycete Trichoderma harzianum (Rifai 1969) which belongs to the family Hypocreaceae. The genus Trichoderma is beneficial in vegetable production due to its ability to outcompete plant-pathogenic microorganisms. It is also a parasite on other fungi and can induce systemic and localized resistance to pathogens in plants (21). T. harzianum is applied to foliage, seeds, or soil but can also be applied to plant wounds as a paste. Another fungal species used in biocontrol products is, e.g., Beauveria bassiana, which is applied by soil treatment or spraying. The biocontrol product Mycostop contains the actinomycete Streptomyces griseoviridis strain K61. Actinomycetes are Gram-positive bacteria that reproduce through spore release. S. griseoviridis can suppress a range of seed- and soilborne fungal pathogens through the release of antibiotics (28).The aim of this study was to investigate growers'' exposure to MPCAs and other bioaerosol components during the growth season of greenhouse tomatoes. We monitored the exposure to dust, microorganisms, endotoxin, and β-glucans from the time of application of MPCAs by drip irrigation in late winter to tomato harvest in late spring.     

Multiple Interaction Domains in FtsL,a Protein Component of the Widely Conserved Bacterial FtsLBQ Cell Division Complex

A bioinformatic analysis of nearly 400 genomes indicates that the overwhelming majority of bacteria possess homologs of the Escherichia coli proteins FtsL, FtsB, and FtsQ, three proteins essential for cell division in that bacterium. These three bitopic membrane proteins form a subcomplex in vivo, independent of the other cell division proteins. Here we analyze the domains of E. coli FtsL that are involved in the interaction with other cell division proteins and important for the assembly of the divisome. We show that FtsL, as we have found previously with FtsB, packs an enormous amount of information in its sequence for interactions with proteins upstream and downstream in the assembly pathway. Given their size, it is likely that the sole function of the complex of these two proteins is to act as a scaffold for divisome assembly.The division of an Escherichia coli cell into two daughter cells requires a complex of proteins, the divisome, to coordinate the constriction of the three layers of the Gram-negative cell envelope. In E. coli, there are 10 proteins known to be essential for cell division; in the absence of any one of these proteins, cells continue to elongate and to replicate and segregate their chromosomes but fail to divide (29). Numerous additional nonessential proteins have been identified that localize to midcell and assist in cell division (7-9, 20, 25, 34, 56, 59).A localization dependency pathway has been determined for the 10 essential division proteins (FtsZ→FtsA/ZipA→FtsK→FtsQ→FtsL/FtsB→FtsW→FtsI→FtsN), suggesting that the divisome assembles in a hierarchical manner (29). Based on this pathway, a given protein depends on the presence of all upstream proteins (to the left) for its localization and that protein is then required for the localization of the downstream division proteins (to the right). While the localization dependency pathway of cell division proteins suggests that a sequence of interactions is necessary for divisome formation, recent work using a variety of techniques reveals that a more complex web of interactions among these proteins is necessary for a functionally stable complex (6, 10, 19, 23, 24, 30-32, 40). While numerous interactions have been identified between division proteins, further work is needed to define which domains are involved and which interactions are necessary for assembly of the divisome.One subcomplex of the divisome, composed of the bitopic membrane proteins FtsB, FtsL, and FtsQ, appears to be the bridge between the predominantly cytoplasmic cell division proteins and the predominantly periplasmic cell division proteins (10). FtsB, FtsL, and FtsQ share a similar topology: short amino-terminal cytoplasmic domains and larger carboxy-terminal periplasmic domains. This tripartite complex can be divided further into a subcomplex of FtsB and FtsL, which forms in the absence of FtsQ and interacts with the downstream division proteins FtsW and FtsI in the absence of FtsQ (30). The presence of an FtsB/FtsL/FtsQ subcomplex appears to be evolutionarily conserved, as there is evidence that the homologs of FtsB, FtsL, and FtsQ in the Gram-positive bacteria Bacillus subtilis and Streptococcus pneumoniae also assemble into complexes (18, 52, 55).The assembly of the FtsB/FtsL/FtsQ complex is important for the stabilization and localization of one or more of its component proteins in both E. coli and B. subtilis (11, 16, 18, 33). In E. coli, FtsB and FtsL are codependent for their stabilization and for localization to midcell, while FtsQ does not require either FtsB or FtsL for its stabilization or localization to midcell (11, 33). Both FtsL and FtsB require FtsQ for localization to midcell, and in the absence of FtsQ the levels of full-length FtsB are significantly reduced (11, 33). The observed reduction in full-length FtsB levels that occurs in the absence of FtsQ or FtsL results from the degradation of the FtsB C terminus (33). However, the C-terminally degraded FtsB generated upon depletion of FtsQ can still interact with and stabilize FtsL (33).While a portion of the FtsB C terminus is dispensable for interaction with FtsL and for the recruitment of the downstream division proteins FtsW and FtsI, it is required for interaction with FtsQ (33). Correspondingly, the FtsQ C terminus also appears to be important for interaction with FtsB and FtsL (32, 61). The interaction between FtsB and FtsL appears to be mediated by the predicted coiled-coil motifs within the periplasmic domains of the two proteins, although only the membrane-proximal half of the FtsB coiled coil is necessary for interaction with FtsL (10, 32, 33). Additionally, the transmembrane domains of FtsB and FtsL are important for their interaction with each other, while the cytoplasmic domain of FtsL is not necessary for interaction with FtsB, which has only a short 3-amino-acid cytoplasmic domain (10, 33).In this study, we focused on the interaction domains of FtsL. We find that, as with FtsB, the C terminus of FtsL is required for the interaction of FtsQ with the FtsB/FtsL subcomplex while the cytoplasmic domain of FtsL is involved in recruitment of the downstream division proteins. Finally, we provide a comprehensive analysis of the presence of FtsB, FtsL, and FtsQ homologs among bacteria and find that the proteins of this complex are likely more widely distributed among bacteria than was previously thought.     

Treatment of Fungal Bioaerosols by a High-Temperature,Short-Time Process in a Continuous-Flow System

Airborne fungi, termed fungal bioaerosols, have received attention due to the association with public health problems and the effects on living organisms in nature. There are growing concerns that fungal bioaerosols are relevant to the occurrence of allergies, opportunistic diseases in hospitals, and outbreaks of plant diseases. The search for ways of preventing and curing the harmful effects of fungal bioaerosols has created a high demand for the study and development of an efficient method of controlling bioaerosols. However, almost all modern microbiological studies and theories have focused on microorganisms in liquid and solid phases. We investigated the thermal heating effects on fungal bioaerosols in a continuous-flow environment. Although the thermal heating process has long been a traditional method of controlling microorganisms, the effect of a continuous high-temperature, short-time (HTST) process on airborne microorganisms has not been quantitatively investigated in terms of various aerosol properties. Our experimental results show that the geometric mean diameter of the tested fungal bioaerosols decreased when they were exposed to increases in the surrounding temperature. The HTST process produced a significant decline in the (1→3)-β-d-glucan concentration of fungal bioaerosols. More than 99% of the Aspergillus versicolor and Cladosporium cladosporioides bioaerosols lost their culturability in about 0.2 s when the surrounding temperature exceeded 350°C and 400°C, respectively. The instantaneous exposure to high temperature significantly changed the surface morphology of the fungal bioaerosols.Fungi are omnipresent in indoor and outdoor environments (2, 28, 39). Most fungi are dispersed through the release of spores into the air, a phenomenon known to be driven by two kinds of energy (17): the energy provided by the fungus itself and the energy provided by external sources, such as air currents, rain, gravity, or changes in temperature and nutritional sources. Of these various mechanisms of fungal particle release, dispersal by air currents is the most prevalent mechanism for indoor fungal particles (19, 31). These airborne fungal spores, termed fungal bioaerosols, are resistant to environmental stresses and are adapted to airborne transport.Fungal bioaerosols constitute the major component of ambient airborne microorganisms (23, 50, 51). Several studies have reported that the concentration of fungal bioaerosols is relevant to the occurrence of human diseases and public health problems associated with acute toxic effects, allergies (3, 18), and asthma (4, 5, 13, 48). Fungal bioaerosols are of particular concern in healthcare facilities, where they can cause major infectious complications as opportunistic pathogens in patients with an immunodeficiency (9). For instance, invasive mycoses can affect patients undergoing high-dose chemotherapy for hematological malignancies associated with a prolonged period of neutropenia; they can also affect solid-organ transplant recipients. Despite all diagnostic and therapeutic efforts, the outcome of an invasive fungal infection is often fatal (with a mortality rate of around 50% for aspergillosis) (37). The main fungal genera responsible for these infections are as follows: Aspergillus spp., Fusarium spp., Scedosporium spp., and Mucorales spp. (10, 12, 20). However, virtually any filamentous fungus can be a pathogen (22, 41). In the hospital environment, possible sources of airborne nosocomial infection include ventilation or air-conditioning systems, decaying organic material, dust, water, food, ornamental plants, and building materials in and around hospitals (1).One of the major bioaerosols of concern is (1→3)-β-d-glucans, which comprises up to 60% of the cell wall of most fungal organisms. The (1→3)-β-d-glucans are glucose polymers with a variable molecular weight and a degree of branching (49). The results of several studies about the exposure of subjects to airborne (1→3)-β-d-glucans suggest that these agents play a role in bioaerosol-induced inflammatory responses and resulting respiratory symptoms, such as a dry cough, phlegmy cough, hoarseness, and atopy (11, 44). In addition, given that many epidemiological studies have reported that (1→3)-β-d-glucan has strong immuno-modulating effects (42, 47), (1→3)-β-d-glucan is an important parameter for exposure assessment by itself and as a surrogate component for fungi (16).To prevent the adverse health effects of fungal bioaerosols, we must ensure that control methods for airborne fungal spores are studied and developed. However, despite the necessity of controlling fungal bioaerosols, few studies have focused on such control mechanisms. The most common control methods are UV irradiation and electric ion emission. Given that UV irradiation is known to have a germicidal effect, several studies have examined how UV irradiation affects the viability of bioaerosols (35, 42). However, although UV irradiation can be easily applied by simply installing and turning on a UV lamp, the 254-nm-wavelength UV light produces ozone and radicals, which cause harmful effects to surrounding humans. Electric ion emission has also been studied as a means of controlling bioaerosols (21, 27). When the efficiency of the filter is increased, the efficacy of respiratory protection devices against bioaerosols can be enhanced. Although electric ions decrease the viability of airborne bacteria (25), the generation of the ions produces ozone, a pollutant, and also causes electric charges to accumulate on surrounding surfaces.Recently, heat treatment of indoor air using thermal processes has been considered a safe, effective, and environment-friendly method; it does not produce ozone or use ion or filter media. A thermal heating process has long been considered a suitable and reliable method for controlling microorganisms. Two types of heat are generally used, moist heat and dry heat. Moist heat utilizes steam under pressure, whereas dry heat involves high-temperature exposure without additional moisture. Several types of heat treatment are currently used for killing microorganisms. The treatments include incineration, Tyndallization, pasteurization, and autoclaving (32). However, most of these technologies were originally limited to controlling microorganisms in liquid or on material surfaces. In addition, they may not be adequate for controlling bioaerosols because the continuous surrounding environment of bioaerosols is significantly different from the conditions in liquid and on solid surfaces. Therefore, it is necessary to find adequate and practical conditions for controlling bioaerosols. Thus far, several investigations regarding the use of thermal processes against bioaerosols have been reported. Some of these studies have targeted airborne bacteria spores widely used as surrogates for biological warfare agents (8, 34), while others have focused on environmental parameters for the culture and survival of various vegetative cells (14, 29, 46). However, in these studies novel techniques for aerosols, such as measuring and analyzing aerosol particle size, distributions, and concentrations, were not utilized. In addition, to the best of our knowledge, there has been no study on the use of a thermal process for controlling fungal bioaerosols in continuous airflow. Fungal bioaerosols were found to be very resistant to a thermal environment in previous studies.In this study, we investigated the thermal heating effects on the physical, chemical, and biological properties of fungal bioaerosols using a high-temperature, short-time (HTST) sterilization process. The HTST process, a type of thermal heating process, is based on high-temperature stresses for very short periods. Although this thermal process has been used for the microbial decontamination of seeds and dried, powdered products, such as pharmaceuticals and heat-sensitive drink and food, it can be also applied to the control of an airborne microorganism in a continuous-flow system, such as a heating, ventilation, and air-conditioning system (15, 33, 38). When the fungal bioaerosol was passed through a thermal electric heating system, the fungal spores were exposed to various temperatures for short periods. Then, we examined the bioaerosol and aerosol characteristics, including aerosol size distribution, culturability, (1→3)-β-d-glucan production, and surface morphology, using a novel technique for sampling and measuring aerosols.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Identification and Characterization of gshA,a Gene Encoding the Glutamate-Cysteine Ligase in the Halophilic Archaeon Haloferax volcanii

Halophilic archaea were found to contain in their cytoplasm millimolar concentrations of γ-glutamylcysteine (γGC) instead of glutathione. Previous analysis of the genome sequence of the archaeon Halobacterium sp. strain NRC-1 has indicated the presence of a sequence homologous to sequences known to encode the glutamate-cysteine ligase GshA. We report here the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii and show that H. volcanii gshA directs in vivo the synthesis and accumulation of γGC. We also show that the H. volcanii gene when expressed in an Escherichia coli strain lacking functional GshA is able to restore synthesis of glutathione.Many organisms contain millimolar concentrations of low-molecular-weight thiol compounds that participate in a number of important biological functions involving thiol-disulfide exchanges (7). In particular, they serve to maintain an intracellular reducing environment, to provide reducing power for key reductive enzymes, to combat the effects of oxidative and disulfide stress, and to detoxify xenobiotic compounds (7). Glutathione (GSH), a cysteine-containing tripeptide, l-γ-glutamyl-l-cysteinylglycine, is the best-characterized low-molecular-weight thiol (7, 19, 21). GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (3, 21). The γ-carboxyl group of l-glutamate and the amino group of l-cysteine are ligated by the enzyme glutamylcysteine (GC) ligase EC 6.3.2.2 (GshA, encoded by gshA), which is then condensed with glycine in a reaction catalyzed by GSH synthetase (GshB, encoded by gshB) to form GSH (10, 38). GSH is found primarily in gram-negative bacteria and eukaryotes and only rarely in gram-positive bacteria (26). Fahey and coworkers showed that GSH is absent from the high-GC gram-positive actinomycetes which produce, as the major low-molecular-weight thiol, mycothiol, 1-d-myo-inosityl-2-(N-acetyl-l-cysteinyl)-amido-2-deoxy-α-d-glucopyranoside (13, 26-28, 35). GSH is also absent in Archaea. In Pyrococcus furiosus, coenzyme A SH (CoASH) is the main thiol (11), whereas in Halobacterium salinarum, γGC is the predominant thiol and the organism possesses bis-γGC reductase activity (30, 36). Similarly, Leuconostoc kimchi and Leuconostoc mesenteroides, gram-positive lactic acid bacterial species, were recently found to contain γGC rather than GSH (15). To date, these are the sole procaryotic species reported to naturally produce γGC but not GSH (6, 30). In this report, we describe the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii. Copley and Dhillon (6) previously identified, using bioinformatic tools, an open reading frame (ORF) (gene VNG1397C) in Halobacterium sp. strain NRC-1 with limited sequence relatedness to known GshA proteins (6). However, no genetic or biochemical evidence was presented to substantiate their conclusion. Here, we show that Haloferax volcanii strain DS2 (1, 25) contains an ORF that directs in vivo the synthesis and accumulation of γGC. We also show that the H. volcanii ORF, when expressed in Escherichia coli lacking functional GshA, is able to restore synthesis of GSH.     

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

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.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 smaller forespore compartment and a larger mother cell compartment, each containing 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 (11). Mother cell lysis releases the mature spore, which is dormant and capable of surviving in harsh environments for many years (16). When spores encounter an aqueous environment containing nutrients, they can germinate and grow as vegetative cells (23).Recently, interest in B. anthracis spores has intensified in response to their use as agents of bioterrorism. Of particular interest has been the outermost exosporium layer, which serves as a semipermeable barrier excluding potentially harmful macromolecules (9, 26) and as a vital first point of contact with the immune system of an infected host (14, 18, 32). The exosporium of B. anthracis and closely related species such as Bacillus cereus and Bacillus thuringiensis is a prominent structure comprised of a paracrystalline basal layer and an external hair-like nap (2). The basal layer contains approximately 20 different proteins (22, 25), while the filaments of the nap are formed by trimers of a single collagen-like glycoprotein called BclA (4, 27). The central region of BclA contains a large number of GXX repeats, mostly GTP triplets, and this region varies in length in naturally occurring strains of B. anthracis, resulting in hair-like naps of differing lengths (24, 28). Multiple copies of two O-linked oligosaccharides, a trisaccharide and a pentasaccharide, are attached to the protein component of BclA. The pentasaccharide side chains appear to be attached to threonine residues within the central region, while the trisaccharide side chains are attached to presently undefined residues in the protein (7).The precise structure of the trisaccharide side chain has not been determined, but its sequence is 3-O-methyl-l-rhamnose-l-rhamnose-N-acetylgalactosamine (GalNAc) (7). Except for a single glycosidic linkage, the structure of the pentasaccharide is known. Its structure 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-rhamnopyranosyl-(1→?)-N-acetylgalactosamine (7). Both oligosaccharides are attached to the BclA protein backbone through GalNAc residues. The pentasaccharide sugar 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose, which was given the trivial name anthrose, has been found only in B. anthracis strains and a limited number of highly pathogenic strains of B. cereus and B. thuringiensis (7, 8). For that reason, anthrose has joined other exosporium components as targets for the detection of B. anthracis spores and as new targets for therapeutic intervention in anthrax (6, 26, 29).In view of the potential importance of the BclA oligosaccharides, especially the anthrose-containing pentasaccharide, we have undertaken a comprehensive study of their biosynthesis. This effort involves identifying the biosynthetic genes for the three component sugars, anthrose, rhamnose, and GalNAc, as well as the genes involved in assembling the oligosaccharides and attaching them to the protein backbone of BclA. We recently reported the identification of a four-gene anthrose biosynthetic operon (8). A four-gene rhamnose biosynthetic operon has also been identified (24). This paper describes the identification of the gene encoding the UDP-N-acetylglucosamine (GlcNAc) 4-epimerase necessary for GalNAc biosynthesis. It also describes a surprising alternative BclA oligosaccharide biosynthetic pathway, which is active only in the absence of the UDP-GlcNAc 4-epimerase. Finally, this paper reports a biphasic pattern of expression of the gene encoding this epimerase during sporulation.     

Identification of a Terminal Rhamnopyranosyltransferase (RptA) Involved in Corynebacterium glutamicum Cell Wall Biosynthesis

A bioinformatics approach identified a putative integral membrane protein, NCgl0543, in Corynebacterium glutamicum, with 13 predicted transmembrane domains and a glycosyltransferase motif (RXXDE), features that are common to the glycosyltransferase C superfamily of glycosyltransferases. The deletion of C. glutamicum NCgl0543 resulted in a viable mutant. Further glycosyl linkage analyses of the mycolyl-arabinogalactan-peptidoglycan complex revealed a reduction of terminal rhamnopyranosyl-linked residues and, as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues, which was fully restored upon the complementation of the deletion mutant by NCgl0543. As a result, we have now termed this previously uncharacterized open reading frame, rhamnopyranosyltransferase A (rptA). Furthermore, an analysis of base-stable extractable lipids from C. glutamicum revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase.A common feature of members of the Corynebacterineae is that they possess an unusual cell wall dominated by a heteropolysaccharide termed an arabinogalactan (AG), which is linked to both mycolic acids and peptidoglycan, forming the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex (5, 10, 12, 15, 24, 25, 34). The formation of the arabinan domain in the mAGP complex, consisting mainly of α1→5, α1→3, and β1→2 glycosyl linkages, results from the subsequent addition of arabinofuranose (Araf) from the lipid-linked sugar donor β-d-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) by a set of unique membrane-bound arabinofuranosyltransferases (5, 7, 12, 18, 34).The deletion of Corynebacterium glutamicum emb (emb_Cg) (4) and a chemical analysis of the cell wall revealed a novel truncated AG structure possessing only terminal Araf residues with a corresponding loss of cell wall-bound mycolic acids (4). The presence of a novel enzyme responsible for “priming” the galactan domain for further elaboration by Emb_Cg proteins led to the identification of AftA, which belongs to the glycosyltransferase C (GT-C) superfamily (5). Recently, additional GT-C enzymes have been identified, termed AftB, which is responsible for the attachment of terminal β(1→2) Araf residues (34), and AftC, which is involved in AG branching (12) before decoration with mycolic acids, both of which are conserved within the Corynebacterineae (12, 34). It is clear that additional glycosyltransferases involved in both AG and lipoarabinomannan biosynthesis still remain to be identified. Indeed, Liu and Mushegian (22) identified 15 members of the GT-C superfamily residing in the Corynebacterineae, representing candidates involved in the biosynthesis of cell wall-related glycans and lipoglycans (22). We have continued our earlier studies (5, 12, 34) to identify genes required for the biosynthesis of the core structural elements of the mAGP complex by studying mutants of C. glutamicum and the orthologous genes and enzymes of Mycobacterium tuberculosis.A particularly interesting feature of C. glutamicum is the presence of terminal rhamnopyranose (t-Rhap) residues attached to the C2 position of α(1→5)-linked Araf residues in the arabinan domain of AG (4). The biological function of these residues remains to be clarified; nevertheless, they are a feature of the corynebacterial cell wall, and the biosynthesis of which needs to be addressed. The current paradigm of AG biosynthesis follows a linear pathway which is built upon a decaprenyl pyrophosphate lipid carrier. The unique disaccharide linker and galactan domain is synthesized by a variety of GT-A and GT-B family glycosyltransferases, all of which utilizing a nucleotide diphosphate-activated sugar substrate for transferase activity. It has been hypothesized by us (3, 5) and others (8) that a major shift in the biosynthetic machinery takes place upon the initiation of arabinan polymerization. AftA, Emb, AftC, and AftB all belong to the GT-C family of glycosyltransferases, all of which utilize DPA as the sole lipid-activated phosphosugar donor for arabinose transfer into the cell wall. Since t-Rhap residues are present in the arabinan component of the cell wall, the enzyme(s) responsible for its addition is likely to belong to the GT-C family of glycosyltransferases and, as determined through deduction, is one which utilizes a lipid-phosphate-derived rhamnose substrate similar to DPA. Herein, we present the putative protein NCgl0543 as a distinct t-Rhap of the GT-C superfamily, which is responsible for the transfer of t-Rhap residues to the arabinan domain to form the branched 2,5-linked Araf motifs of C. glutamicum. In addition, we have identified a novel decaprenyl-monophosphorylrhamnose and discuss its role in substrate presentation for AG biosynthesis in C. glutamicum.