The Germination-Specific Lytic Enzymes SleB,CwlJ1, and CwlJ2 Each Contribute to Bacillus anthracis Spore Germination and Virulence

The bacterial spore cortex is critical for spore stability and dormancy and must be hydrolyzed by germination-specific lytic enzymes (GSLEs), which allows complete germination and vegetative cell outgrowth. We created in-frame deletions of three genes that encode GSLEs that have been shown to be active in Bacillus anthracis germination: sleB, cwlJ1, and cwlJ2. Phenotypic analysis of individual null mutations showed that the removal of any one of these genes was not sufficient to disrupt spore germination in nutrient-rich media. This finding indicates that these genes have partially redundant functions. Double and triple deletions of these genes resulted in more significant defects. Although a small subset of ΔsleB ΔcwlJ1 spores germinate with wild-type kinetics, for the overall population there is a 3-order-of-magnitude decrease in the colony-forming efficiency compared with wild-type spores. ΔsleB ΔcwlJ1 ΔcwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both ΔsleB ΔcwlJ1 and ΔsleB ΔcwlJ1 ΔcwlJ2 spores are significantly attenuated, but are not completely devoid of virulence, in a mouse model of inhalation anthrax. Although unable to germinate in standard nutrient-rich media, spores lacking SleB, CwlJ1, and CwlJ2 are able to germinate in whole blood and serum in vitro, which may explain the persistent low levels of virulence observed in mouse infections. This work contributes to our understanding of GSLE activation and function during germination. This information may result in identification of useful therapeutic targets for the disease anthrax, as well as provide insights into ways to induce the breakdown of the protective cortex layer, facilitating easier decontamination of resistant spores.Bacillus anthracis, a gram-positive spore-forming bacterium, is the causative agent of anthrax. The dormant spore form is the infectious particle and produces three different forms of the disease depending on the route of entry into a suitable host (8). When spores enter through a skin lesion and when they are ingested, they cause cutaneous and gastrointestinal anthrax, respectively. Spores entering through the lungs cause the most severe form of the disease, inhalation anthrax, which is often fatal even with aggressive antibiotic therapy (1, 8, 34). Because true pneumonias are rarely seen in victims, it is believed that inhaled spores do not germinate in the lung but are phagocytosed by alveolar macrophages and germinate intracellularly en route to the mediastinal lymph nodes, which leads to dissemination, septicemia, toxemia, and often death (1, 34). It has been shown that the spores are able to germinate and the bacteria are able to multiply inside macrophages both in cell culture and in the lungs of challenged animals (7, 11, 28, 29).Independent of the route of infection, spore germination inside a susceptible host is essential for disease. The highly stable spore form of the bacterium can remain viable under harsh environmental conditions for many decades (32). However, a spore can form a rapidly dividing vegetative cell upon entry into a host and recognition of specific chemical signals, or germinants, through specialized germinant receptors (32). The spore cortex, a thick layer of modified peptidoglycan (PG), contributes much of the spore''s environmental resistance as it is necessary to maintain dehydration of the spore core (25). This protective barrier is broken down following the activation of germination-specific lytic enzymes (GSLEs), allowing full core rehydration and cell outgrowth (32). Experimentally, germination can also be triggered by nongerminant treatments, such as lysozyme treatment, high pressure, exogenous Ca²⁺-dipicolinic acid treatment, and treatment with cationic surfactants (32). Several of these treatments likely cause spore cortex hydrolysis, triggering spore germination. This indicates the importance of cortex degradation in the spore germination process.Bacterial cell wall PG consists of polysaccharide chains of repeating N-acetylglucosamine and N-acetylmuramic acid, joined by β(1,4) glycosidic bonds (25). This basic structure is modified in several ways in spore cortex PG. In one major modification, 50% of the muramic acid residues (alternating every other residue) are converted to muramic-δ-lactam residues (25). This modification is essential for the specificity of GSLEs for degrading the cortex and prevents degradation of the bacterial cell wall during cortex hydrolysis (21).Previous work on the role of GSLEs in Bacillus subtilis and, recently, in B. anthracis has shown that the enzymes SleB and CwlJ have partially redundant roles and are necessary together for full cortex hydrolysis and spore germination (6, 14). SleB is a lytic transglycosylase that, when activated by an unknown mechanism, hydrolyzes the bond between N-acetylmuramic acid and N-acetylglucosamine (5). In both B. subtilis and B. anthracis, the sleB gene is found in a bicistronic operon with ypeB. Although the function of YpeB is not known, deletion of ypeB prevents SleB activity in spore germination, and sleB and ypeB mutants have similar phenotypes (5). Expression of both gene products is necessary for the presence of SleB in the cortex and inner membrane of mature spores (2, 5).Although no specific enzymatic activity has been attributed to CwlJ, it is required for full germination and it shares a homologous catalytic domain with SleB (20). In B. subtilis and Bacillus cereus, cwlJ is found in an operon with gerQ. Similar to the finding that ypeB is necessary for a functional SleB protein, gerQ is required for CwlJ activity (26). The B. anthracis genome contains two homologs of cwlJ (designated cwlJ1 and cwlJ2 [14]), whereas a single copy is present in B. subtilis and B. cereus. As it is in the related species, cwlJ1 is found in an operon with gerQ, but cwlJ2 is in a different locus and is not in an operon with a gerQ homolog (14). It has been shown that CwlJ is localized to the spore coat and that it is necessary for spore germination with exogenous Ca²⁺-dipicolinic acid treatment (3, 24).GSLE activation represents a critical step in the complex process of germination. The relatively small number of genes involved and the apparent essential nature of their activity make them attractive targets for new therapeutics, as well as environmental decontamination compounds. The objective of this study was to test by using genetic analysis the role of the GSLE genes sleB, cwlJ1, and cwlJ2 in B. anthracis spore germination. Mutants lacking these three genes were tested to determine their effects on in vitro germination kinetics and colony-forming efficiency. Additionally, the virulence of these mutant strains was examined by comparing mutant and wild-type spores in an in vivo mouse model of inhalational anthrax.     

Role of the gerI Operon of Bacillus cereus 569 in the Response of Spores to Germinants

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to l-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to l-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to l-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to l-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in l-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of l-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.Bacillus species have the ability, under certain nutrient stresses, to undergo a complex differentiation process resulting in the formation of a highly resistant dormant endospore (6). These spores can then persist in the environment for prolonged periods until a sensitive response mechanism detects specific environmental conditions, initiating the processes of germination and outgrowth (9, 21, 25). Germination can be initiated by a variety of agents (12), including nutrients, enzymes, or physical factors, such as abrasion or hydrostatic pressure.The molecular genetics of spore germination has been most extensively studied in Bacillus subtilis 168 (21). B. subtilis spores can be triggered to germinate in response to either l-alanine or to a combination (29) of asparagine, glucose, fructose, and potassium ions (AGFK). Mutants of B. subtilis which are defective in germination responses to one or to both types of germinant have been isolated previously (20, 27). Analysis of these mutants suggests that the germinants interact with separate germinant-specific complexes within the spore (21). This in some way leads to activation of components of the germination apparatus common to both responses, such as germination-specific cortex lytic enzymes, leading in turn to complete germination of the spore (10, 22). The mutations within the gerA operon of B. subtilis specifically block germination initiated by l-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded in the operon suggest that these proteins could be membrane associated, and they are the most likely candidates to represent the germinant receptor for alanine (21).The amino acid l-alanine has been identified as a common but not universal germinant in a variety of Bacillus species, often requiring the presence of adjuncts such as electrolytes and sugars. Ribosides, such as inosine, represent another type of common germinant, although many species are unable to germinate rapidly in response to these without the addition of l-alanine (9).The food-borne pathogen Bacillus cereus is a major cause of food poisoning of an emetic and diarrheal type (13, 16). The germination and growth of Bacillus cereus spores during food storage can lead to food spoilage and the potential to cause food poisoning (16). B. cereus has been shown to germinate in response to l-alanine and to ribosides (11, 18, 23). Spore germination can be triggered by l-alanine alone, but at high spore densities this response becomes inhibited by d-alanine, generated by the alanine racemase activity associated with the spores (8, 11). This auto-inhibition of l-alanine germination can be reduced by the inclusion of a racemase inhibitor (O-carbamyl-d-serine) with the germinating spores (11).Inosine is the most effective riboside germinant for B. cereus T, while adenosine and guanosine are less potent (28). The rate of riboside-triggered germination has been reported to be enhanced dramatically by the addition of l-alanine (18). It is unclear whether ribosides can act as a sole germinant, or whether there is an absolute requirement for l-alanine (28).An attempt has been made to analyze genetically the molecular components of the germination apparatus in B. cereus in order to dissect the germination responses of this species and to determine whether riboside-induced germination involves components related to those already described for amino acid and sugar germinants in B. subtilis.     

SleC Is Essential for Germination of Clostridium difficile Spores in Nutrient-Rich Medium Supplemented with the Bile Salt Taurocholate

Clostridium difficile is the major cause of infectious diarrhea and a major burden to health care services. The ability of this organism to form endospores plays a pivotal role in infection and disease transmission. Spores are highly resistant to many forms of disinfection and thus are able to persist on hospital surfaces and disseminate infection. In order to cause disease, the spores must germinate and the organism must grow vegetatively. Spore germination in Bacillus is well understood, and genes important for this process have recently been identified in Clostridium perfringens; however, little is known about C. difficile. Apparent homologues of the spore cortex lytic enzyme genes cwlJ and sleB (Bacillus subtilis) and sleC (C. perfringens) are present in the C. difficile genome, and we describe inactivation of these homologues in C. difficile 630Δerm and a B1/NAP1/027 clinical isolate. Spores of a sleC mutant were unable to form colonies when germination was induced with taurocholate, although decoated sleC spores formed the same number of heat-resistant colonies as the parental control, even in the absence of germinants. This suggests that sleC is absolutely required for conversion of spores to vegetative cells, in contrast to CD3563 (a cwlJ/sleB homologue), inactivation of which had no effect on germination and outgrowth of C. difficile spores under the same conditions. The B1/NAP1/027 strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 was found to sporulate more slowly and produce fewer spores than 630Δerm. Furthermore, fewer {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic C. difficile isolates.The Gram-positive anaerobe Clostridium difficile causes diarrheal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis (8, 30). This organism is resistant to various broad-spectrum antibiotics and capitalizes on disruption of the normal intestinal flora to colonize and cause disease symptoms through the action of toxins A and B (16, 40). While these toxins are the principal virulence factors, the ability of the organism to produce endospores is necessary for disease transmission.Clostridial spores are extremely resistant to all kinds of chemical and physical agents and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants (35). Thus, the spores shed in fecal matter are very difficult to eradicate and can persist on contaminated surfaces in health care facilities for extended periods of time (35). This leads to infection or reinfection of cohabitating individuals through inadvertent ingestion of infected material (10, 32). Once in the anaerobic environment of the gut, spores presumably germinate to form toxin-producing vegetative cells and, in susceptible individuals, diarrheal disease.Spore germination is defined as the events that result in the irreversible loss of spore characteristics. However, current mechanistic knowledge of the germination process is based principally on data derived from studying Bacillus subtilis. Little is known about spore germination in clostridia and, in particular, in C. difficile. Germination is initiated when the bacterial spore senses specific effectors, termed germinants. These effectors can include nutrients, cationic surfactants, peptidoglycan, and a 1:1 chelate of pyridine-2,6-dicarboxylic acid (dipicolinic acid) and Ca²⁺ (CaDPA) (23, 34, 36). Spores of B. subtilis can germinate in response to nutrients through the participation of three sensory receptors located in the spore inner membrane, GerA, GerB, and GerK (23). After activation, the events include the release of monovalent cations (H⁺, K⁺, and Na⁺) and CaDPA (accounting for approximately 10% of the spore dry weight) (36). The third major step in germination involves hydrolysis of the spore peptidoglycan (PG) cortex. It is during this hydrolysis that the previously low water content of the spore is restored to the water content of a normal vegetative cell and the core is able to expand, which in turn allows enzyme activity, metabolism, and spore outgrowth (36).CwlJ and SleB are two specific spore cortex-lytic enzymes (SCLEs) involved in Bacillus cortex hydrolysis, which break down PG containing muramic-δ-lactam (28). SleB has been shown to localize in both the inner and outer layers of B. subtilis spores through interaction of the enzyme peptidoglycan-binding motif and the δ-lactam structure of the cortex (7, 19) and in association with YpeB, which is required for sleB expression during sporulation (4, 7). SleB is a lytic transglycosylase muramidase, but so far its mode of activation is unknown (21). CwlJ is localized to the spore coat during sporulation (3) and is required for CaDPA-induced germination in B. subtilis. Activation can be due to either CaDPA released from the spore core at the onset of germination or exogenous CaDPA (22). Neither enzyme is individually essential for complete cortex hydrolysis during nutrient germination, although inactivation of both cwlJ and sleB in B. subtilis results in a spore unable to complete this process (15). The role of SleL has recently been studied in Bacillus anthracis. Mutants unable to produce this enzyme are still able to germinate, but the process is retarded (18).The SCLEs of Clostridium are less well studied than those of Bacillus. The SCLEs SleC (20) and SleM (6) have been identified in Clostridium perfringens, and a recent study demonstrated that SleC is required during germination for complete cortex hydrolysis (26). Although SleM can degrade spore cortex peptidoglycan and inactivation of both sleC and sleM decreased the ability of spores to germinate more than inactivation of sleC alone did, SleM was not essential (26). It has also been shown that the germination-specific serine protease CspB is essential for cortex hydrolysis and converts the inactive pro-SleC found in dormant spores to an active enzyme (24). So far, there has been no detailed study of any gene responsible for spore germination in C. difficile, although genes showing homology to cwlJ and sleB of B. subtilis (CD3563) and sleC of C. perfringens (CD0551) have now been identified in the C. difficile 630 genome (33).With germinant receptors in C. difficile yet to be identified, the mechanism by which the spores sense a suitable environment for germination is unclear. Recent studies have suggested that this process may involve the interaction of C. difficile with bile. Taurocholate has been shown to enhance recovery of C. difficile spores in nutrient-rich medium (42), and it has been proposed that glycine and taurocholate act as cogerminants (38), while chenodeoxycholate inhibits C. difficile spore germination (39).The emergence of C. difficile B1/NAP1/027 strains has increased the burden on health care services worldwide. Such strains have been shown to produce higher levels of toxin in the laboratory than many other types of strains (41), although the mechanism behind this production is not fully understood. However, while the observed higher levels of toxin production is doubtless important, perhaps the recent attention given to B1/NAP1/027 strains has focused too much on toxins. As spores represent the infectious stage of C. difficile, processes such as spore germination may also contribute to the greater virulence of these strains. In this study we evaluated the sporulation and germination efficiencies of an “epidemic” B1/NAP1/027 C. difficile strain ({"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, isolated from the Stoke Mandeville outbreak in 2004 and 2005) and the “nonepidemic” strain 630Δerm (14). We then constructed strains with mutations in CD3563 (a cwlJ/sleB homologue) and a sleC homologue to analyze the role of these genes in the germination of C. difficile spores.     

Peptidoglycan Structural Dynamics during Germination of Bacillus subtilis 168 Endospores

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant l-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic δ-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.The extreme heat resistance of dormant bacterial endospores has made them an important problem in the production of safe foodstuffs (3). The spore cell wall peptidoglycan is considered to play a major role in the maintenance of heat resistance and dormancy (6). Bacillus subtilis spore peptidoglycan is composed of two layers. A thin, inner layer called the primordial cell wall retains the basic vegetative cell peptidoglycan structure. The primordial cell wall represents 2 to 4% of the total endospore peptidoglycan, is not digested during germination, and serves as the initial cell wall during outgrowth (2, 5, 25, 29). The outer thick layer of peptidoglycan, known as the cortex, is characterized by several unique spore-specific features. Approximately 50% of the muramic acid residues in the glycan strands are present in the δ-lactam form (2, 24). Muramic acid side chains are composed of 26 and 23% of tetrapeptide and single l-alanine, respectively (2).Despite their extreme dormancy and thermostability, bacterial endospores retain an alert sensory mechanism enabling them to respond within minutes to the presence of specific germinants. Spores of B. subtilis respond to at least two different types of germinative stimuli: (i) l-alanine and (ii) a combination of l-asparagine, glucose, fructose, and KCl (AGFK) (34). The germination response is initiated by the interaction of a receptor protein with specific germinants which triggers the loss of spore-specific properties and the transformation of a dormant resistant bacterial spore into a metabolically active vegetative cell. The germination process is characterized by sequential, interrelated biochemical events. The specific hydrolysis of peptidoglycan in the spore cortex layer is an essential event in germination (2, 25). Its degradation removes the physical constraints of the cortex and allows core expansion and outgrowth (9, 25). As a consequence of cortex hydrolysis, peptidoglycan fragments can be detected in the germination exudate (13, 33).A number of bacterial spore germination-specific cortex-lytic enzymes (GSLEs) have been reported to be involved in cortex hydrolysis (9, 18–20). A gene homologous to that encoding the GSLE from Bacillus cereus has been identified and inactivated in B. subtilis, and the resulting mutant germinates more slowly than the wild type (22). Recently a germination-specific muramidase isolated from a germination extract of Clostridium perfringens S40 has been purified and characterized (4).GSLEs have a high substrate specificity, requiring intact spore cortex for activity (9, 23). The muramidase from C. perfringens S40, however, hydrolyzes cortical fragments but has a strict requirement for the presence of the muramic δ-lactam residues (4). Thus, the GSLEs are highly specialized and may exist as proforms which are specifically activated during germination (9).Very little is known about the mechanism by which the cortex is hydrolyzed during germination and the autolytic enzymes involved. Muropeptide analysis provides a method for fine chemical structural determination of spore cortex (2, 24, 25). In this paper, we report the use of muropeptide analysis to determine the peptidoglycan structural dynamics which occur during spore germination of B. subtilis 168 and the evidence for a number of different enzyme activities.     

Nuclear Dynamics during Germination,Conidiation, and Hyphal Fusion of Fusarium oxysporum

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.Fusarium oxysporum is a soilborne pathogen that causes substantial losses in a wide variety of crops (12) and has been reported as an emerging human pathogen (36, 38). Similar to other fungal pathogens (18), the early stages of interaction between F. oxysporum and the host are crucial for the outcome of infection (11). Key processes occurring during these initial stages include spore germination, adhesion to the host surface, establishment of hyphal networks through vegetative hyphal fusion, differentiation of infection hyphae, and penetration of the host (53). Surprisingly, very little is known about the cytology of basic processes, such as spore germination and hyphal development, which play key roles during infection by F. oxysporum.F. oxysporum produces three types of asexual spores: microconidia, macroconidia, and chlamydospores (9, 26). Germination usually represents the first step in the colonization of a new environment, including the host. Once dormancy is broken, spores undergo a defined set of morphogenetic changes that lead to the establishment of a polarized growth axis and the emergence of one or multiple germ tubes (reviewed by d''Enfert and Hardham [10, 19]). In certain fungi, such as Aspergillus nidulans, germ tube emergence and septum formation are subject to precise spatial controls and are tightly coordinated with nuclear division (20, 22, 34, 42, 54). In contrast, in spores from other filamentous fungi, such as macroconidia of Fusarium graminearum, nuclear division is not required for the emergence of germ tubes (21, 48). During hyphal growth, multinucleate fungi display distinct mitotic patterns, such as asynchronous nuclear division in Neurospora crassa and Ashbya gossypii (15, 16, 29, 30, 33, 49), parasynchronous in A. nidulans (7, 15, 23, 46), and synchronous in Ceratocystis fagacearum (1, 15).Vegetative hyphal fusion, or anastomosis, is a common developmental process during the life cycle of filamentous fungi that is thought to serve important functions in intrahyphal communication, nutrient transport, and colony homeostasis (41). F. oxysporum undergoes anastomosis (8, 25, 32, 40), and although this process is not strictly required for plant infection, it appears to contribute to efficient colonization of the root surface (39).The aim of this study was to explore nuclear dynamics during different developmental stages of F. oxysporum that are of key relevance during the establishment of infection. They include germination of microconidia, vegetative hyphal development, and conidiation, as well as vegetative hyphal fusion during colony establishment. Fusion PCR-mediated gene targeting (55) was used to C-terminally label histone H1 in F. oxysporum (FoH1) with either green fluorescent protein (GFP) or the cherry variant (ChFP), allowing us to perform, for the first time, live-cell analysis of nuclear dynamics in this species. Our study revealed distinct patterns of nuclear divisions in F. oxysporum. Moreover, we report, for the first time in an ascomycete, that hyphal fusion initiates a series of nuclear events, including mitosis in the fusing hypha and nuclear migration into the receptor hypha, followed by degradation of the resident nucleus.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by d,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent d,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.Peptidoglycan (PG) is the structural element of the bacterial cell wall which determines cell shape and which resists the turgor pressure within the cell. The bacterial endospores produced by species of Bacillus, Clostridium, and several other bacterial genera are modified cells that are able to survive long periods and extreme conditions in a dormant, relatively dehydrated state. The PG wall within the endospore is required for maintenance of the dehydrated state (10, 11), which is the major determinant of spore heat resistance (2, 17, 22). Spore PG appears to be comprised of two distinct though contiguous layers. The thin inner layer, the germ cell wall, appears to have a structure similar to that of the vegetative wall and serves as the initial cell wall of the germinated spore (1, 20, 21, 31). The thicker outer layer, the spore cortex, has a modified structure which may determine its ability to carry out roles specific to the spore, and is rapidly degraded during spore germination (1, 20, 35, 37). The most dramatic of the cortex structural modifications results in partial cleavage or complete removal of ∼75% of the peptide side chains from the glycan strands. Loss of these peptides limits the cross-linking potential of the PG and results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20, 36). This low degree of cross-linking has been predicted to give spore PG a flexibility that allows it to have a role in attainment of spore core dehydration (14, 34) in addition to its clear role in maintenance of dehydration. We are studying the structure and mechanism of synthesis of spore PG in an attempt to discern the roles of this structure and its individual components in determining spore properties.A family of proteins called the penicillin-binding proteins (PBPs) polymerizes PG on the external surface of the cell membrane (reviewed in reference 7). The high-molecular-weight (high-MW) members of this family (generally ≥60 kDa) carry the transglycosylase and transpeptidase activities involved in polymerization and cross-linking of the glycan strands. The low-MW PBPs have commonly been found to possess d,d-carboxypeptidase activity. This activity can remove the terminal d-alanine of the peptide side chains and thereby prevent the side chain from serving as a donor in the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes: dacA (33), dacB (4), dacC (19), dacF (38), pbpE (23), and pbpX (accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z99112","term_id":"32468745","term_text":"Z99112"}}Z99112). The four dac gene products exhibit very high sequence similarity to proven d,d-carboxypeptidases, and this activity has been demonstrated in vitro for the dacA and dacB products, PBP5 (12) and PBP5* (32), respectively. The sequences of the pbpE and pbpX products are more distantly related, and no activity has yet been established or ruled out for them.PBP5 is the major penicillin-binding and d,d-carboxypeptidase activity found in vegetative cells (12). Although dacA expression declines significantly during sporulation, a significant amount of PBP5 remains during the time of spore PG synthesis (29). A dacA-null mutation results in no obvious effects on vegetative growth, sporulation, spore characteristics, or spore germination (3, 33). However, loss of PBP5 does result in a reduction of cleavage of peptide side chains from the tetrapeptide to the tripeptide form in the spore PG (20). PBP5* is expressed only during sporulation and only in the mother cell compartment of the sporangium, under the control of the RNA polymerase ς^E subunit (4, 5, 28, 29). A dacB-null mutation leading to loss of this d,d-carboxypeptidase results in a fourfold increase in the effective cross-linking of the spore PG (1, 20, 22). This structural change is accompanied by only slight decreases in spore core dehydration and heat resistance (3, 22). The suspected d,d-carboxypeptidase activities of the products of the dacC and dacF genes have not been demonstrated. The latter two genes are expressed only during the postexponential growth phase: dacC is expressed during early stationary phase under the control of ς^H (19) and dacF is expressed only within the forespore under the control of ς^F (27, 38). Null mutations effecting either gene result in no obvious phenotype and no change in spore PG structure (19, 38).The multiplicity of these proteins in sporulating cells and the lack of effect of loss of some of them suggested redundancy of function among these proteins, a situation observed previously with PBPs of a high-MW class (25, 30, 39). In order to examine this possibility we have constructed mutants lacking multiple low-MW PBPs and have examined their sporulation efficiency, spore PG structure, spore heat resistance and wet density, and spore germination and outgrowth. The present study demonstrates a role for the dacF gene product in synthesis of spore PG, and we also present a model for the roles of the dacB and dacF gene products in spore PG formation.     

Identification and Validation of Specific Markers of Bacillus anthracis Spores by Proteomics and Genomics Approaches

Bacillus anthracis is the causative bacteria of anthrax, an acute and often fatal disease in humans. The infectious agent, the spore, represents a real bioterrorism threat and its specific identification is crucial. However, because of the high genomic relatedness within the Bacillus cereus group, it is still a real challenge to identify B. anthracis spores confidently. Mass spectrometry-based tools represent a powerful approach to the efficient discovery and identification of such protein markers. Here we undertook comparative proteomics analyses of Bacillus anthracis, cereus and thuringiensis spores to identify proteoforms unique to B. anthracis. The marker discovery pipeline developed combined peptide- and protein-centric approaches using liquid chromatography coupled to tandem mass spectrometry experiments using a high resolution/high mass accuracy LTQ-Orbitrap instrument. By combining these data with those from complementary bioinformatics approaches, we were able to highlight a dozen novel proteins consistently observed across all the investigated B. anthracis spores while being absent in B. cereus/thuringiensis spores. To further demonstrate the relevance of these markers and their strict specificity to B. anthracis, the number of strains studied was extended to 55, by including closely related strains such as B. thuringiensis 9727, and above all the B. cereus biovar anthracis CI, CA strains that possess pXO1- and pXO2-like plasmids. Under these conditions, the combination of proteomics and genomics approaches confirms the pertinence of 11 markers. Genes encoding these 11 markers are located on the chromosome, which provides additional targets complementary to the commonly used plasmid-encoded markers. Last but not least, we also report the development of a targeted liquid chromatography coupled to tandem mass spectrometry method involving the selection reaction monitoring mode for the monitoring of the 4 most suitable protein markers. Within a proof-of-concept study, we demonstrate the value of this approach for the further high throughput and specific detection of B. anthracis spores within complex samples.Bacillus anthracis is a highly virulent bacterium, which is the etiologic agent of anthrax, an acute and often lethal disease of animals and humans (1). According to the Centers for Disease Control and Prevention, B. anthracis is classified as a category A agent, the highest rank of potential bioterrorism agents (http://www.bt.cdc.gov/agent/agentlist-category.asp). The infectious agent of anthrax, the spore, was used as a bioterrorism weapon in 2001 in the United States when mailed letters containing B. anthracis spores caused 22 cases of inhalational and/or cutaneous anthrax, five of which were lethal (2). These events have emphasized the need for rapid and accurate detection of B. anthracis spores.Bacillus anthracis is a member of the genus Bacillus, Gram-positive, rod-shaped bacteria characterized by the ability to form endospores under aerobic or facultative anaerobic conditions (3). The genus Bacillus is a widely heterogeneous group encompassing 268 validly described species to date (http://www.bacterio.net/b/bacillus.html, last accessed on August 9^th 2013). B. anthracis is part of the B. cereus group which consists of six distinct species: B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 5). The latter three species are generally regarded as nonpathogenic whereas B. cereus and B. thuringiensis could be opportunistic or pathogenic to mammals or insects (5, 6). B. cereus is a ubiquitous species that lives in soil but is also found in foods of plant and animal origin, such as dairy products (7). Its occurrence has also been linked to food poisoning and it can cause diarrhea and vomiting (6, 8). B. thuringiensis is primarily an insect pathogen, also present in soil, and often used as a biopesticide (9).B. anthracis is highly monomorphic, that is, shows little genetic variation (10), and primarily exists in the environment as a highly stable, dormant spore in the soil (1). Specific identification of B. anthracis is challenging because of its high genetic similarity (sequence similarity >99%) with B. cereus and B. thuringiensis (5, 11). The fact that these closely related species are rather omnipresent in the environment further complicates identification of B. anthracis. The main difference among these three species is the presence in B. anthracis of the two virulence plasmids pXO1 and pXO2 (1), which are responsible for its pathogenicity. pXO1 encodes a tripartite toxin (protective antigen (PA), lethal factor (LF), and edema factor (EF)) which causes edema and death (1), whereas pXO2 encodes a poly-γ-d-glutamate capsule which protects the organism from phagocytosis (1). B. anthracis identification often relies on the detection of the genes encoded by these two plasmids via nucleic acid-based assays (12–14). Nevertheless, the occasionally observed loss of the pXO2 plasmid within environmental species may impair the robustness of detection (1). In addition, in recent years a series of findings has shown that the presence of pXO1 and pXO2 is not a unique feature of B. anthracis. Indeed, Hu et al. have demonstrated that ∼7% of B. cereus/B. thuringiensis species can have a pXO1-like plasmid and ∼1.5% a pXO2-like plasmid (15). This was particularly underlined for some virulent B. cereus strains (i.e. B. cereus strains G9241, B. cereus biovar anthracis strains CA and CI) (16–20).Because of these potential drawbacks, the use of chromosome-encoded genes would be preferable for the specific detection of B. anthracis. Such genes (rpoB, gyrA, gyrB, plcR, BA5345, and BA813) have been reported as potential markers (21–25), but concerns have also been raised about their ability to discriminate B. anthracis efficiently from closely related B. cereus strains (26). Ahmod et al. have recently pointed out, by in silico database analysis, that a specific sequence deletion (indel) occurs in the yeaC gene and exploited it for the specific identification of B. anthracis (27). Nevertheless, a few B. anthracis strains (e.g. B. anthracis A1055) do not have this specific deletion and so may lead to false-negative results (27).In the last few years, protein profiling by MS, essentially based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS), has emerged as an alternative (or a complement) to genotypic or phenotypic methods for the fast and efficient identification of microorganisms (28, 29). Such an approach is based on the reproducible acquisition of global bacterial protein fingerprints/patterns. The combination of MS-based protein patterns and chemometric/bioinformatic tools has been demonstrated to efficiently differentiate members of the B. cereus group from other Bacillus species (30). However, the specific discrimination of B. anthracis from the closely related B. cereus and B. thuringiensis remains difficult (30). This study of Lasch and coworkers, performed on vegetative cells, identified a few ribosomal and spore proteins as being responsible for this clustering (30). Closer inspection of the data revealed that B. anthracis identification was essentially based on one particular isoform of the small acid-soluble spore protein B (SASP-B)¹ (30–34), which is exclusively expressed in spores, as the samples were shown to contain residual spores. However, the specificity of SASP-B has recently been questioned as the published genomes of B. cereus biovar anthracis CI and B. thuringiensis BGSC 4CC1 strains have been shown to share the same SASP-B isoform as B. anthracis (35). Altogether these results underline that the quest for specific markers of B. anthracis needs to be pursued.Mass spectrometry also represents a powerful tool for the discovery and identification of protein markers (36, 37). In the case of B. anthracis, this approach has more commonly been used for the comprehensive characterization of given bacterial proteomes. For example, the proteome of vegetative cells with variable plasmid contents has been extensively studied (38–40), as the proteomes of mature spores (41, 42) and of germinating spores (43, 44). Only one recent study, based on a proteo-genomic approach, was initiated to identify protein markers of B. anthracis (45). In this work, potential markers were characterized but using a very limited number of B. cereus group strains (three B. cereus and two B. thuringiensis). Moreover, this study was done on vegetative cells, whereas the spore proteome is drastically different. To our knowledge, no study has characterized and validated relevant protein markers specific to B. anthracis spores, which constitute the dissemination form of B. anthracis and are often targeted by first-line immunodetection methods (46).Here we report comparative proteomics analyses of Bacillus anthracis/cereus/thuringiensis spores, undertaken to identify proteoforms unique to B. anthracis. Preliminary identification was performed on a limited set of Bacillus species both at the peptide (after enzymatic digestion) and protein levels by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a high resolution/high mass accuracy LTQ-Orbitrap instrument. The pertinence of 11 markers was further demonstrated using proteomics and genomics approaches on a representative larger set of up to 55 different strains, including the closely related B. cereus biovar anthracis CI, CA, and B. thuringiensis 9727. Lastly, as a proof-of-concept study, we also report for four B. anthracis markers the implementation of a targeted LC-MS/MS method using selected reaction monitoring (SRM), based on the extension of a previous one focused on SASP-B (35). Preliminary results regarding method usefulness for the high throughput and accurate detection of B. anthracis spores in complex samples were also obtained and will be reported herein.