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Bacterial SPOR domains target proteins to the divisome by binding septal peptidoglycan (PG) at sites where cell wall amidases have removed stem peptides. These PG structures are referred to as denuded glycans. Although all characterized SPOR domains bind denuded glycans, whether there are differences in affinity is not known. Here we use isothermal titration calorimetry (ITC) to determine the relative PG glycan binding affinity (Kd) of four Escherichia coli SPOR domains and one Cytophaga hutchinsonii SPOR domain. We found that the Kd values ranged from approximately 1 µM for E. coli DamXSPOR and C. hutchinsonii CHU2221SPOR to about 10 µM for E. coli FtsNSPOR. To ask whether these differences in PG binding affinity are important for SPOR domain protein function, we constructed and characterized a set of DamX and FtsN “swap” proteins. As expected, all SPOR domain swap proteins localized to the division site, and in the case of FtsN all of the heterologous SPOR domains supported cell division. But for DamX only the high-affinity SPOR domain from CHU2221 supported normal function in cell division. In summary, different SPOR domains bind denuded PG glycans with different affinity, which appears to be very important for the function of some SPOR domain proteins (e.g., DamX) but not others (e.g., FtsN).ImportanceSPOR domain proteins are prominent components of the cell division apparatus in a wide variety of bacteria. The primary function of SPOR domains is to target proteins to the division site, which they accomplish by binding to septal peptidoglycan. But whether SPOR domains have any functions beyond septal targeting is unknown. Here we show that SPOR domains vary in their PG binding affinities and, at least in the case of the E. coli cell division protein DamX, having a high-affinity SPOR domain contributes to proper function.

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to l-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA, gerAB, and gerAC. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA*) that carries a mutation in gerAA and show that it constitutively activates germination even in the presence of a wild-type copy of gerA. Using an enrichment strategy to screen for suppressors of gerA*, we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one (gerAB[E105K]) reduces the GerA complex’s ability to respond to l-alanine, while another (gerAB[F259S]) disrupts the germinant signal downstream of l-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway. IMPORTANCE Endospore formers are a broad group of bacteria that can enter dormancy upon starvation and exit dormancy upon sensing the return of nutrients. How dormant spores sense and respond to these nutrients is poorly understood. Here, we identify a key step in the signal transduction pathway that is activated after spores detect the amino acid l-alanine. We present a model that provides a more complete picture of this process that is critical for allowing dormant spores to germinate and resume growth.

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile σV is required for lysozyme resistance and σV is activated in response to lysozyme. Once activated σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here we analyze the contribution of individual genes in the csfV regulon to lysozyme resistance. Using CRISPR-Cas9 mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also use CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile.ImportanceClostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here we use CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile. We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation and PdaV, encoded as part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile, σV is required for lysozyme resistance, and σV is activated in response to lysozyme. Once activated, σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σV regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find that pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile. IMPORTANCEClostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile. We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Bacterial endospores can survive harsh environmental conditions and long-term dormancy in the absence of nutrients, but can rapidly germinate under favorable conditions. In the present study, we employed transposon sequencing (Tn-seq) to identify genes with previously uncharacterized roles in spore germination. Identified genes that encoded spore inner membrane proteins were chosen for study of defined mutants, which exhibited delayed germination in several assays in response to varying germinants. Significantly slowed release of DPA indicated that mutants were affected in Stage I of germination. Several mutants exhibited phenotypic traits consistent with failure of a GerA germinant receptor-mediated response, while others appeared to have a more general loss of response to varied germinants. Use of a gerA-lacZ transcriptional fusion and quantitative western blotting of GerAC allowed mutants to be classified based upon normal or decreased gerA transcription and normal or reduced GerA accumulation. Fourteen genes were identified to have newly described roles within Bacillus spore germination. A more complete understanding of this process can contribute to the development of better spore decontamination procedures.

Bacterial endospores produced by Bacillus and Clostridium species can remain dormant and highly resistant to environmental insults for long periods, but they can also rapidly germinate in response to a nutrient-rich environment. Multiple proteins involved in sensing and responding to nutrient germinants, initiating solute and water transport, and accomplishing spore wall degradation are associated with the membrane surrounding the spore core. In order to more fully catalog proteins that may be involved in spore germination, as well as to identify protein changes taking place during germination, unbiased proteomic analyses of membrane preparations isolated from dormant and germinated spores of Bacillus anthracis and Bacillus subtilis were undertaken. Membrane-associated proteins were fractionated by SDS-PAGE, gel slices were trypsin digested, and extracted peptides were fractionated by liquid chromatography and analyzed by matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry. More than 500 proteins were identified from each preparation. Bioinformatic methods were used to characterize proteins with regard to membrane association, cellular function, and conservation across species. Numerous proteins not previously known to be spore associated, 6 in B. subtilis and 68 in B. anthracis, were identified. Relative quantitation based on spectral counting indicated that the majority of spore membrane proteins decrease in abundance during the first 20 min of germination. The spore membranes contained several proteins thought to be involved in the transport of metal ions, a process that plays a major role in spore formation and germination. Analyses of mutant strains lacking these transport proteins implicated YloB in the accumulation of calcium within the developing forespore. IMPORTANCE Bacterial endospores can remain dormant and highly resistant to environmental insults for long periods but can also rapidly germinate in response to a nutrient-rich environment. The persistence and subsequent germination of spores contribute to their colonization of new environments and to the spread of certain diseases. Proteins of Bacillus subtilis and Bacillus anthracis were identified that are associated with the spore membrane, a position that can allow them to contribute to germination. A set of identified proteins that are predicted to carry out ion transport were examined for their contributions to spore formation, stability, and germination. Greater knowledge of spore formation and germination can contribute to the development of better decontamination strategies.

Mounting evidence suggests that d-amino acids play previously underappreciated roles in diverse organisms. In bacteria, even d-amino acids that are absent from canonical peptidoglycan (PG) may act as growth substrates, as signals, or in other functions. Given these proposed roles and the ubiquity of d-amino acids, the paucity of known d-amino-acid-responsive transcriptional control mechanisms in bacteria suggests that such regulation awaits discovery. We found that DarR, a LysR-type transcriptional regulator (LTTR), activates transcription in response to d-Asp. The d-Glu auxotrophy of a Vibrio fischerimurI::Tn mutant was suppressed, with the wild-type PG structure maintained, by a point mutation in darR This darR mutation resulted in the overexpression of an adjacent operon encoding a putative aspartate racemase, RacD, which compensated for the loss of the glutamate racemase encoded by murI Using transcriptional reporters, we found that wild-type DarR activated racD transcription in response to exogenous d-Asp but not upon the addition of l-Asp, l-Glu, or d-Glu. A DNA sequence typical of LTTR-binding sites was identified between darR and the divergently oriented racD operon, and scrambling this sequence eliminated activation of the reporter in response to d-Asp. In several proteobacteria, genes encoding LTTRs similar to DarR are linked to genes with predicted roles in d- and/or l-Asp metabolism. To test the functional similarities in another bacterium, darR and racD mutants were also generated in Acinetobacter baylyi In V. fischeri and A. baylyi, growth on d-Asp required the presence of both darR and racD Our results suggest that multiple bacteria have the ability to sense and respond to d-Asp.IMPORTANCE d-Amino acids are prevalent in the environment and are generated by organisms from all domains of life. Although some biological roles for d-amino acids are understood, in other cases, their functions remain uncertain. Given the ubiquity of d-amino acids, it seems likely that bacteria will initiate transcriptional responses to them. Elucidating d-amino acid-responsive regulators along with the genes they control will help uncover bacterial uses of d-amino acids. Here, we report the discovery of DarR, a novel LTTR in V. fischeri that mediates a transcriptional response to environmental d-Asp and underpins the catabolism of d-Asp. DarR represents the founding member of a group of bacterial homologs that we hypothesize control aspects of aspartate metabolism in response to d-Asp and/or to d-Asp-containing peptides.

Aims The goal of this work was to determine conditions under which external application of a spore germination-specific lytic enzyme (GSLE) can increase the germination efficiency of spore populations. Methods and Results The Bacillus anthracis GSLE SleB was applied to native and coat-disrupted B. anthracis and Bacillus subtilis spores. SleB was inactive on native spores but was able to trigger rapid germination of coat-disrupted spores. Using spores lacking their GSLEs or their germinant receptors to model poorly germinating spores, SleB application was able to increase colony-forming efficiency 100-fold for native spores and >1000-fold for coat-disrupted spores. SleB effects on GSLE-deficient spores were greater than on germinant receptor-deficient spores. Conclusions SleB treatment can increase spore germination efficiency. The greater effect of SleB on coat-disrupted spores is presumably due to the greater access afforded to the cortex. However, SleB apparently gained access to the cortex of native spores after they responded to nutrients and completed stage I of germination, which may result in the disruption of coat structure. Significance and Impact of the Study Treatment of spore populations with a GSLE can increase germination efficiency. Such a treatment might be utilized to increase the rapid activation of industrial spore-based products.

Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students’ understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice. Such two-tiered CIs afford an opportunity for faculty to explore the student thinking behind the common misconceptions represented by their choice of a distractor. In this study, we specifically sought to probe the misconceptions that students hold prior to beginning an introductory microbiology course (i.e., preconceptions). Faculty-learning communities at two research-intensive universities used the validated Host-Pathogen Interaction Concept Inventory (HPI-CI) to reveal student preconceptions. Our method of deep analysis involved communal review and discussion of students’ explanations for their CI answer choice. This approach provided insight valuable for curriculum development. Here the process is illustrated using one question from the HPI-CI related to the important topic of antibiotic resistance. The frequencies with which students chose particular multiple-choice responses for this question were highly correlated between institutions, implying common underlying misconceptions. Examination of student explanations using our analysis approach, coupled with group discussions within and between institutions, revealed patterns in student thinking to the participating faculty. Similar application of a two-tiered concept inventory by general microbiology instructors, either individually or in groups, at other institutions will allow them to better understand student thinking related to key concepts in their curriculum.

The infectious agent of the disease anthrax is the spore of Bacillus anthracis . Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis , and YpeB, a protein necessary for SleB stability and function. The results indicate that Δ sleB and Δ ypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup.

The study aimed at investigating Gram-positive and Gram-negative bacteria in moldy and non-moldy homes, as defined by the home’s Environmental Relative Moldiness Index (ERMI) value. The ERMI values were determined from floor dust samples in 2010 and 2011 and homes were classified into low (5) ERMI groups based on the average ERMI values as well as 2011 ERMI values. Dust and air samples were collected from the homes in 2011 and all samples were analyzed for Gram-positive and Gram-negative bacteria using QPCR assays, endotoxin by the LAL assay, and N-acetyl-muramic acid using HPLC. In addition, air samples were analyzed for culturable bacteria. When average ERMI values were considered, the concentration and load of Gram-positive bacteria determined with QPCR in house dust, but not air, were significantly greater in high ERMI homes than in low ERMI homes. Furthermore, the concentration of endotoxin, but not muramic acid, in the dust was significantly greater in high ERMI than in low ERMI homes. In contrast, when ERMI values of 2011 were considered, Gram-negative bacteria determined with QPCR in air, endotoxin in air, and muramic acid in dust were significantly greater in high ERMI homes. The results suggest that both short-term and long-term mold contamination in homes could be linked with the bacterial concentrations in house dust, however, only the current mold status was associated with bacterial concentrations in air. Although correlations were found between endotoxin and Gram-negative bacteria as well as between muramic acid and Gram-positive bacteria in the entire data set, diverging associations were observed between the different measures of bacteria and the home moldiness. It is likely that concentrations of cells obtained by QPCR and concentrations of cell wall components are not equivalent and represent too broad categories to understand the bacterial composition and sources of the home microbiota.

Rare lipoprotein A (RlpA) is a widely conserved outer membrane protein of unknown function that has previously only been studied in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. Here we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in low osmotic strength media. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from an rlpA deletion mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called 'naked glycans'). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6-anhydro N-acetylmuramic acid ends. RlpA did not degrade sacculi from wild-type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Thus, RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall.

SPOR domains are about 75 amino acids long and probably bind septal peptidoglycan during cell division. We mutagenized 33 amino acids with surface-exposed side chains in the SPOR domain from an Escherichia coli cell division protein named FtsN. The mutant SPOR domains were fused to Tat-targeted green fluorescent protein ((TT)GFP) and tested for septal localization in live E. coli cells. Lesions at the following 5 residues reduced septal localization by a factor of 3 or more: Q251, S254, W283, R285, and I313. All of these residues map to a β-sheet in the published solution structure of FtsN(SPOR). Three of the mutant proteins (Q251E, S254E, and R285A mutants) were purified and found to be defective in binding to peptidoglycan sacculi in a cosedimentation assay. These results match closely with results from a previous study of the SPOR domain from DamX, even though these two SPOR domains share

Decades of study have revealed the fine chemical structure of the bacterial peptidoglycan cell wall, but the arrangement of the peptidoglycan strands within the wall has been challenging to define. The application of electron cryotomography (ECT) and new methods for fluorescent labelling of peptidoglycan are allowing new insights into wall structure and synthesis. Two articles in this issue examine peptidoglycan structures in the model Gram-positive species Bacillus subtilis. Beeby et al. combined visualization of peptidoglycan using ECT with molecular modelling of three proposed arrangements of peptidoglycan strands to identify the model most consistent with their data. They argue convincingly for a Gram-positive wall containing multiple layers of peptidoglycan strands arranged circumferentially around the long axis of the rod-shaped cell, an arrangement similar to the single layer of peptidoglycan in similarly shaped Gram-negative cells. Tocheva et al. examined sporulating cells using ECT and fluorescence microscopy to demonstrate the continuous production of a thin layer of peptidoglycan around the developing spore as it is engulfed by the membrane of the adjacent mother cell. The presence of this peptidoglycan in the intermembrane space allows the refinement of a model for engulfment, which has been known to include peptidoglycan synthetic and lytic functions.

SPOR domains are present in thousands of bacterial proteins and probably bind septal peptidoglycan (PG), but the details of the SPOR-PG interaction have yet to be elucidated. Here we characterize the structure and function of the SPOR domain for an Escherichia coli division protein named DamX. Nuclear magnetic resonance revealed the domain comprises a four-stranded antiparallel β-sheet buttressed on one side by two α-helices. A third helix, designated α3, associates with the other face of the β-sheet, but this helix is relatively mobile. Site-directed mutagenesis revealed the face of the β-sheet that interacts with α3 is important for septal localization and binding to PG sacculi. The position and mobility of α3 suggest it might regulate PG binding, but although α3 deletion mutants still localized to the septal ring, they were too unstable to use in a PG binding assay. Finally, to assess the importance of the SPOR domain in DamX function, we constructed and characterized E. coli mutants that produced DamX proteins with SPOR domain point mutations or SPOR domain deletions. These studies revealed the SPOR domain is important for multiple activities associated with DamX: targeting the protein to the division site, conferring full resistance to the bile salt deoxycholate, improving the efficiency of cell division when DamX is produced at normal levels, and inhibiting cell division when DamX is overproduced.

Bacterial endospores possess multiple integument layers, one of which is the cortex peptidoglycan wall. The cortex is essential for the maintenance of spore core dehydration and dormancy and contains structural modifications that differentiate it from vegetative cell peptidoglycan and determine its fate during spore germination. Following the engulfment stage of sporulation, the cortex is synthesized within the intermembrane space surrounding the forespore. Proteins responsible for cortex synthesis are produced in both the forespore and mother cell compartments. While some of these proteins also contribute to vegetative cell wall synthesis, others are sporulation specific. In order for the bacterial endospore to germinate and resume metabolism, the cortex peptidoglycan must first be degraded through the action of germination-specific lytic enzymes. These enzymes are present, yet inactive, in the dormant spore and recognize the muramic-δ-lactam modification present in the cortex. Germination-specific lytic enzymes across Bacillaceae and Clostridiaceae share this specificity determinant, which ensures that the spore cortex is hydrolyzed while the vegetative cell wall remains unharmed. Bacillus species tend to possess two redundant enzymes, SleB and CwlJ, capable of sufficient cortex degradation, while the clostridia have only one, SleC. Additional enzymes are often present that cannot initiate the cortex degradation process, but which can increase the rate of release of small fragments into the medium. Between the two families, the enzymes also differ in the enzymatic activities they possess and the mechanisms acting to restrict their activation until germination has been initiated.

Heat-resistant endospore formation plays an important role in Clostridium perfringens -associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of l -arginine-poly( l -aspartic acid), to β-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium , but their role has not been defined. To determine the function of cyanophycin in C. perfringens , a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase. IMPORTANCE Clostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria.

A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3–3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3–3 cross-links as opposed to 16% 3–3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella’s environmental resistance. Published version

Three strains of lactic acid bacteria (LAB) were isolated from the faeces of apparently healthy wild Canada geese (Branta canadensis) in 2010 by cultivating faecal LAB on Rogosa SL agar under aerobic conditions. These three isolates were found to share 99.9 % gene sequence similarity of their 16S rRNA, their 16S–23S intergenic transcribed spacer region (ITS), partial 23S rRNA, rpoB, rpoC, rpoA and pheS gene sequences. However, the three strains exhibited lower levels of sequence similarity of these genetic targets to all known LAB, and the phylogenetically closest species to the geese strains were Lactobacillus casei , Lactobacillus paracasei , Lactobacillus rhamnosus and Lactobacillus saniviri . In comparison to L. casei ATCC 393T, L. paracasei ATCC 25302T, L. rhamnosus ATCC 7469T and L. saniviri DSM 24301T, the novel isolates reacted uniquely in tests for cellobiose, galactose, mannitol, citric acid, aesculin and dextrin, and gave negative results in tests for l-proline arylamidase and l-pyrrolydonyl-arylamidase, and in the Voges-Proskauer test. Biochemical tests for cellobiose, aesculin, galactose, gentiobiose, mannitol, melezitose, ribose, salicin, sucrose, trehalose, raffinose, turanose, amygdalin and arbutin could be used for differentiation between L. saniviri and the novel strains. On the basis of phenotypic and genotypic characteristics, and phylogenetic data, the three isolates represent a novel species of the genus Lactobacillus , for which the name Lactobacillus brantae sp. nov. is proposed. The type strain is SL1108T ( = ATCC BAA-2142T = LMG 26001T = DSM 23927T) and two additional strains are SL1170 and SL60106.

Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-δ-lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft. Proteins 2012;. © 2012 Wiley Periodicals, Inc.

The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic enzyme composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain. Derivatives of SleL containing both, one or no LysM domains were purified and characterized. SleL is incapable of digesting intact cortical PG of either decoated spores or purified spore sacculi. However, SleL derivatives can hydrolyse fragmented PG substrates containing muramic-δ-lactam recognition determinants. The muropeptides that result from SleL hydrolysis are the products of N-acetylglucosaminidase activity. These muropeptide products are small and readily released from the cortex matrix. Loss of the LysM domain(s) decreases both PG binding and hydrolysis activity but these domains do not appear to determine specificity for muramic-δ-lactam. When the SleL derivatives are expressed in vivo, those proteins lacking one or both LysM domains do not associate with the spore. Instead, these proteins remain in the mother cell and are apparently degraded. SleL with both LysM domains localizes to the coat or cortex of the endospore. The information revealed by elucidating the role of SleL and its domains in B. anthracis sporulation and germination is important in designing new spore decontamination methods. By exploiting germination-specific lytic enzymes, eradication techniques may be greatly simplified.

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca 2+ -dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore's integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca 2+ -DPA-induced germination. SleL is an N -acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.

SPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli . The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus , localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.

Strains of Clostridium perfringens that cause acute food poisoning have been shown to produce spores that are significantly more heat resistant than those of other strains. Previous studies demonstrated that the spore core density and the ratio of spore cortex peptidoglycan relative to the germ cell wall were factors that correlated with the heat resistance of a C. perfringens spore. To further evaluate these relationships, mutant strains of C. perfringens SM101 were constructed with null mutations in dacF, encoding a D,D-carboxypeptidase, and in the spmA-spmB operon, which is involved in spore core dehydration. The dacF mutant was shown to produce less spore cortex peptidoglycan and had a corresponding decrease in spore heat resistance. The spmA-spmB strain produced highly unstable spores with significantly lower core densities and increased heat sensitivity, which were easily destroyed during treatments affecting the spore coat layers. These results support the previous assertion that a threshold core density as well as a high ratio of cortex peptidoglycan relative to the germ cell wall contribute to the formation of a more heat-resistant spore in this species.

Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL ( yaaH) , which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N -acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N -acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL -encoded N -acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.

Pressure-induced germination and inactivation of three strains of B. cereus spores by high hydrostatic pressures between 100 and 550 MPa at 40°C for 15 min was studied. In sterile distilled water, spore germination remained stable with pressures up to 300 MPa, and increased at pressures between 300 and 500 MPa, with maximum germination and inactivation obtained at 550 MPa. Of the 3 strains, spores from the crabmeat isolate were found to be the most resistant to germination and inactivation by pressure. In fresh crabmeat the B. cereus spores were more resistant to inactivation, but germination was improved compared to spores suspended in distilled water. Fresh crabmeat inoculated with B. cereus spores, treated with high pressure (550 MPa, 40°C, 15 min) and then stored at 4 and 12°C for 31 days showed that B. cereus was not a competitive organism in the presence of crabmeat microflora. Some pressure-sensitive microflora were inactivated in the treated crabmeat, but B. cereus survived through the en...

Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that found in other species. During germination, B. anthracis released peptidoglycan fragments into the surrounding medium more quickly than some other species. A major lytic enzymatic activity was a glucosaminidase, probably YaaH, that cleaved between Nacetylglucosamine and muramic--lactam. An epimerase activity previously proposed to function on spore peptidoglycan was not detected, and it is proposed that glucosaminidase products were previously misidentified as epimerase products. Spore cortex lytic enzymes and their regulators are attractive targets for development of germination inhibitors to kill spores and for development of activators to cause loss of resistance properties for decontamination of spore release sites. Bacterial endospores are metabolically dormant and are highly resistant to many treatments that rapidly kill vegetative cells. The resistance properties are dependent on the relative dehydration of the spore core or cytoplasm and the high concentrations of calcium dipicolinic acid (DPA) and other core solutes (26). When exposed to a nutrient-rich environment, spores initiate germination and outgrowth into vegetative cells. Nutrient germinant molecules bind to Ger protein family receptors in the inner forespore membrane surrounding the spore core and trigger biophysical changes (20). The spore becomes partially rehydrated as water enters the spore core, and the calcium DPA that was in the core is released into the environment. To complete the germination process and resume metabolism (11, 24), the spore must go through a second stage, which is more biochemical (20). This involves activation of spore cortex peptidoglycan (PG) lytic enzymes, which are present in an inactive state within the dormant spore. As the cortex is degraded, the cell expands to its full size and regains full metabolic activity. Due to the key role that cortex lytic enzymes play in germination, they are potential targets for spore decontamination methods that could involve either irreversible inactivation or gratuitous activation of the enzymes. The latter would lead to premature spore germination into vegetative forms that are much more easily killed. PG consists of glycan strands with alternating residues of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that are cross-linked by peptide side chains located on the NAM residues (Fig. 1). While the PG structure in vegetative cells can vary between species, primarily in the amino acid composition of the peptide side chains, spore PG structure has been found to be highly conserved across species (3, 30). The peptide side chains contain L-alanine, D-glutamate, meso-diaminopimelic acid (Dpm), and D-alanine. The cross-linking occurs between the Dpm on one strand and the D-alanine of a tetrapeptide on an adjacent strand. The spore PG is composed of the inner, germ cell wall layer (10 to 20% of the PG), which resembles the PG of vegetative cells (6, 19), and the cortex, which has important and unique chemical modifications (2, 3, 5, 23, 28, 30). In the cortex, approximately 50% of the muramic acid residues (the residues on every alternate disaccharide) have their peptide side chains completely removed, and these NAM residues are converted to muramic--lactam. In the Bacillus species previously examined, 25% of the cortex muramic acid residues have tetrapeptide side chains, while the majority of the remaining residues have their peptides cleaved to single L-alanine residues. Both the shortening and the removal of peptide side chains cause a decrease in cross-linking

The endospores formed by strains of type A Clostridium perfringens that produce the C. perfringens enterotoxin (CPE) are known to be more resistant to heat and cold than strains that do not produce this toxin. The high heat resistance of these spores allows them to survive the cooking process, leading to a large number of food-poisoning cases each year. The relative importance of factors contributing to the establishment of heat resistance in this species is currently unknown. The present study examines the spores formed by both CPE + and CPE − strains for factors known to affect heat resistance in other species. We have found that the concentrations of DPA and metal ions, the size of the spore core, and the protoplast-to-sporoplast ratio are determining factors affecting heat resistance in these strains. While the overall thickness of the spore peptidoglycan was found to be consistent in all strains, the relative amounts of cortex and germ cell wall peptidoglycan also appear to play a role in the heat resistance of these strains.

Curli fibers are produced by some Escherichia coli cells in response to environmental stimuli. These extracellular proteins enhance the cell's ability to form biofilms on various abiotic surfaces. E. coli 0157:H7 cells readily attach to a variety of fruit and vegetable surfaces. It is not known whether the expression of curli influences the cell's ability to attach to produce surfaces. In this study, the effect of curli expression on the cell's overall hydrophobicity, charge, and ability to attach to cut and whole iceberg lettuce surfaces was examined. All strains, regardless of curli expression, attached preferentially to the cut edges of lettuce (P < 0.05). The curli-producing cells of E. coli 0157:H7 strain E0018 attached in significantly greater numbers to both cut and whole lettuce pieces than did the non-curli-producing E0018 cells (P < 0.05); however, no significant attachment differences were observed between the curli-producing and non-curli-producing cells of E. coli 0157:H7 strains 43894 and 43895. All curli-producing E. coli 0157:H7 strains were significantly more hydrophobic (P < 0.01); however, no association between the cells' hydrophobic characteristics and lettuce attachment was observed. Overall surface charge of the cells did not differ among strains or curli phenotypes. Results indicate that overall hydrophobicity and cell charge in E. coli 0157:H7 strains do not influence attachment to iceberg lettuce surfaces. The presence of curli may not have any influence on attachment of E. coli 0157:H7 cells to produce items. Additional factorsmay influence the attachment of E. coli 0157:H7 to plant surfaces and should be further examined.

SummaryRecent discoveries of regulated cell death in bacteria have led to speculation about possible benefits that apoptosis-like pathways may confer to single-celled organisms. However, establishing how these pathways provide increased ecological fitness has remained difficult to determine. Here, we report a pathway in Bacillus subtilis in which regulated cell death maintains the fidelity of sporulation through selective removal of cells that misassemble the spore envelope. The spore envelope, which protects the dormant spore’s genome from environmental insults, uses the protein SpoIVA as a scaffold for assembly. We found that disrupting envelope assembly activates a cell death pathway wherein the small protein CmpA acts as an adaptor to the AAA+ ClpXP protease to degrade SpoIVA, thereby halting sporulation and resulting in lysis of defective sporulating cells. We propose that removal of unfit cells from a population of terminally differentiating cells protects against evolutionary deterioration and ultimately loss of the sporulation program.

We determined global transcriptional responses of Escherichia coli K-12 to sulfur (S)- or nitrogen (N)-limited growth in adapted batch cultures and cultures subjected to nutrient shifts. Using two limitations helped to distinguish between nutrient-specific changes in mRNA levels and common changes related to the growth rate. Both homeostatic and slow growth responses were amplified upon shifts. This made detection of these responses more reliable and increased the number of genes that were differentially expressed. We analyzed microarray data in several ways: by determining expression changes after use of a statistical normalization algorithm, by hierarchical and k-means clustering, and by visual inspection of aligned genome images. Using these tools, we confirmed known homeostatic responses to global S limitation, which are controlled by the activators CysB and Cbl, and found that S limitation propagated into methionine metabolism, synthesis of FeS clusters, and oxidative stress. In addition, we identified several open reading frames likely to respond specifically to S availability. As predicted from the fact that the ddp operon is activated by NtrC, synthesis of cross-links between diaminopimelate residues in the murein layer was increased under N-limiting conditions, as was the proportion of tripeptides. Both of these effects may allow increased scavenging of N from the dipeptide d -alanine- d -alanine, the substrate of the Ddp system.

The Bacillus subtilis transcription factor σ E directs the expression of a regulon of 262 genes, but null mutations in only a small fraction of these genes severely impair sporulation. We have previously reported that mutations in seven σ E -controlled genes cause a mild (2- to 10-fold) defect in sporulation. In this study, we found that pairwise combinations of some of these seven mutations led to strong synthetic sporulation phenotypes, especially those involving the ytrHI operon and ybaN . Double mutants of ybaN and ytrH and of ybaN and ytrI had >10,000-fold lower sporulation efficiencies than the wild type. Thin-section electron microscopy revealed a block in cortex formation for the ybaN ytrH double mutant and coat defects for the ybaN single and ybaN ytrI double mutants. Sporulating cells of a ybaN ytrI double mutant and of a ybaN ytrHI triple mutant exhibited a pronounced loss of dipicolinic acid (DPA) between hours 8 and 24 of sporulation, in contrast to the constant levels seen for the wild type. An analysis of the spore cortex peptidoglycans of the ybaN ytrI and ybaN ytrHI mutants showed striking decreases in the levels of total muramic acid by hour 24 of sporulation. These data, along with the loss of DPA in the mutants, suggest that the developing spores were unstable and that the cortex underwent degradation late in sporulation. The existence of otherwise hidden sporulation pathways indicates that functional redundancy may mask the role of hitherto unrecognized sporulation genes.

Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic δ-lactam. Production of muramic δ-lactam in Bacillus subtilis requires removal of a peptide side chain from the N -acetylmuramic acid residue by a cwlD -encoded muramoyl- l -Alanine amidase. Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK . We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of pdaA ( yfjS ), which encodes a putative deacetylase, has recently been shown to also be required for muramic δ-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl l -Alanine amidase activity but no muramic δ-lactam formation. Expression of PdaA alone in E. coli had no effect on E. coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic δ-lactam. CwlD and PdaA are necessary and sufficient for muramic δ-lactam production, and no other B. subtilis gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate.

The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH ( ykuA ) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.

Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.

Aims: To determine the effect of sporulation temperature on Bacillus subtilis spore resistanceand spore composition.Methods and Results: Bacillus subtilis spores prepared at temperatures from 22 to 48 C hadidentical amounts of dipicolinic acid and small, acid-soluble proteins but the core water contentwas lower in spores prepared at higher temperatures. As expected from this latter finding,spores prepared at higher temperatures were more resistant to wet heat than were sporesprepared at lower temperatures. Spores prepared at higher temperatures were also moreresistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidizedwater, Sterilox. However, spores prepared at high and low temperatures exhibited nearlyidentical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores preparedat different temperatures showed very little difference in structure with only a small, albeitsignificant, increase in the percentage of muramic acid with a crosslink in spores prepared athigher temperatures. In contrast, there were readily detectable differences in the levels of coatproteins in spores prepared at different temperatures and the levels of at least one coat protein,CotA, fell significantly as the sporulation temperature increased. However, this latter changewas not due to a reduction in cotA gene expression at higher temperatures.Conclusions: The temperature of sporulation affects a number of spore properties, includingresistance to many different stress factors, and also results in significant alterations in the sporecoat and cortex composition.Significance and Impact of the Study: The precise conditions for the formation ofB. subtilis spores have a large effect on many spore properties.

A modified peptidoglycan (PG) wall is required for maintenance of spore core dehydration and the accompanying metabolic dormancy and heat resistance. Production of the spore PG depends on the cooperative expression of gene products within both the mother cell and forespore compartments of the sporangium. Structural elements that differentiate spore PG from vegetative cell PG include the presence of the modified sugar muramic-delta-lactam and a low level of peptide cross-links between the glycan strands. Detailed analyses of PG structure in dormant spores and in developing forespores of wild-type and mutant strains are providing data on factors required for introduction of these modifications and the importance of these structural elements in determining spore properties. Muramic-delta-lactam is not required for spore core dehydration but serves as a specificity factor for spore germination lytic enzymes. Cross-linking of spore PG can vary over a relatively wide range without significantly effecting spore core dehydration but does have an influence on the rate of spore germination and outgrowth. Future studies will examine how the two cells within the sporangium coordinate the production of this unique structure between themselves and how specific spore PG structural modifications are produced and participate in determining spore resistance properties.

Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein inBacillusspecies plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified inBacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. AB. anthracisstrain lacking the HtrC protease did not generate the same stable YpeB products. InB. anthracisandBacillus subtilishtrCmutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observedin vivo, and this cleavage was stimulated by Mn2+or Ca2+ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation.

Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. Although they lack flagella, C. perfringens bacteria can still migrate across surfaces using a type of gliding motility that involves the formation of filaments of bacteria lined up in an end-to-end conformation. In strain SM101, hypermotile variants are often found arising from the edges of colonies on agar plates. Hypermotile cells are longer than wild-type cells, and video microscopy of their gliding motility suggests that they form long, thin filaments that move rapidly away from a colony, analogously to swarmer cells in bacteria with flagella. To identify the cause(s) of the hypermotility phenotype, the genome sequences of normal strains and their direct hypermotile derivatives were determined and compared. Strains SM124 and SM127, hypermotile derivatives of strains SM101 and SM102, respectively, contained 10 and 6 single nucleotide polymorphisms (SNPs) relative to their parent strains. While SNPs were located in different genes in the two sets of strains, one feature in common was mutations in cell division genes, an ftsI homolog in strain SM124 ( CPR _ 1831 ) and a minE homolog in strain SM127 ( CPR _ 2104 ). Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model explaining the principles underlying the hypermotility phenotype is presented.

Rare lipoprotein A (RlpA) is a widely-conserved outer membrane protein of unknown function that has previously only been studied in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. Here we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in low osmotic strength media. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from an rlpA deletion mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called “naked glycans”). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6-anhydro N-acetylmuramic acid ends. RlpA did not degrade sacculi from wild-type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Thus, RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall.

Gross peptidoglycan structure and mechanisms of synthesis are conserved across all bacteria with differences arising in the amino acid composition of peptide side chains, the presence and composition of peptide cross bridges involved in cross-linking, lengths of glycan strands, and levels of acetylation and deacetylation of the glycan strands. Synthesis of precursor molecules utilized for polymerization of peptidoglycan strands takes place within the cell cytoplasm. Analysis of peptidoglycan synthesis in developing spores indicates that the germ cell wall is the first spore peptidoglycan synthesized and that its structure is stable through the remainder of spore development. The majority of the spore peptidoglycan, the cortex, appears to take place from the mother cell side of the inter-membrane space, being laid down in successive layers around the germ cell wall. Observations that cell wall autolysis is enhanced by ionophores or uncouplers have led to proposals that autolysins may be controlled by the proton motive force. Surface layers (S-layers) have been demonstrated to affect attachment of extracellular amylase to Bacillus stearoihermophilus, phage attachment to Lactobacillus helveticus, protection from predatory bacteria in Cauhbacter crescentus, and virulence of Campylobacter fetus. Assignment of capsule synthesis function to Cps14E genes must be made with caution, as some of these gene products also have significant similarity to proteins involved in anionic polymer synthesis or implicated in spore surface protein glycosylation. Recent studies have begun to elucidate important details of the chemical complexity of the cell wall and how it is synthesized, modified, and hydrolyzed.

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.

Clostridium difficile is a clinically important pathogen and the most common cause of hospital-acquired infectious diarrhea. Expression of the C. difficile gene csfV , which encodes σ V , an extracytoplasmic function σ factor, is induced by lysozyme, which damages the peptidoglycan of bacteria. Here we show that σ V is required for lysozyme resistance in C. difficile . Using microarray analysis, we identified the C. difficile genes whose expression is dependent upon σ V and is induced by lysozyme. Although the peptidoglycan of wild-type C. difficile is intrinsically highly deacetylated, we have found that exposure to lysozyme leads to additional peptidoglycan deacetylation. This lysozyme-induced deacetylation is dependent upon σ V . Expression of pdaV , which encodes a putative peptidoglycan deacetylase, was able to increase lysozyme resistance of a csfV mutant. The csfV mutant strain is severely attenuated compared to wild-type C. difficile in a hamster model of C. difficile -associated disease. We conclude that the σ V signal transduction system, which senses the host innate immune defense enzyme lysozyme, is required for lysozyme resistance and is necessary during C. difficile infection.

Bacterial spores are protected from the environment by a proteinaceous coat and a layer of specialized peptidoglycan called the cortex. In Bacillus subtilis , the attachment of the coat to the spore surface and the synthesis of the cortex both depend on the spore protein SpoIVA. To identify functionally important amino acids of SpoIVA, we generated and characterized strains bearing random point mutations of spoIVA that result in defects in coat and cortex formation. One mutant resembles the null mutant, as sporulating cells of this strain lack the cortex and the coat forms a swirl in the surrounding cytoplasm instead of a shell around the spore. We identified a second class of six mutants with a partial defect in spore assembly. In sporulating cells of these strains, we frequently observed swirls of mislocalized coat in addition to a coat surrounding the spore, in the same cell. Using immunofluorescence microscopy, we found that in two of these mutants, SpoIVA fails to localize to the spore, whereas in the remaining strains, localization is largely normal. These mutations identify amino acids involved in targeting of SpoIVA to the spore and in attachment of the coat. We also isolated a large set of mutants producing spores that are unable to maintain the dehydrated state. Analysis of one mutant in this class suggests that spores of this strain accumulate reduced levels of peptidoglycan with an altered structure.

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.