Mining transcriptome data: Utilization of environmentally regulated promoters for protein expression and purification in Clostridium perfringens.

Clostridium perfringens is a Gram-positive pathogen with low GC content. To identify genes that are transcribed at higher levels when the bacteria grow on a surface, we used RNA-seq in a previous study to measure global transcript levels of cells grown in three types of media on both plates and in liquid culture. We found the arcABDC-argR operon is induced >1000-fold when the cells were grown on plates than in liquid brain heart infusion (BHI). In addition, the pyrBICFZDE operon was transcribed >1000-fold higher in liquid BHI than on plates. Biochemical analysis of C. perfringens proteins is usually accomplished by cloning and expressing the relevant genes in Escherichia coli, a Gram-negative bacterium. Here we utilize both the arcA and pyrB promoters to express and purify proteins from C. perfringens plate and liquid-grown cultures, respectively. Three mg of the His-tagged cytoplasmic protein PilM were obtained when the pilM gene was expressed in cells grown on 10 BHI plates using the arcA promoter. Using the pyrB promoter, 0.85 mg of the C. perfringens His-tagged secreted toxin collagenase was purified from the culture supernatant of 500 ml of cells grown in liquid BHI. In the process of constructing clones, we found we can transform C. perfringens strain HN13 directly with DNA from an in vitro ligation mix, bypassing E. coli. These environmentally regulated promoters can be used to express clostridial or other low GC content genes for protein purification without the addition of an inducer molecule.

Mechanical limitation of bacterial motility mediated by growing cell chains.

Contrasting most known bacterial motility mechanisms, a bacterial sliding motility discovered in at least two gram-positive bacterial families does not depend on designated motors. Instead, the cells maintain end-to-end connections following cell divisions to form long chains and exploit cell growth and division to push the cells forward. To investigate the dynamics of this motility mechanism, we constructed a mechanical model that depicts the interplay of the forces acting on and between the cells comprising the chain. Due to the exponential growth of individual cells, the tips of the chains can, in principle, accelerate to speeds faster than any known single-cell motility mechanism can achieve. However, analysis of the mechanical model shows that the exponential acceleration comes at the cost of an exponential buildup in mechanical stress in the chain, making overly long chains prone to breakage. Additionally, the mechanical model reveals that the dynamics of the chain expansion hinges on a single non-dimensional parameter. Perturbation analysis of the mechanical model further predicts the critical stress leading to chain breakage and its dependence on the non-dimensional parameter. Finally, we developed a simplistic population-expansion model that uses the predicted breaking behavior to estimate the physical limit of chain-mediated population expansion. Predictions from the models provide critical insights into how this motility depends on key physical properties of the cell and the substrate. Overall, our models present a generically applicable theoretical framework for cell-chain-mediated bacterial sliding motility and provide guidance for future experimental studies on such motility.

Holin-Dependent Secretion of the Large Clostridial Toxin TpeL by Clostridium perfringens.

Large clostridial toxins (LCTs) are secreted virulence factors found in several species, including Clostridioides difficile, Clostridium perfringens, Paeniclostridium sordellii, and Clostridium novyi LCTs are large toxins that lack a secretion signal sequence, and studies by others have shown that the LCTs of C. difficile, TcdA and TcdB, require a holin-like protein, TcdE, for secretion. The TcdE gene is located on the pathogenicity locus (PaLoc) of C. difficile, and holin-encoding genes are also present in the LCT-encoded PaLocs from P. sordellii and C. perfringens However, the holin (TpeE) associated with the C. perfringens LCT TpeL has no homology and a different membrane topology than TcdE. In addition, TpeE has a membrane topology identical to that of the TatA protein, which is the core of the twin-arginine translocation (Tat) secretion system. To determine if TpeE was necessary and sufficient to secrete TpeL, the genes from a type C strain of C. perfringens were expressed in a type A strain of C. perfringens, HN13, and secretion was measured using Western blot methods. We found that TpeE was required for TpeL secretion and that secretion was not due to cell lysis. Mutant forms of TpeE lacking an amphipathic helix and a charged C-terminal domain failed to secrete TpeL, and mutations that deleted conserved LCT domains in TpeL indicated that only the full-length protein could be secreted. In summary, we have identified a novel family of holin-like proteins that can function, in some cases, as a system of protein secretion for proteins that need to fold in the cytoplasm.IMPORTANCE Little is known about the mechanism by which LCTs are secreted. Since LCTs are major virulence factors in clostridial pathogens, we wanted to define the mechanism by which an LCT in C. perfringens, TpeL, is secreted by a protein (TpeE) lacking homology to previously described secretion-associated holins. We discovered that TpeE is a member of a widely dispersed class of holin proteins, and TpeE is necessary for the secretion of TpeL. TpeE bears a high degree of similarity in membrane topology to TatA proteins, which form the pore through which Tat secretion substrates pass through the cytoplasmic membrane. Thus, the TpeE-TpeL secretion system may be a model for understanding not only holin-dependent secretion but also how TatA proteins function in the secretion process.

Plasmalogen Biosynthesis by Anaerobic Bacteria: Identification of a Two-Gene Operon Responsible for Plasmalogen Production in Clostridium perfringens.

Plasmalogens are vinyl ether-containing lipids produced by mammals and bacteria. The aerobic biosynthetic pathway in eukaryotes and bacteria is known, but the anaerobic pathway has remained a mystery. Here, we describe a two-gene operon (plasmalogen synthase, pls) responsible for plasmalogen production in the anaerobic bacterium Clostridium perfringens. While aerobic plasmalogen biosynthesis involves an oxidative conversion of an ether to a vinyl ether, anaerobic plasmalogen biosynthesis uses the reductive conversion of an ester to an aldehyde equivalent. Heterologous expression of the C. perfringens pls operon in E. coli conferred the ability to produce plasmalogens. The pls operon is predicted to encode a multidomain complex similar to benzoyl-CoA reductase/hydroxylacyl-CoA dehydratase (BCR/HAD) enzymes. Versions of this operon can be found in a wide range of obligate and facultative anaerobic bacteria, including many human gut microbes.

Comparison of the Pathogenicity of Five Clostridium perfringens Isolates Using an Eimeria maxima Coinfection Necrotic Enteritis Disease Model in Commercial Broiler Chickens.

Clostridium perfringens (CP) is the etiologic agent of necrotic enteritis (NE) in broiler chickens that is responsible for massive economic losses in the poultry industry in response to voluntary reduction and withdrawal of antibiotic growth promoters. Large variations exist in the CP isolates in inducing intestinal NE lesions. However, limited information is available on CP isolate genetics in inducing NE with other predisposing factors. This study investigated the ability of five CP isolates from different sources to influence NE pathogenesis by using an Eimeria maxima (EM) coinfection NE model: Str.13 (from soil), LLY_N11 (healthy chicken intestine), SM101 (food poisoning), Del1 (netB+tpeL-) and LLY_Tpel17 (netB+tpeL+) for NE-afflicted chickens. The 2-wk-old broiler chickens were preinfected with EM (5 × 103 oocysts) followed by CP infection (around 1 × 109 colony-forming units per chicken). The group of the LLY_Tpel17 isolate with EM coinfection had 25% mortality. No mortality was observed in the groups infected with EM alone, all CP alone, or dual infections of EM/other CP isolates. In this model of EM/CP coinfections, the relative percentages of body weight gain showed statistically significant decreases in all EM/CP groups except the EM/SM101 group when compared with the sham control group. Evident gut lesions were only observed in the three groups of EM/LLY_N11, EM/Del1, and EM/LLY_Tpel17, all of which possessed an essential NE pathogenesis locus in their genomes. Our studies indicate that LLY_Tpel17 is highly pathogenic to induce severe gut lesions and would be a good CP challenge strain for studies investigating pathogenesis and evaluating the protection efficacy for antibiotic alternative approaches.

An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response.

Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(ß1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.

Changes in the expression of genes encoding type IV pili-associated proteins are seen when Clostridium perfringens is grown in liquid or on surfaces.

BACKGROUND: Clostridium perfringens is a Gram-positive anaerobic pathogen that causes multiple diseases in humans and animals. C. perfringens lack flagella but have type IV pili (TFP) and can glide on agar surfaces. When C. perfringens bacteria are placed on surfaces, they become elongated, flexible and have TFP on their surface, traits not seen in liquid-grown cells. In addition, the main pilin in C. perfringens TFP, PilA2, undergoes differential post-translational modification when grown in liquid or on plates. To understand the mechanisms underlying these phenotypes, bacteria were grown in three types of liquid media and on agar plates with the same medium to compare gene expression using RNA-Seq. RESULTS: Hundreds of genes were differentially expressed, including transcriptional regulatory protein-encoding genes and genes associated with TFP functions, which were higher on plates than in liquid. Transcript levels of TFP genes reflected the proportion of each protein predicted to reside in a TFP assembly complex. To measure differences in rates of translation, the Escherichia coli reporter gene gusA gene (encoding ß-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. ß-glucuronidase expression was then measured in cells grown in liquid or on plates. ß-glucuronidase activity was proportional to mRNA levels in liquid-grown cells, but not plate-grown cells, suggesting significant levels of post-transcriptional regulation of these TFP-associated genes occurs when cells are grown on surfaces. CONCLUSIONS: This study reveals insights into how a non-flagellated pathogenic rod-shaped bacterium senses and responds to growth on surfaces, including inducing transcriptional regulators and activating multiple post-transcriptional regulatory mechanisms associated with TFP functions.

Membrane Proteomes and Ion Transporters in Bacillus anthracis and Bacillus subtilis Dormant and Germinating Spores.

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.

Expansion of the Clostridium perfringens toxin-based typing scheme.

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, ß-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not ß-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.

Using a Concept Inventory to Reveal Student Thinking Associated with Common Misconceptions about Antibiotic Resistance.

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.

Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.

The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium.IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in polymerizing the pilin, PilA2, into a pilus. The assembly ATPase PilB2 and its cognate membrane protein partner, PilC2, were purified. PilB2 bound the intracellular signal molecule c-di-GMP. Increased levels of intracellular c-di-GMP led to increased polymerization of PilA2, indicating that Gram-positive bacteria use this molecule to regulate pilus synthesis. These findings provide valuable information for understanding how pathogenic clostridia regulate TFP to cause human diseases.

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly.

UNLABELLED: 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 ß-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.

NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens.

Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell.

Genomic analyses of Clostridium perfringens isolates from five toxinotypes.

Clostridium perfringens can be isolated from a range of environments, including soil, marine and fresh water sediments, and the gastrointestinal tracts of animals and humans. Some C. perfringens strains have attractive industrial applications, e.g., in the degradation of waste products or the production of useful chemicals. However, C. perfringens has been most studied as the causative agent of a range of enteric and soft tissue infections of varying severities in humans and animals. Host preference and disease type in C. perfringens are intimately linked to the production of key extracellular toxins and on this basis toxigenic C. perfringens strains have been classified into five toxinotypes (A-E). To date, twelve genome sequences have been generated for a diverse collection of C. perfringens isolates, including strains associated with human and animal infections, a human commensal strain, and a strain with potential industrial utility. Most of the sequenced strains are classified as toxinotype A. However, genome sequences of representative strains from each of the other four toxinotypes have also been determined. Analysis of this collection of sequences has highlighted a lack of features differentiating toxinotype A strains from the other isolates, indicating that the primary defining characteristic of toxinotype A strains is their lack of key plasmid-encoded extracellular toxin genes associated with toxinotype B to E strains. The representative B-E strains sequenced to date each harbour many unique genes. Additional genome sequences are needed to determine if these genes are characteristic of their respective toxinotypes.

Hypermotility in Clostridium perfringens strain SM101 is due to spontaneous mutations in genes linked to cell division.

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.

Levels of germination proteins in Bacillus subtilis dormant, superdormant, and germinating spores.

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.

Type IV pili in Gram-positive bacteria.

Type IV pili (T4P) are surface-exposed fibers that mediate many functions in bacteria, including locomotion, adherence to host cells, DNA uptake (competence), and protein secretion and that can act as nanowires carrying electric current. T4P are composed of a polymerized protein, pilin, and their assembly apparatuses share protein homologs with type II secretion systems in eubacteria and the flagella of archaea. T4P are found throughout Gram-negative bacterial families and have been studied most extensively in certain model Gram-negative species. Recently, it was discovered that T4P systems are also widespread among Gram-positive species, in particular the clostridia. Since Gram-positive and Gram-negative bacteria have many differences in cell wall architecture and other features, it is remarkable how similar the T4P core proteins are between these organisms, yet there are many key and interesting differences to be found as well. In this review, we compare the two T4P systems and identify and discuss the features they have in common and where they differ to provide a very broad-based view of T4P systems across all eubacterial species.

Biochemistry and physiology of the ß class carbonic anhydrase (Cpb) from Clostridium perfringens strain 13.

The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the ß class, the first ß class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO2 and 4.8 × 106 s⁻¹ M⁻¹, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO2. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO2 in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO2 fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.

Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding motility.

Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility.

Expression of a Clostridium perfringens type IV pilin by Neisseria gonorrhoeae mediates adherence to muscle cells.

Clostridium perfringens is an anaerobic, Gram-positive bacterium that causes a range of diseases in humans, including lethal gas gangrene. We have recently shown that strains of C. perfringens move across the surface of agar plates by a unique type IV pilus (TFP)-mediated social motility that had not been previously described. Based on sequence homology to pilins in Gram-negative bacteria, C. perfringens appears to have two pilin subunits, PilA1 and PilA2. Structural prediction analysis indicated PilA1 is similar to the pseudopilin found in Klebsiella oxytoca, while PilA2 is more similar to true pilins found in the Gram-negative pathogens Pseudomonas aeruginosa and Neisseria gonorrhoeae. Strains of N. gonorrhoeae that were genetically deficient in the native pilin, PilE, but supplemented with inducible expression of PilA1 and PilA2 of C. perfringens were constructed. Genetic competence, wild-type twitching motility, and attachment to human urogenital epithelial cells were not restored by expression of either pilin. However, attachment to mouse and rat myoblast (muscle) cell lines was observed with the N. gonorrhoeae strain expressing PilA2. Significantly, wild-type C. perfringens cells adhered to mouse myoblasts under anaerobic conditions, and adherence was 10-fold lower in a pilT mutant that lacked functional TFP. These findings implicate C. perfringens TFP in the ability of C. perfringens to adhere to and move along muscle fibers in vivo, which may provide a therapeutic approach to limiting this rapidly spreading and highly lethal infection.