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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.

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, pis) 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 pis 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. National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01AT009708, F32GM122233, F32AT010415] Published version We thank H. Goldfine (University of Pennsylvania) for kindly providing insights from his laboratory's work on plasmalogens in anaerobic bacteria, and for inspiring and supporting us early on in our research efforts. We acknowledge funding support from the National Institutes of Health (Grant Nos. R01AT009708 (J.C. and R.J.X.), F32GM122233 (D.R.J.), and F32AT010415 (C.D.C.).

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 β-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. β-glucuronidase expression was then measured in cells grown in liquid or on plates. β-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.

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 induced1000-fold when the cells were grown on plates than in liquid brain heart infusion (BHI). In addition, the pyrBICFZDE operon was transcribed1000-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.

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.

[Image: see text] 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.

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-(β1→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.

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.

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.

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.

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.

Clostridium perfringens is a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working with C. perfringens , a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is an Escherichia coli-C. perfringens shuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of gene bgaL ( bgaR -P bgaL ). To measure transcription at the bgaL promoter in pKRAH1, the E. coli reporter gene gusA , encoding β-glucuronidase, was placed downstream of the P bgaL promoter to make plasmid pAH2. When transformed into three strains of C. perfringens , pAH2 exhibited lactose-inducible expression. C. perfringens strain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated gene bglR , encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express the yfp - pilB gene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility in C. perfringens . Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in a pilC mutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

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.

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.

Clostridium perfringens is the most common cause of clostridial myonecrosis (gas gangrene). Polymorphonuclear cells (PMNs) appear to play only a minor role in preventing the onset of myonecrosis in a mouse animal model of the disease (unpublished results). However, the importance of macrophages in the host defense against C. perfringens infections is still unknown. Two membrane-active toxins produced by the anaerobic C. perfringens , alpha-toxin (PLC) and perfringolysin O (PFO), are thought to be important in the pathogenesis of gas gangrene and the lack of phagocytic cells at the site of infection. Therefore, C. perfringens mutants lacking PFO and PLC were examined for their relative cytotoxic effects on macrophages, their ability to escape the phagosome of macrophages, and their persistence in mouse tissues. C. perfringens survival in the presence of mouse peritoneal macrophages was dependent on both PFO and PLC. PFO was shown to be the primary mediator of C. perfringens -dependent cytotoxicity to macrophages. Escape of C. perfringens cells from phagosomes of macrophage-like J774-33 cells and mouse peritoneal macrophages was mediated by either PFO or PLC, although PFO seemed to play a more important role in escape from the phagosome in peritoneal macrophages. At lethal doses (10 9 ) of bacteria only PLC was necessary for the onset of myonecrosis, while at sublethal doses (10 6 ) both PFO and PLC were necessary for survival of C. perfringens in mouse muscle tissue. These results suggest PFO-mediated cytotoxicity toward macrophages and the ability to escape macrophage phagosomes may be important factors in the ability of C. perfringens to survive in host tissues when bacterial numbers are low relative to those of phagocytic cells, e.g., early in an infection.

Clostridium perfringensis a Gram-positive, anaerobic bacterium that is the most common cause of gas gangrene (clostridial myonecrosis) in humans.C. perfringensproduces a variety of extracellular toxins that are thought to be the major virulence factors of the organism. However,C. perfringenshas recently been shown to have the ability to survive in a murine macrophage-like cell line, J774-33, even under aerobic conditions. In J774-33 cells,C. perfringenscan escape the phagosome and gain access to the cytoplasm. Since the receptor that is used for phagocytosis can determine the fate of an intracellular bacterium, we used a variety of inhibitors of specific receptors to identify those used by J774-33 cells to phagocytoseC. perfringens. It was found that the scavenger receptor and mannose receptor(s) were involved in the phagocytosis ofC. perfringens. In the presence of complement, the complement receptor (CR3) was also involved in the binding and/or uptake ofC. perfringens. Since the receptor inhibition studies indicated that the scavenger receptor played a major role in phagocytosis,C. perfringensbinding studies were performed with a Chinese hamster ovary (CHO) cell line expressing the mouse SR-A receptor. The cell line expressing the SR-A receptor showed a significant increase inC. perfringensbinding in comparison to the non-transfected CHO cells. In the absence of opsonizing antibodies, the Fc receptor was not used to phagocytoseC. perfringens. Forcing the macrophages to use a specific receptor by using combinations of different receptor inhibitors led to only a slight increase in co-localization of intracellularC. perfringenswith the late endosome-lysosome marker LAMP-1. Carbohydrate analysis ofC. perfringensstrain 13 extracellular polysaccharide confirmed the presence of mannose and negatively charged residues of glucuronic acid, which may provide the moieties that promote binding to the mannose and scavenger receptors, respectively.

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.

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 perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N -acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N -acetylmannosamine-6-phosphate to N -acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from C. perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned. The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78. The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C. perfringens is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose. With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which β-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.

The cydAB operon of Escherichia coli encodes the cytochrome d oxidase complex, one of two aerobic terminal oxidases that catalyses the oxidation of ubiquinol-8 and the reduction of oxygen to water. This enzyme has a higher affinity for oxygen than the cytochrome o oxidase complex and accumulates as oxygen becomes limiting. Expression of the cydAB operon is microaerobically controlled by the ArcA/ArcB two-component regulatory system and by Fnr. To understand how ArcA and Fnr contribute to this control, a set of cyd-lacZ reporter fusions were constructed and analysed in vivo. Two cydAB promoters, designated P1 and P2, were identified by primer extension analysis and are located 288 and 173 bp upstream of the start of cydA translation respectively. Transcription from promoter P1 was shown to be regulated by both Fnr and ArcA in response to anaerobiosis. DNasel footprint experiments revealed the locations of two Fnr binding sites at the P1 promoter: one is centred at the start of cyd transcription, while the other is positioned 53.5 bp upstream. A single ArcA-phosphate binding site of 49 bp, centred 93 bp upstream of promoter P1, was identified to be sufficient for the activation of cydAB expression. Based on the results of the in vitro and in vivo studies, a working model for ArcA activation and Fnr repression of cydAB transcription is proposed.

SUMMARY 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.

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 β class, the first β 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 K m and k cat / K m values of 3.1 mM CO 2 and 4.8 × 10 6 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 CO 2 . 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 CO 2 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 CO 2 fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.

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.

Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5-15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis, and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens, Clostridium botulinum and Clostridium tetani, has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces < or = 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli, and this protein catalysed DPA formation in vitro. The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.

Clostridium perfringens is the third most frequent cause of bacterial food poisoning annually in the United States. Ingested C. perfringens vegetative cells sporulate in the intestinal tract and produce an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. Studies of Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors σ F , σ E , σ G , and σ K coordinate much of the developmental process. The C. perfringens cpe gene, encoding CPE, is transcribed from three promoters, where P1 was proposed to be σ K dependent, while P2 and P3 were proposed to be σ E dependent based on consensus promoter recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens . With the sigE and sigK mutants, gusA fusion assays indicated that there was no expression of cpe in either mutant. Results from gusA fusion assays and immunoblotting experiments indicate that σ E -associated RNA polymerase and σ K -associated RNA polymerase coregulate each other's expression. Transcription and translation of the spoIIID gene in C. perfringens were not affected by mutations in sigE and sigK , which differs from B. subtilis , in which spoIIID transcription requires σ E -associated RNA polymerase. The results presented here show that the regulation of developmental events in the mother cell compartment of C. perfringens is not the same as that in B. subtilis and Clostridium acetobutylicum .

The predominant organizational state of bacteria in nature is biofilms. Biofilms have been shown to increase bacterial resistance to a variety of stresses. We demonstrate for the first time that the anaerobic gram-positive pathogen Clostridium perfringens forms biofilms. At the same concentration of glucose in the medium, optimal biofilm formation depended on a functional CcpA protein. While the ratio of biofilm to planktonic growth was higher in the wild type than in a ccpA mutant strain in middle to late stages of biofilm development, the bacteria shifted from a predominantly biofilm state to planktonic growth as the concentration of glucose in the medium increased in a CcpA-independent manner. As is the case in some gram-negative bacteria, type IV pilus (TFP)-dependent gliding motility was necessary for efficient biofilm formation, as demonstrated by laser confocal and electron microscopy. However, TFP were not associated with the bacteria in the biofilm but with the extracellular matrix. Biofilms afforded C. perfringens protection from environmental stress, including exposure to atmospheric oxygen for 6 h and 24 h and to 10 mM H 2 O 2 for 5 min. Biofilm cells also showed 5- to 15-fold-increased survival over planktonic cells after exposure to 20 μg/ml (27 times the MIC) of penicillin G for 6 h and 24 h, respectively. These results indicate C. perfringens biofilms play an important role in the persistence of the bacteria in response to environmental stress and that they may be a factor in diseases, such as antibiotic-associated diarrhea and gas gangrene, that are caused by C. perfringens .

The fnr gene product, FNR, is a global regulator of anaerobic gene expression in Escherichia coli. When E. coli is switched from aerobic to anaerobic growth conditions, cytochrome o (cyoABCDE) and d oxidase (cydAB) genes are repressed and the anaerobic terminal reductase genes, including nitrate (narGHJI), dimethyl sulfoxide/trimethylamine (dmsABC), and fumarate (frdABCD) reductase, are induced. To determine if certain amino acid residues are essential for FNR to function in this regulatory process, site-directed mutations were introduced into the fnr gene. The resulting mutant proteins were assayed in vivo for their ability to either activate dmsA'-'lacZ and frdA'-'lacZ gene expression, or repress expression of a cyoA'-'lacZ gene fusion. The fnr mutants were grouped into four classes. Class I exhibited a severe decrease in the ability to either activate or repress fnr-dependent gene expression. Mutations in four of the five cysteine residues in the FNR protein were in this class. The sole exception was an FNR Cys16----Ser "mutant" that exhibited normal activity. Class II mutations caused a mild reduction in FNR-dependent activation or repression while Class III mutations conferred a modest increase in the ability of the FNR protein to activate gene expression under aerobic conditions (i.e. FNR*). Finally, Class IV mutations lowered the modest aerobic FNR transcriptional activation function proportionally more than the anaerobic FNR activity. These findings identify an essential role for the NH2 terminus of the FNR protein in its various activities in anaerobic gene regulation.

Clostridium perfringens is a common cause of the fatal disease gas gangrene (myonecrosis). Established gas gangrene is notable for a profound absence of neutrophils and monocytic cells (phagocytes), and it has been suggested that the bactericidal activities of these cells play an insignificant role in controlling the progression of the infection. However, large inocula of bacteria are needed to establish an infection in experimental animals, suggesting phagocytes may play a role in inhibiting the initiation of gangrene. Examination of tissue sections of mice infected with a lethal (1 x 10(9)) or sublethal (1 x 10(6)) inoculum of C. perfringens revealed that phagocyte infiltration in the first 3 h postinfection was inhibited with a lethal dose but not with a sublethal dose, indicating that exclusion of phagocytes begins very early in the infection cycle. Experiments in which mice were depleted of either circulating monocytes or neutrophils before infection with C. perfringens showed that monocytes play a role in inhibiting the onset of gas gangrene at intermediate inocula but, although neutrophils can slow the onset of the infection, they are not protective. These results suggest that treatments designed to increase monocyte infiltration and activate macrophages may lead to increased resistance to the initiation of gas gangrene.

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.

Bacteria can swim in liquid media by flagellar rotation and can move on surfaces via gliding or twitching motility. One type of gliding motility involves the extension, attachment and retraction of type IV pili (TFP), which pull the bacterium towards the site of attachment. TFP-dependent gliding motility has been seen in many Gram-negative bacteria but not in Gram-positive bacteria. Recently, the genome sequences of three strains of Clostridium perfringens have been completed and we identified gene products involved in producing TFP in each strain. Here we show that C. perfringens produces TFP and moves with an unusual form of gliding motility involving groups of densely packed cells moving away from the edge of a colony in curvilinear flares. Mutations introduced into the pilT and pilC genes of C. perfringens abolished motility and surface localization of TFP. Genes encoding TFP are also found in the genomes of all nine Clostridium species sequenced thus far and we demonstrated that Clostridium beijerinckii can move via gliding motility. It has recently been proposed that the Clostridia are the oldest Eubacterial class and the ubiquity of TFP in this class suggests that a Clostridia-like ancestor possessed TFP, which evolved into the forms seen in many Gram-negative species.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique “genomic islands” identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

Human diabetics frequently suffer delayed wound healing, increased susceptibility to localized and systemic infections, and limb amputations as a consequence of the disease. Lower-limb infections in diabetic patients are most often polymicrobial, involving mixtures of aerobic, facultative anaerobic, and anaerobic bacteria. The purpose of this study is to determine if these organisms contribute to synergy in polymicrobial infections by using diabetic mice as an in vivo model. The model was the obese diabetic mouse strain BKS.Cg- m +/+ Lepr db /J, a model of human type 2 diabetes. Young (5- to 6-week-old) prediabetic mice and aged (23- to 24-week-old) diabetic mice were compared. The mice were injected subcutaneously with mixed cultures containing Escherichia coli , Bacteroides fragilis , and Clostridium perfringens . Progression of the infection (usually abscess formation) was monitored by examining mice for bacterial populations and numbers of white blood cells at 1, 8, and 22 days postinfection. Synergy in the mixed infections was defined as a statistically significant increase in the number of bacteria at the site of injection when coinfected with a second bacterium, compared to when the bacterium was inoculated alone. E. coli provided strong synergy to B. fragilis but not to C. perfringens. C. perfringens and B. fragilis provided moderate synergy to each other but only in young mice. B. fragilis was anergistic (antagonistic) to E. coli in coinfections in young mice at 22 days postinfection. When age-matched nondiabetic mice (C57BLKS/J) were used as controls, the diabetic mice exhibited 5 to 35 times the number of CFU as did the nondiabetic mice, indicating that diabetes was a significant factor in the severity of the polymicrobial infections.

Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens . In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens , which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens , glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe - gusA fusion ( gusA is an Escherichia coli gene encoding the enzyme β-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).

Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischaemic tissues become contaminated with C. perfringens vegetative cells or spores. An aerotolerant anaerobe, C. perfringens quickly multiplies in ischaemic tissues and spreads to healthy areas, leading to a high level of morbidity and mortality. As a species, the bacterium can synthesize 13 different toxins, and these are thought to be the major virulence factors of the disease. However, we present evidence here that C. perfringens can also persist inside macrophages, under aerobic conditions, by escaping the phagosome into the cytoplasm. C. perfringens was not killed by the cells of a clone (J774-33) of the macrophage-like murine cell line J774A.1 under aerobic or anaerobic conditions, whereas the non-pathogenic bacterium Bacillus subtilis was killed by J774-33 cells under both conditions. Electron microscopy images showed that C. perfringens cells were intact and resided mostly in the cytoplasm of J774-33 cells, whereas B. subtilis was in the phagosome. Immunofluorescence microscopy showed that intracellular C. perfringens bacteria failed to co-localize with the late endosome-lysosomal marker glycoprotein LAMP-1, whereas B. subtilis did co-localize with LAMP-1. C. perfringens also appeared to escape the phagosome of both activated and unactivated mouse peritoneal macrophages, but not as efficiently as was seen with the J774-33 cell line. In addition, cytochalasin D was used to show that phagocytosis of C. perfringens was dependent on actin polymerization and that the bacteria attach to J774-33 cells at distinct areas of the cell membrane. We propose that the ability to escape the phagosome and persist inside macrophages is an important factor in the early stages of a gangrene infection, when bacterial numbers are low and phagocytic cells are present.

Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringens enterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the cpe promoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression of cpe , translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli , which codes for β-glucuronidase; E. coli-C. perfringens shuttle vectors carrying the fusions were introduced into C. perfringens by electroporation. In addition, in vitro transcription assays were performed with the cpe promoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis , the same compartment in which enterotoxin is synthesized in C. perfringens.

Publisher Summary The early studies led to investigations into the physiological and genetic basis for enterotoxin production by sporulating C. perfringens cells. Using DS medium, Duncan constructed a fairly precise kinetic analysis of sporulation, enterotoxin synthesis, and release of the mature spore by the mother cell during the growth and sporulation cycle of C. perfringens type A strain NCTC 8798. Genetic evidence in support of the coregulation of sporulation and Clostridium perfringens enterotoxin (CPE) synthesis was provided by the analysis of spontaneous and chemical sporulation mutants of CPE+ C. perfringens type A strain NCTC 8798. During the sporulation cycle, CPE synthesis appears to be regulated at the transcriptional level. The levels of both cpe mRNA and protein rose rapidly in synchrony, about 1 hour after the cells entered stationary phase, indicating that the increase in CPE expression as cells enter sporulation results from increased transcription of the cpe gene. Expression of CPE in Escherichia coli is useful as a heterologous host system for identifying gene products that regulate cpe expression. CPE expression is detected by the E. coli transformants, even though naturally nonenterotoxigenic C. perfringens transformants carrying the same shuttle plasmid are able to produce significant levels of CPE.

The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state. To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis. In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth. The altered fur gene was overexpressed in E. coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures. An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein. The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer. In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription. The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site. A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.

Clostridium perfringens is a source of food poisoning in humans and animals because of production of a potent enterotoxin (CPE). To study the regulation of the cpe gene in C. perfringens, we cloned and sequenced the cpe promoter regions and N-terminal domains from three strains. The cpe promoter region from one strain contained a 45-bp insertion compared with previously published sequences. This insertion was also found in two (of five) other Cpe+ strains. cpe gene expression in C. perfringens was measured by using translational fusions of each promoter type to the Escherichia coli gusA gene, which codes for beta-glucuronidase. For either promoter type, cpe-gusA expression was undetectable throughout exponential growth but increased dramatically at the beginning of the stationary phase. To measure cpe expression in Bacillus subtilis, cpe-gusA fusions were integrated into the B. subtilis chromosome. Both types of promoter exhibited moderate expression during exponential growth; cpe expression increased threefold at the beginning of the stationary phase. Transcriptional start sites were determined by primer extension and in vitro transcription assays. For C. perfringens, both types of promoter gave the same 5' end, 197 bp upstream of the translation start (50 bp downstream of the 45-bp insertion). In B. subtilis, however, the 5' end was internal to the 45-bp insertion, suggesting the use of a different promoter than that utilized by C. perfringens.

Selenomonas ruminantium HD4 produced significant quantities of d- and l-lactate from glucose in batch culture. Both isomers also supported growth if fumarate was present. In glucose-limited continuous culture, d-lactate was detected in the medium only at fast dilution rates. In continuous-culture-grown cells, only a cytoplasmic NAD-dependent l-lactate dehydrogenase (LDH) and a membrane-associated NAD-independent l-LDH were detected; activity of the soluble enzyme was twice as high at the fast dilution rate as at the slow dilution rate. Lactate racemase was also detected; its activity was 4-fold higher at the fast dilution rate. The presence of racemase explains why d-lactate was made and used by this organism.

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.

We have applied a model that permits the estimation of the sensitivity of flux through branch point enzymes (D. C. LaPorte, K. Walsh, and D. E. Koshland, J. Biol. Chem. 259:14068-14075, 1984) in order to analyze the control of flux through the lactate-acetate branch point of Selenomonas ruminantium grown in glucose-limited continuous culture. At this branch point, pyruvate is the substrate of both the NAD-dependent L-(+)-lactate dehydrogenase (LDH) and the pyruvate:ferredoxin oxidoreductase (PFOR). The LDH was purified, and it exhibited positive cooperativity for the binding of pyruvate. The LDH had an [S].5 for pyruvate of 0.43 mM, a Hill coefficient of 2.4, and a K' equal to 0.13 mM. The PFOR, assayed in cell extracts, exhibited Michaelis-Menten kinetics for pyruvate, with a Km of 0.49 mM. Carbon flux through the LDH and the PFOR increased 80-fold and 3-fold, respectively, as the dilution rate was increased from 0.07 to 0.52 h-1 in glucose-limited continuous culture. There was nearly a twofold increase, from 6.5 to 11.2 mumol min-1 mg of protein-1 in the specific activity (i.e., maximum velocity) of the LDH at dilution rates of 0.11 and 0.52 h-1, respectively. A flux equation was used to calculate the intracellular concentration of pyruvate; a fourfold increase in pyruvate, from 0.023 to 0.093 mM, was thereby predicted as the dilution rate was increased from 0.07 to 0.52 h-1. When these calculated values of intracellular pyruvate concentration were inserted into the flux equation, the predicted values of flux through the LDH and the PFOR were found to match closely the flux actually measured in the chemostat-grown cells. Thus, the 80-fold increase in flux through the LDH was due to a twofold increase in the maximum velocity of the LDH and a fourfold increase in the intracellular pyruvate concentration. In addition, the flux through the LDH exhibited ultrasensitivity to changes in both the maximum velocity of the LDH and the intracellular concentration of pyruvate. The flux through the PFOR exhibited ultrasensitivity to changes in the maximum velocity of the LDH and hyperbolic sensitivity to changes in the intracellular concentration of pyruvate.