Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.     

Clostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants

Two versatile Clostridium perfringens-Escherichia coli shuttle vectors were constructed. Each plasmid carried a single antibiotic resistance gene which was expressed in both organisms. The plasmid pJIR750 encoded resistance to chloramphenicol and pJIR751 encoded resistance to erythromycin. Each plasmid contained the pUC18-derived multiple cloning site and the lacZ′ gene which enabled direct screening for recombinants in E. coli . These plasmids should prove invaluable for the genetic manipulation of C. perfringens.     

Toxin Plasmids of Clostridium perfringens

SUMMARY

In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch''s postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract.     

Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX

The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site‐specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA‐binding motifs. We have solved the solution structure of residues 1–120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX_1–120, we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into ¹⁵N‐labeled TnpX_1–120 and observed progressive nuclear magnetic resonance chemical shift perturbations using ¹⁵N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data‐driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain. Copyright © 2015 John Wiley & Sons, Ltd.     

Two Novel Membrane Proteins,TcpD and TcpE,Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.     

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation

Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog.     

Molecular genetics of the chloramphenicol-resistance transposon Tn4451 from Clostridium perfringens: the TnpX site-specific recombinase excises a circular transposon molecule

The chloramphenicol-resistance transposon Tn4451 undergoes precise conjugative deletion from its parent plasmid piP401 in Clostridium perfringens and precise spontaneous excision from multicopy plasmids in Escherichia coli. The complete nucleotide sequence of the 6338 bp transposon was determined and it was found to encode six genes. Genetic analysis demonstrated that the largest Tn4451-encoded gene, tnpX, was required for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnpX gene was completely stable in both organisms. Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans-acting site-specific recombinase. Allelic exchange was used to introduce the tnpXΔ allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens. It was also shown by hybridization and polymerase chain reaction (PCR) studies that TnpX-mediated transposon excision resulted in the formation of a circular form of the transposon. The TnpX recombinase was unique because it potentially contained the motifs of two independent site-specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/invertase domain of TnpX was likely to be involved in the excision process by catalysing the formation of a 2bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at the junction of the circular form. The other Tn4451-encoded genes include tnpZ, which appears to encode a second potential site-specific recombinase. This protein has similarity to plasmid-encoded Mob/Pre proteins, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.     

Conjugative Transfer of theEscherichia coli–Clostridium perfringensShuttle Vector pJIR1457 toClostridium botulinumType A Strains

An RP4-oriT shuttle vector pJIR1457 originally developed forClostridium perfringenswas successfully transferred by conjugation fromEscherichia colitoClostridium botulinumtype A strains and to a nontoxigenicC. botulinumtype A–transposon Tn916mutant strain lacking the entire toxin gene cluster. The light chain (LC) of botulinum toxin was highly expressed in the toxin deletion mutant strain from a pJIR1457 construct containing the recombinant botulinal gene for LC. This shuttle vector system will be valuable for genetic analysis ofC. botulinumand will enable genetic manipulation and recombinant expression studies of botulinum neurotoxins as pharmaceutical agents.     

Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

Background

Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.

Methodology/Principal Findings

An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.

Conclusions/Significance

The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.     

A Clostridium perfringens Vector for the Selection of Promoters

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be a useful reporter system for C. perfringens genes.     

tISCpe8, an IS1595-Family Lincomycin Resistance Element Located on a Conjugative Plasmid in Clostridium perfringens

Clostridium perfringens is a normal gastrointestinal organism that is a reservoir for antibiotic resistance genes and can potentially act as a source from which mobile elements and their associated resistance determinants can be transferred to other bacterial pathogens. Lincomycin resistance in C. perfringens is common and is usually encoded by erm genes that confer macrolide-lincosamide-streptogramin B resistance. In this study we identified strains that are lincomycin resistant but erythromycin sensitive and showed that the lincomycin resistance determinant was plasmid borne and could be transferred to other C. perfringens isolates by conjugation. The plasmid, pJIR2774, is the first conjugative C. perfringens R-plasmid to be identified that does not confer tetracycline resistance. Further analysis showed that resistance was encoded by the lnuP gene, which encoded a putative lincosamide nucleotidyltransferase and was located on tISCpe8, a functional transposable genetic element that was a member of the IS1595 family of transposon-like insertion sequences. This element had significant similarity to the mobilizable lincomycin resistance element tISSag10 from Streptococcus agalactiae. Like tISSag10, tISCpe8 carries a functional origin of transfer within the resistance gene, allowing the element to be mobilized by the conjugative transposon Tn916. The similarity of these elements and the finding that they both contain an oriT-like region support the hypothesis that conjugation may result in the movement of DNA modules that are not obviously mobile since they are not linked to conjugation or mobilization functions. This process likely plays a significant role in bacterial adaptation and evolution.There has been increasing concern about the emergence of multiply antibiotic-resistant strains of many common bacterial pathogens. The development of multiple resistance phenotypes has already led to compromises in the ability to successfully treat infected patients and to increased treatment costs (15). The emergence of resistant bacteria is often the result of excessive or inappropriate use of antibiotics and the ability of antibiotic resistance genes to be transferred from resistant to susceptible bacteria, either within a bacterial species, between different species within the same genus, or between different genera (14). Different types of mobile genetic elements, including conjugative plasmids, conjugative transposons, mobilizable plasmids, mobilizable transposons, nonconjugative plasmids, and integrons, may contain the resistance genes (14). All of these elements have the ability to mediate the transfer of resistance genes within and between bacterial cells, either independently or cooperatively, which has significant implications for the transfer and evolution of antibiotic resistance, particularly in pathogenic bacterial species.Clostridium perfringens is a normal gastrointestinal organism that causes food poisoning, necrotic enteritis, and gas gangrene (29). It is a proven reservoir for antibiotic resistance determinants. For example, the catP chloramphenicol resistance determinant, which is located on the Tn4451/Tn4453 family of integrative mobilizable elements in C. perfringens and Clostridium difficile, has been detected in clinical isolates of Neisseria meningitidis (20, 23, 41). Similarly, genetically related variants of the macrolide-lincosamide-streptogramin B (MLS) resistance determinant Erm(B) from C. perfringens have been found in Enterococcus faecalis, Streptococcus agalactiae, and C. difficile (19). It is likely that the C. perfringens determinant is the progenitor of the C. difficile determinant (18, 19, 44). Significantly, both determinants can be transferred into recipient cells by conjugation, although the processes are different (12, 19, 43). The pathogenic clostridia also carry other uncharacterized MLS resistance determinants and can potentially act as a source from which these resistance determinants may be transferred to other bacterial pathogens (10, 18).Lincomycin belongs to the lincosamide group of antibiotics, which also includes clindamycin. The spectrum of activity of lincosamides predominantly encompasses gram-positive bacteria, and these antimicrobial agents are often used for treatment of infections caused by anaerobic bacteria (45). These antibiotics inhibit protein synthesis by blocking the peptidyltransferase site of the 23S rRNA component of the 50S subunit of the bacterial ribosome (17). Although cross-resistance to MLS antibiotics most commonly involves N6 dimethylation of the A2058 residue of 23S rRNA and is catalyzed by an erm-encoded rRNA methyltransferase (24, 34, 47), specific resistance to the lincosamides is the result of modification and inactivation by a lincosamide nucleotidyltransferase encoded by members of the lnu (previously lin) gene family (5, 34, 45). This type of resistance gene is found in staphylococci and streptococci, where it is often located on plasmids or transposons (5, 45).Lincomycin resistance in C. perfringens is relatively common, but it is usually conferred as MLS resistance by erm(B) or erm(Q) genes (10, 11). Recent studies have shown that there has been an increase in lincomycin resistance in C. perfringens strains isolated from chickens in Belgium (28). The researchers reported two strains that conferred resistance to lincomycin and carried the lnu(A) or lnu(B) gene, the first such strains reported for C. perfringens.In the current study we analyzed several multiply antibiotic-resistant isolates of C. perfringens and identified strains that were lincomycin resistant but were susceptible to erythromycin. We characterized these isolates and their lincomycin resistance determinant(s) and showed that resistance could be transferred to other C. perfringens isolates. Detailed analysis of the lincomycin-resistant strain 95-949 showed that resistance was encoded by the lnuP gene, which was located on a transposable genetic element, tISCpe8, that was located on a conjugative plasmid, pJIR2774. This plasmid is the first conjugative C. perfringens R-plasmid to be identified that does not confer tetracycline resistance.     

Resistance Determinants of a Highly Arsenic-Resistant Strain of Leptospirillum ferriphilum Isolated from a Commercial Biooxidation Tank

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.     

Cross-complementation of Clostridium perfringens PLC and Clostridium septicum α-toxin mutants reveals PLC is sufficient to mediate gas gangrene

Clostridium perfringens and Clostridium septicum are the most common causes of clostridial myonecrosis or gas gangrene. Although they mediate a similar disease pathology, they elaborate functionally very different α-toxins. We used a reciprocal complementation approach to assess the contribution of the primary toxin of each species to disease and found that C. perfringens α-toxin (PLC) was able to mediate the gross pathology of myonecrosis even in a C. septicum background, although it could not induce vascular leukostasis. Conversely, while C. septicum α-toxin restored some virulence to a C. perfringens plc mutant, it was less active than in its native background.     

A Role for Tn6029 in the Evolution of the Complex Antibiotic Resistance Gene Loci in Genomic Island 3 in Enteroaggregative Hemorrhagic Escherichia coli O104:H4

In enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104 the complex antibiotic resistance gene loci (CRL) found in the region of divergence 1 (RD1) within E. coli genomic island 3 (GI3) contains bla_TEM-1, strAB, sul2, tet(A)A, and dfrA7 genes encoding resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim respectively. The precise arrangement of antibiotic resistance genes and the role of mobile elements that drove the evolutionary events and created the CRL have not been investigated. We used a combination of bioinformatics and iterative BLASTn searches to determine the micro-evolutionary events that likely led to the formation of the CRL in GI3 using the closed genome sequences of EAHEC O104:H4 strains 2011C-3493 and 2009EL-2050 and high quality draft genomes of EAHEC E. coli O104:H4 isolates from sporadic cases not associated with the initial outbreak. Our analyses indicate that the CRL in GI3 evolved from a progenitor structure that contained an In2-derived class 1 integron in a Tn21/Tn1721 hybrid backbone. Within the hybrid backbone, a Tn6029-family transposon, identified here as Tn6029C abuts the sul1 gene in the 3´-Conserved Segment (-CS) of a class 1 integron generating a unique molecular signature that has only previously been observed in pASL01a, a small plasmid found in commensal E. coli in West Africa. From this common progenitor, independent IS26-mediated events created two novel transposons identified here as Tn6029D and Tn6222 in 2011C-3493 and 2009EL-2050 respectively. Analysis of RD1 within GI3 reveals IS26 has played a crucial role in the assembly of regions within the CRL.     

The Murein Hydrolase of the Bacteriophage φ3626 Dual Lysis System Is Active against All Tested Clostridium perfringens Strains

Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry. As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C. perfringens bacteriophage 3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene. Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin. A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology. Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage λ S holin in λΔSthf. The endolysin gene ply3626 was cloned in Escherichia coli. However, protein synthesis occurred only when bacteria were supplemented with rare tRNA^Arg and tRNA^Ile genes. Formation of inclusion bodies could be avoided by drastically lowering the expression level. Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography. Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme. All 48 tested strains of C. perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected. This highly specific activity towards C. perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities.     

Antisense-RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of Clostridium perfringens Spores to Moist Heat and UV Radiation

Previous work has suggested that a group of α/β-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding α/β-type SASP in this organism. We have now used antisense RNA to decrease levels of α/β-type SASP in C. perfringens spores by ~90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of α/β-type SASP in the resistance of C. perfringens spores.     

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44⁺ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.     

Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes

Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens.Clostridium perfringens is a gram-positive, anaerobic bacterium that forms heat-resistant spores. It is widespread in the soil and commonly found in the gastrointestinal tract of mammals. It has been implicated in several medical conditions in humans, ranging from mild food poisoning to necrotic enteritis and gas gangrene. C. perfringens strains also cause a variety of important diseases in domestic animals, including several enteric syndromes, such as enterotoxemia in cattle, sheep, and pigs, necrotic enteritis in poultry, and typhocolitis in equines (17, 40).Understanding the pathogenesis of these infections is of crucial importance for the development of new tools for the prevention and control of C. perfringens-related diseases. Genetic modification is a valuable approach to identify new virulence factors and to study their role in the pathogenesis of C. perfringens.Since the 1980s, several tools for manipulation of C. perfringens at the molecular level have been developed (1, 5, 28, 35, 38). Among these tools, transposon mutagenesis is a method that is widely used for identification of virulence genes. Until now, the only reproducible method for transposon mutagenesis in C. perfringens was based on Tn916, a tetracycline resistance-encoding conjugative transposon originally isolated from Enterococcus faecalis (10, 11, 13). Tn916 has been used extensively for transposon mutagenesis due to its broad host range and has been proven to be valuable for the identification of genes in C. perfringens (3, 7, 22). Nevertheless, this method has major disadvantages; multiple Tn916 insertion events occur with an incidence of 65% to 75%, severely complicating identification of genes responsible for phenotype changes (3, 7, 19). Furthermore, Tn916 is still active after insertion, resulting in unstable mutants (6, 39, 42). To our knowledge, generation of Tn916-derived transposon mutants in C. perfringens field strains has never been described.Although a variety of transposon mutagenesis methods are available for gram-positive bacteria (4, 37, 41, 43), the inherent species nonspecificity, as well as the lack of mobility of the integrated transposon, makes the bacteriophage Mu-based transposon delivery system a system of choice for a variety of species (16, 26, 46). The Mu transposition approach includes in vitro assembly of a complex between the transposon DNA and the transposase enzyme, the transpososome, followed by delivery of the transpososome into the recipient cells. Once inside a cell, the Mu transpososome becomes activated in the presence of divalent cations, resulting in genomic integration of the delivered transposon. The bacteriophage Mu transposition system is also functional in vitro (15, 32, 33), in contrast to the Tn916 mutagenesis strategy, which is restricted to transposon mobilization in vivo following conjugation or electroporation. Under the optimal in vitro conditions, the Mu transposition reaction requires only the MuA transposase, a mini-Mu transposon, and target DNA as macromolecular components (15).In this study, a novel protocol is described for transposon mutagenesis in C. perfringens that exploits the bacteriophage Mu transposition system. To our knowledge, this report is the first report describing a mutagenesis method generating single-insertion transposon mutants in laboratory and field isolates of C. perfringens. This method is important for the identification of C. perfringens virulence factors involved in the numerous diseases caused by this bacterium.     

Global regulation of gene expression in response to cysteine availability in <Emphasis Type="Italic">Clostridium perfringens</Emphasis>

Background  

Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail.