Plasmid transformation of Clostridium perfringens by electroporation methods

Two electroporation methods were compared and modified to improve the frequencies of transfer of plasmid DNA into Clostridium perfringens. A plasmid shuttle vector, pSB92A2, containing chloramphenicol and ampicillin resistance genes and a clostridial origin of replication isolated from a cryptic C. perfringens plasmid, was constructed and successfully introduced into C. perfringens by both electrotransformation methods. Modifications which improved frequencies by 15-28 fold are described and may improve frequencies sufficiently for some vector/host combinations to consider the future use of more direct cloning strategies for the clostridia.     

Plasmid copy number and stability determination in Clostridium perfringens transformants

The copy number of the streptococcal plasmid pAM beta 1 (26.5 kb), and its deletion derivatives, pVA1 (11 kb) and pVA677 (7.6 kb) contained in Clostridium perfringens 3624A transformants was determined by incorporation of [methyl-3H]thymidine (4 muCi/ml) into chromosomal and plasmid DNA and sizing of the C. perfringens genome using transverse alternating field electrophoresis. Plasmids pAM beta 1, pVA1, and pVA677 were found to be present at 1.0, 97, and 216 copies/cell, respectively. 10.2, 54, and 96% of the initial pAM beta 1-, pVA1- and pVA677-containing transformants, respectively, remained resistant to erythromycin over 220 generations of growth. The results indicate a size-dependent relationship between plasmid stability and plasmid copy number in C. perfringens.     

Genomic map of Clostridium perfringens strain 13

A physical and genetic map of Clostridium perfringens strain 13 was constructed. C. perfringens strain 13 was found to have a 3.1-Mb chromosome and a large 50-kb plasmid, indicating that strain 13 has a relatively small genome among C. perfringens strains. A total of 313 genetic markers were mapped on the chromosome of strain 13. Compared with the physical and genetic map of C. perfringens CPN50, strain 13 had a quite similar genome organization, but with a large deletion (approximately 400 kb) in a particular segment of the chromosome. Among several toxin genes, a beta2 toxin gene that is a novel virulence gene in C. perfringens was found to be located on the 50-kb plasmid.     

Identification and molecular genetic analysis of replication functions of the bacteriocinogenic plasmid pIP404 from Clostridium perfringens

The replication functions of the bacteriocinogenic plasmid pIP404, from Clostridium perfringens, were localized to a 2.8-kb EcoRI-EcoRV fragment by cloning into a vector deficient for replication in Bacillus subtilis. This fragment contains two genes, cop and rep, which encode proteins and an 800-bp noncoding segment of complex structure consisting of multiple tandemly repeated sequences. The Cop protein is involved in copy number control, whereas the rep gene product is essential for plasmid replication. By deletion analysis the minimal origin of replication was defined as the rep gene plus most of the repeated sequences. A powerful promoter producing a 150-nucleotide RNA molecule, RNA1, that could act as an anti-sense RNA to the rep gene was detected in the "origin-like" region. In contrast to most other small plasmids of gram-positive bacteria, pIP404, and its derivatives, does not appear to replicate via a single stranded intermediate in either C. perfringens or B. subtilis.     

Identification of Accessory Genome Regions in Poultry Clostridium perfringens Isolates Carrying the netB Plasmid

Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance.     

High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens

Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO.     

Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens

The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined. The plasmid genome comprises 10,207 bp and has a dA + dT content of 75%. Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified. The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content. Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.     

Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens

Clostridium perfringens is ubiquitous in the environment and causes diseases in man and animals, with syndromes ranging from enteritis, enterotoxemia, and sudden death to food poisoning and gas gangrene. Understanding the epidemiology of these infections and of the evolution of virulence in C. perfringens necessitate an efficient, time and cost effective strain typing method. Multiple-locus variable-number tandem repeat analysis (MLVA) has been applied to typing of other pathogens and we describe here the development of a MLVA scheme for C. perfringens. We characterized five variable tandem repeat (VNTR) loci, four of which are contained within protein encoding genes and screened 112 C. perfringens isolates to evaluate typability, reproducibility, and discriminatory power of the scheme. All the isolates were assigned a MLVA genotype and the technique has excellent reproducibility, with a numerical index of discrimination for the five VNTR loci of 0.995. Thus MLVA is an efficient tool for C. perfringens strain typing, and being PCR based makes it rapid, easy, and cost effective. In addition, it can be employed in epidemiological, ecological, and evolutionary investigations of the organism.     

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.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Pathogenesis of Hobbs' heat-sensitive spore forming Clostridium perfringens type A strain.

Food poisoning in man due to heat-sensitive strains of Cl. perfringens type A appeared to be mediated through enterotoxin synthesized in vivo during sporulation. A minimum of 2.0 X 10(5) vegetative cells suspended in sporulation medium was sufficient to elicit gut-loop response in rabbits. The functional disturbance in the gut as well as the structural changes were progressive and proportional to the size of the inoculum up to a dose limit of 2.0 X 10(7) vegetative cells and beyond this the changes remained steady.     

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

PCR detection of Clostridium perfringens producing different toxins in faeces of goats

A polymerase chain reaction (PCR) was used to identify the genes encoding the major toxins of Clostridium perfringens in faeces of goats. When pure cultures of Cl. perfringens types A, B, C, D and E were used as templates in the PCR, amplicons were observed on the agarose gel as bands at approximately the 247 (alpha primers), 1025 (beta primers), 403 (epsilon primers) and 298 (iota primers) bp level of the DNA marker. When used to identify different types of Cl. perfringens in samples artificially spiked with these micro-organisms, the PCR detected as few as 1–1·5×10² cfu g⁻¹ of the five types of Cl. perfringens tested. The PCR technique allowed the identification and typing of Cl. perfringens strains in faeces of goats, without recourse to other techniques such as the mouse neutralization test.     

Phospholipids of Clostridium perfringens: a reexamination

We have identified phosphatidylethanolamine as one of the major phospholipids of Clostridium perfringens by two dimensional thin layer chromatography of the intact lipids and of their deacylation products and by liquid chromatography followed by mass spectrometry of the intact neutral phospholipid fraction. The principal fatty acids of phosphatidylethanolamine are myristic acid (14:0), lauric acid (12:0), and palmitic acid (16:0) and the major molecular species are 14:0,14:0 (26.3%); 12:0,14:0 (19.0%); 14:0,16:0 (22.4%) and 16:0,16:0 (17.6%). A similar distribution of molecular species was found in the other major phospholipid, O-alanyl phosphatidylglycerol.     

Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly

Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation.     

食品中产气荚膜梭菌LAMP快速检测方法的建立

应用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP), 针对产气荚膜梭菌特有的α毒素(CPa)基因序列分析设计特异性引物, 建立食品中产气荚膜梭菌特异性快速检测方法。研究结果表明, 所设计的引物具有良好的特异性, 5株产气荚膜梭菌均能扩增出特异性片段, 而13株非产气荚膜梭菌均未扩增出相应片段, 无假阴性或假阳性情况出现。同时, 该方法可在1 h内完成反应, 且检测灵敏度达到10 fg/μL。该方法为产气荚膜梭菌的快速检测提供了一种重要的技术手段。