Molecular Analysis of an Extrachromosomal Element Containing the C2 Toxin Gene Discovered in Clostridium botulinum Type C

Clostridium botulinum cultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including the c2I and c2II genes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were σ factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.Clostridium botulinum is a gram-positive, spore-forming, anaerobic bacterium. Cultures of this species produce poisonous botulinum neurotoxins (BoNTXs) that are lethal to humans and animals and are classified into seven types, types A to G, based on the antigenicity of the BoNTXs produced (actually, the cultures producing type G toxin were recently classified in a new species, Clostridium argentinens [40]).C. botulinum type C and D cultures produce a binary C2 toxin in addition to C (or C1) and D BoNTXs; this additional toxin consists of two nonlinked proteins, C2I and C2II (28), that occur independently in the culture supernatant and are not chemically joined to each other. The C. botulinum C2 toxin used here is a representative of the family of binary actin-ADP-ribosylating toxins, which includes, in addition to C2 toxin, the Clostridium perfringens iota toxin, Clostridium difficile toxin, Clostridium spiroforme toxin, and the vegetative insecticidal proteins from Bacillus cereus (4).The enzyme component of C2 toxin (C2I) ADP ribosylates G-actin at arginine 177 (1). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding-translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (5). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. Subsequently, C2I translocates across the membrane into the cytosol through these C2IIa pores.The production of C1 and D BoNTXs is governed by bacteriophages (12, 13, 18, 19, 26), and both toxin genes have been cloned from the corresponding phage DNAs (16, 21). Recently, we determined the whole-genome sequence of a type C toxin-converting phage (c-st) genome (31). Eklund et al. reported that C2 toxin toxigenicity (mouse lethality) became clear when strains were cultured in fortified egg-meat medium and the culture supernatants were treated with trypsin (12). They also reported that C2 toxin production was not related to the BoNTX-converting phages; some non-BoNTX-producing cells produced C2 toxin. Fujii et al. (15) and Kimura et al. (22) determined the whole nucleotide sequences of the c2I and c2II genes and speculated that these genes might be located on the bacterial chromosome (15, 22).In this study, we determined that these genes are present not on the cell chromosome but on a large plasmid; we first speculated that this was this was the case based on the results of both pulsed-field gel electrophoresis (PFGE) and Southern hybridization analysis and then confirmed it by determining the complete nucleotide sequence of the plasmid. Since the plasmid was extremely unstable, we could not purify the complete plasmid DNA; therefore, we determined the whole-genome sequence by using the inverse PCR method, which enables rapid determination of the flanking regions of unknown sequences and determination of unidentified sequences in the genome, and the primer-walking method. This is the first case in which an entire DNA sequence of a large plasmid was determined using only these two procedures. The process used to determine the whole-plasmid DNA sequence and several interesting features of the plasmid are described below.     

猴D型反转录病毒巢式PCR检测方法的建立

目的建立SRV-1巢式PCR检测方法并进行初步应用。方法针对SRV-1env基因的保守区序列,设计特异性引物,以感染SRV-1 Raji细胞提取出的含有前病毒DNA的基因组DNA为模板,进行巢式PCR反应。扩增产物测序后与GenBank报道的序列进行同源比对。将DNA样本进行10倍梯度稀释,以检测巢式PCR反应的灵敏度。使用该方法对正常Raji细胞以及感染SIV、STLV的外周血淋巴细胞DNA样本进行扩增,检测该方法的特异性。用建立的巢式PCR方法检测40份储存猴血标本。结果使用巢式PCR扩增出的特异片段经测序分析,结果证实与GenBank报道的序列一致。所建立的巢式PCR检测法检测限度可达1.5×10-3ng/μL,而且方法特异。用此方法检测40份猴血标本,未检测到阳性标本。结论初步建立SRV-1的巢式PCR检测方法,该方法灵敏、特异,为SRV-1的检测提供了一个快速、有效的手段。     

C型产气荚膜梭菌β1毒素基因表达

利用PCR技术,从C型产气荚膜梭菌染色体DNA中扩增出β1毒素基因,然后用限制性核酸内切酶BamHI和EcoRI对其进行双酶切处理,回收0．95kb的β1毒素基因片段,最后将其定向克隆在事先经同样内切酶处理的载体pET-28c中相应位点上,转化至受体菌B121(DE3)qh。经BamHI和Eco RI双酶切鉴定和核苷酸序列测定证实,构建的重组质粒pETXBl含有B毒素基因,并且具有正确的基因序列和阅读框架。重组菌株BI21(DE3)(pETXB1)经IPIG诱导后,其表达产物经ELISA检测和SDS-PAGE分析,结果表明重组菌株可以高效表达β1毒素蛋白,该蛋白占菌体总蛋白相对含量的12．24％。     

Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10⁴ times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.     

In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay.

A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.     

Kinetics of the Thermal Inactivation of Type E Clostridium botulinum Toxin

Rate of inactivation curves for the "free" toxin, prototoxin, or activated toxin in crude filtrates of Clostridium botulinum type E were nonlinear, consisting of a fast inactivating rate followed by a slow inactivating rate. Thermodynamic parameters were calculated over a temperature range of 125 to 145 F (51.7 to 62.8 C) for the two different inactivation rates. Energy of activation was low at the lower temperature and high at the higher temperature. The thermal requirement for inactivating similar concentrations of the "free" toxin, prototoxin, or activated toxin was considered to be the same.     

Detection of Clostridium botulinum Toxin by Local Paralysis Elicited with Intramuscular Challenge

Clostridium botulinum toxin can be identified by a characteristic, acute local paralysis that follows its injection into the gastrocnemius ("calf" muscle) of mice. The local botulism can be elicited with slightly less than one-tenth the toxin amount that is needed to kill mice by the intraperitoneal (i.p.) challenge route. The practical sensitivities of the intramuscular (i.m.) versus i.p. tests are about equal, however, because maximum sample volume injectable i.m. is 0.1 ml as compared to the 0.5-ml range that can be given i.p. i.m. injection of 10 or more mouse i.p. mean lethal doses causes paralysis in about 1 h, and an i.m. injection of about 0.5 i.p. mean lethal doses causes paralysis in 3 to 4 h. Toxin neutralization by homologous type of antitoxin only can be demonstrated with an incubated mixture of toxin and antitoxin. Although not as convenient as the i.p. method for routine use to detect botulinum toxin, the i.m. method has characteristics which could make it a useful supplement to the presently accepted i.p. procedure.     

Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.     

绵羊肺腺瘤病特异性PCR及套式PCR诊断方法的建立

绵羊肺腺瘤是由外源性反转录病毒JSRV引起的接触性传染的肺肿瘤性疾病。感染羊没有针对JSRV的循环抗体,但在感染羊的肺肿瘤组织、淋巴网状系统及外周血单核细胞中可检测到外源性前病毒exJSRV及JSRV转录产物。健康羊基因组中存在15~20拷贝的内源性前病毒enJSRV,内外源性前病毒的结构基因高度相似,而在U3区存在差别,因而,设计了针对exJSRVU3区的特异性引物并在国内首次建立了一步法特异性PCR检测法及套式PCR检测法。以700ng健康羊基因组DNA为背景,梯度稀释阳性质粒pJSRV-LTR作为模板,比较两种方法的敏感性,结果表明套式法的敏感性是一步法的10倍以上,套式法也是目前可用于检测感染羊血液样品的唯一方法,可望在绵羊肺腺瘤病的流行病学调查及防控方面起重要作用。     

Molecular Composition of the 16S Toxin Produced by a Clostridium botulinum Type D Strain, 1873

The 16S toxin was purified from a Clostridium botulinum type D strain 1873 (D-1873). Furthermore, the entire nucleotide sequences of the genes coding for the 16S toxin were determined. It became clear that the purified D-1873 16S toxin consists of neurotoxin, nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA), and that HA consists of four subcomponents, HA1, HA2, HA3a, and HA3b, the same as type D strain CB16 (D-CB16) 16S toxin. The nucleotide sequences of the nontoxic components of these two strains were also found to be identical except for several bases. However, the culture supernatant and the purified 16S toxin of D-1873 showed little HA activity, unlike D-CB16, though the fractions successively eluted after the D-1873 16S toxin peak from an SP-Toyopearl 650S column showed a low level of HA activity. The main difference between D-1873 and D-CB16 HA molecules was the mobility of the HA1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore it was presumed that the loss of HA activity of D-1873 16S toxin might be caused by the differences of processing HA after the translation.     

The Growth and Toxin Production of Clostridium botulinum Type E in Certain Vacuum Packed Fish

The growth and toxin production of Clostridium botulinum type E in various types of vacuum packed fish products was tested, with particular reference to the time/temperature relationship of storage. Toxin production was most rapid in herring although smoking retarded its development. With as small an inoculum as 10² spores/pack, fresh herring became toxic after storage for 15 days at 5°. Irradiation of three species of fish after inoculation with Cl. botulinum type E showed that spores surviving radiation germinated and produced toxin more rapidly than an equivalent concentration of spores in nonirradiated fish.     

Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.     

Analysis of the Botulinum Neurotoxin Type F Gene Clusters in Proteolytic and Nonproteolytic Clostridium botulinum and Clostridium barati

Comparison of genes encoding type F botulinum neurotoxin progenitor complex in strains of proteolytic Clostridium botulinum strain Langeland, nonproteolytic Clostridium botulinum strain 202F, and Clostridium barati strain ATCC 43256 reveals an identical organization of genes encoding a protein of molecular mass of approx. 47 kDa (P-47), nontoxic-nonhemagglutinin (NTNH) and botulinum toxin (BoNT). Although homology between the protein components of the complexes encoded by these different species all producing botulinum neurotoxin type F is considerable (approx. 69–88% identity), exceptionally high homology is observed between the C-termini of the P-47s (approx. 96% identity) and the NTNHs (approx. 94% identity) encoded by Clostridium botulinum type F strain Langeland and Clostridium botulinum type A strain Kyoto. Such a region of extremely high sequence identity is strongly indicative of recombination in these strains synthesizing botulinum neurotoxins of different antigenic types. Received: 13 April 1998 / Accepted: 9 May 1998     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.     

Clostridium botulinum Type F in the Soil of Argentina

One strain of Clostridium botulinum belonging to type F has been isolated from a soil sample taken in Buenos Aires Province, Argentina. Comparative studies carried out with strain Langeland of C. botulinum type F have shown a few differences between the two strains.     

Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10² cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10⁻² spore/g for types A, B, and F to 10⁻¹ spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.