Levels Of Toxin Production Research Articles

Effect of sub-MIC values of metronidazole, ciprofloxacin, and imipenem on the growth and toxin production in Clostridioides difficile.

This study intends to investigate the effect of sub-minimum inhibitory concentration (sub-MIC) of metronidazole, ciprofloxacin, and imipenem on the growth and toxin production in Clostridioides difficile. C. difficile is the most common causative agent of hospital-acquired diarrhea. Toxin production in C. difficile appears to be a critical process for induction of the disease. Several factors such as antibiotics can facilitate growth and toxin production in C. difficile. Five C. difficile strains were grown with and without sub-MIC concentrations of metronidazole, ciprofloxacin, and imipenem (0.5x MIC). The bacterial growth was measured by density at OD620 nm in 0, 4, 8, 12 and 24 h post inoculation. Toxin production was detected using ELISA in culture supernatants as well as in cell pellet. The five strains showed minor growth variations in the presence and absence of antibiotic sub-MIC values, except for metronidazole, in which the sub-MIC concentration reduced the growth rate of the resistant isolate in comparison with the control without antibiotic. There were no significant variations in the levels of toxin production with the sub-MIC values of antibiotics examined in comparison with antibiotic-free controls. However, the amount of toxin production in the culture supernatant was greater than in the cell pellet. The findings of this study suggested that sub-MIC concentrations of antibiotics may have minor effects on bacterial growth and toxin production of C. difficile. Taken together, these findings suggest that presence of antimicrobial agents increased expression levels of certain genes, particularly virulence genes, which may help C. difficile to survive.

Factors related to water quality and thresholds for microcystin concentrations in subtropical Brazilian reservoirs

ABSTRACTStudies of the environmental factors that lead to cyanotoxic blooms in tropical/subtropical reservoirs are limited. While technological advances for water treatment and remediation techniques can mitigate the effects of cyanobacterial blooms, the identification of initial drivers and tipping points for minimizing cyanotoxin concentrations could be more cost effective. We studied factors related to microcystin (MC) concentrations and estimated thresholds to limit MC using a water quality dataset from 6 subtropical Brazilian reservoirs. Reservoirs varied by water chemistry and toxin concentrations (MC yearly means <0.1–17 µg/L). Phytoplankton community composition in most reservoirs was dominated by cyanobacteria, and both nitrogen-fixing and non-fixing genera were common. We found positive correlations (p < 0.05, Spearman’s rank) between MC and nutrients (ρ = 0.48–0.74) and chlorophyll a (Chl-a, ρ = 0.65) and a negative correlation between MC and total nitrogen/total phosphorus (TN/TP, ρ = −0.49). While no association between MC and water temperature was observed, MC normalized to Chl-a values were positively related to higher temperatures and lower wind velocities, indicating warmer and more stable waters had a higher probability of toxic blooms. We found thresholds for MC ≥0.1 and ≥1 µg/L related to cyanobacterial cell counts of 20 060–136 165 cells/mL, respectively, and for turbidity (5–12 NTU) and TP (0.016–0.028 mg/L), respectively. We considered these MC concentrations (0.1 and 1 µg/L) to account for different levels of toxin production. The thresholds were different from those published for temperate ecosystems but not consistently lower or higher. TN/TP molar ratios favored toxin concentration when ≤121, suggesting that less phosphorus in relation to nitrogen is necessary to allow MC production in subtropical freshwaters. High phosphorus concentrations can increase concentration of cyanotoxins in subtropical reservoirs, and our data confirm that global warming could exacerbate problems associated with toxic algal blooms.

Carbon storage regulator CsrA plays important roles in multiple virulence-associated processes of Clostridium difficile

The carbon storage regulator CsrA is a global regulator that controls multiple virulence-associated processes including host cell invasion, virulence secretion, quorum sensing, biofilm formation, and motility in many pathogenic bacteria. However, the roles of CsrA in Clostridium difficile still remain unclear. In this study, a C. difficile strain overexpressing csrA was constructed to investigate its effects on multiple virulence associated processes. Overexpression of csrA resulted in flagella defect and poor motility in C. difficile 630Δerm, suggesting that CsrA involves in the regulation of flagellum synthesis. The levels of toxin production were increased in the C. difficile 630Δerm overexpressing of csrA. Moreover, csrA overexpression enhanced the adherence ability to Caco-2 cells and solvent production of C. difficile 630Δerm. Altogether, CsrA of C. difficile participates in multiple virulence processes including toxin production, motility, and adherence, and in the regulation of carbon metabolism. These results enhance our understanding of the regulatory functions of CsrA and reveal that CsrA is an important regulator in C. difficile contributing to virulence regulation.

Exotoxin diversity of Staphylococcus aureus isolated from milk of cows with subclinical mastitis in Central Russia

Mastitis, a major veterinary problem widespread in many regions, is caused mainly by Staphylococcus spp. However, there is no current reliable information about the role of Staphylococcus aureus and their toxins in the development of mastitis in cows in the territory of the Russian Federation. The aim of this investigation was to determine the profile of exotoxins of S. aureus from cow milk from farms of Central Russia. A total of 60 isolates of S. aureus were obtained from milk samples of cows with the subclinical form of mastitis. The exotoxin genes were identified using 2 types of PCR assays. The diversity of enterotoxin genes was studied by multiplex PCR. The percentage occurrence of enterotoxin genes was as follows: sea, 53.3%; seb, 3.3%; sec, 50%; sed, 4%; see, 46.6%; seg, 70%; sei, 10%; selp, 3.3%; and tsst1, 1.6%. The seh gene was not detected. The genes of pore-forming toxins and phenol-soluble modulins were identified by singleplex PCR and consisted of the following: hlA, 70%; lucS, 46.6%; psmA, 81.6%; psmB, 95%; and hld, 78.3%. The most abundant genes were psm (psmB, 95%), which codes for pore-forming toxins, and seg (70%), which codes for enterotoxins. The production of some enterotoxins in bacterial culture medium was detected by ELISA. The level of toxin production was near 1 ng/mL for SEA, SEE, SEG, SEI, SELP, and TSST-1 and reached a maximal level of 18 ng/mL for SEE. In the present work, we show that subclinical mastitis in cows is associated with S. aureus in the central region of the Russian Federation. Most of the isolates containing enterotoxin genes also had cytotoxin genes.

Growth Patterns of Clostridium difficile – Correlations with Strains, Binary Toxin and Disease Severity: A Prospective Cohort Study

A broad spectrum of symptoms has been associated with C. difficile infection (CDI). Several studies indicate that toxin-production correlates with growth rates of C. difficile. This study aimed to correlate growth rates of C. difficile with disease severity and strain characteristics. From 01/2003 to 10/2011, strains from a prospective cohort of all inpatients with CDI at the University Hospital Basel, Switzerland were analyzed regarding binary toxin, presence of the tcdC deletion and ribotype. Isothermal microcalorimetry was performed to determine growth rates, quantified by the Gompertz function. Ordered logistic regression models were used to correlate disease severity with strain features and clinical characteristics. Among 199 patients, 31 (16%) were infected with binary toxin-producing strains, of which the tcdC gene-deletion nt117 was detected in 9 (4%). Disease severity was classified as mild in 130 patients (65.3%), as severe in 59 patients (29.7%) and as severe/complicated in 10 patients (5.0%). Growth rates were inversely associated with disease severity in univariable (OR 0.514, 95%CI 0.29–0.91, p = 0.023) and multivariable analyses (OR 0.51, 95%CI 0.26–0.97, p = 0.040). While none of the strain characteristics such as presence of the tcdC gene deletion or binary toxin predicted CDI severity, growth rates were inversely correlated with disease severity. Further investigations are needed to analyze growth-regulators and respective correlations with the level of toxin production in C. difficile, which may be important determinants of disease severity.

Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels.

The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

RAPD analysis of genetic diversity among the isolates of Aspergillus flavus from different hosts and locations

Aflatoxin contamination is a major problem in maize, groundnut, chillies, cotton and tree nuts. These aflatoxins are low molecular weight toxic and carcinogenic secondary metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius. In the present study, a total of 11 isolates of A. flavus isolated from groundnut, maize and chilli collected from different locations of Tamil Nadu, India were tested for their ability to produce aflatoxin B1 (AFB1) in vitro by indirect competitive enzyme-linked immunosorbent assay. The results show that the isolates vary in their level of toxin production. The amount of AFB1 produced by the toxigenic isolates of A. flavus ranged from 6.6 to 108.1 ng ml−1. Among the various isolates of A. flavus, the isolate VKR produced the highest amount (108.1 ng ml−1) of AFB1. The isolates viz. CBE1, CBE2, BSR1, BSR3 and BSR4 were found to be non-toxigenic. The genetic variability among these isolates was assessed by Random amplified polymorphic DNA (RAPD) analysis. DNA fragments of between 0.15 and 3.0 kb were obtained using 13 random primers, and each isolate differed in the size and number of PCR products indicating considerable polymorphism. Cluster analysis using Unweighted Pair Group Method with Arithmetic Mean clearly separated the isolates into four main clusters confirming the genetic diversity among the isolates of A. flavus. Both toxigenic and non-toxigenic isolates were intermingled in these four groups, indicating that no relationship exists between RAPD profile and the production of aflatoxin by A. flavus.

Characterization of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B1, while 47.2% aflatoxins B1 and B2. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson’s correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB1 and AFB2, but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.

Assessment of Severity ofClostridium difficileInfection

The increasing incidence, severity and associated mortality of Clostridium difficile infection (CDI) continues to be a cause of health care concern. The new strain of C difficile (B1/NAP1/027) is associated with high levels of toxin production and more severe disease with poorer outcomes (1). Risk factors for CDI include the use of medications including antibiotics, gastric acid-suppressing agents and nonsteroidal anti-inflammatory drugs, chemotherapeutic agents, inflammatory bowel disease, post-transplantation patients, postsurgical patients, peripartum patients, prolonged length of stay in a health care setting and elderly patients (2). The Centers for Disease Control and Prevention (USA) definition of severe CDI include admission to an intensive care unit as a result of complications of CDI, surgery due to toxic megacolon, colonic perforation or refractory colitis or death attributable to CDI, all within 30 days of diagnosis (3). Clinicians need to be able to assess the severity of CDI early in the course of the disease to tailor medical management or anticipate surgery. Several severity scores have been proposed; however, none have been rigorously validated (4). The Hines VA CDI severity score was reported to best predict severe CDI with poor outcome. This score included fever, hypotension, elevated white blood cell count and radiological findings on computed tomography scan (thickened colonic wall, colonic dilation or ascites). These factors need to be assessed soon after a positive test for C difficle toxin and within three days of therapy commencement. A score of 3 or more indicated severe CDI. In a recent study of 184 patients with CDI (4), the independent risk factors for severe CDI were abdominal distension, fever, leukocytosis and hypoalbuminemia (plasma albumin level less than 30 mg/L). Version 1 of The University of Pittsburgh Medical Center (USA) Index was the next best score to correlate with severe CDI, and this included abdominal pain, radiological or clinical demonstration of ileus and leukocytosis (4). In a study from Toronto (Ontario), published in the current issue of The Canadian Journal of Gastroenterology (pages 368–372), Manek et al (5) reported two modifiable factors predictive of severe CDI – the use of exacerbating antibiotics and the nonuse of vancomycin in the initial therapeutic regimen. The four nonmodifiable factors identified were leukocytosis, systolic hypotension, confusion and repeat CDI. Vancomycin, therefore, should be considered first-line therapy in patients with moderate to severe CDI. It is inappropriate in these patients to wait until failure of metronidazole, given the rapid pace of the illness and early onset of complications. Early discontinuation of exacerbating antibiotics is equally important. New agents effective against CDI, such as fidoxamicin, may provide further options for treatment of severe CDI (6). It is important to identify moderate to severe CDI early to commence treatment with vancomycin and withdraw all incriminating drugs and avoid exposure to further antibiotics. Identification of moderate to severe CDI is essentially a clinical diagnosis, but may be aided by a computed tomography scan of the abdomen. Colonoscopy or flexible sigmoidoscopy is unnecessary, although the presence of pseudomembranous colitis indicates severe CDI (Box 1). Identification of the strain B1/NAP1/027 is generally not available at an early stage to influence management decisions. BOX 1 Indicators of moderate to severe Clostridium difficile infection Leukocytosis (white blood cell count >20×109/L) Plasma albumin level <30 g/L Creatinine level >50% of baseline Hypotension (systolic blood pressure <100 mmHg) Fever (temperature >38°C) Abdominal pain and distension Radiological evidence of colonic dilation, ascites or ileus

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1α partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B 1 and B 2 production levels. Among the isolated strains, 96 were identified as Fusarium verticillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1α sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verticillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r = 0.95 and r = 0.79 (correlation of FUM1 with FB 1 and FB 2, respectively, P < 0.0001); r = 0.93 and r = 0.78 (correlation of FUM19 with FB 1 and FB 2, respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity.

Influence of Tigecycline on Expression of Virulence Factors in Biofilm-Associated Cells of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infections are complicated by the ability of the organism to grow in surface-adhered biofilms on a multitude of abiotic and biological surfaces. These multicellular communities are notoriously difficult to eradicate with antimicrobial therapy. Cells within the biofilm may be exposed to a sublethal concentration of the antimicrobial due to the metabolic and phenotypic diversity of the biofilm-associated cells or the protection offered by the biofilm structure. In the present study, the influence of a sublethal concentration of tigecycline on biofilms formed by an epidemic MRSA-16 isolate was investigated by transcriptome analysis. In the presence of the drug, 309 genes were upregulated and 213 genes were downregulated by more than twofold in comparison to the levels of gene regulation detected for the controls not grown in the presence of the drug. Microarray data were validated by real-time reverse transcription-PCR and phenotypic assays. Tigecycline altered the expression of a number of genes encoding proteins considered to be crucial for the virulence of S. aureus. These included the reduced expression of icaC, which is involved in polysaccharide intercellular adhesin production and biofilm development; the upregulation of fnbA, clfB, and cna, which encode adhesins which attach to human proteins; and the downregulation of the cap genes, which mediate the synthesis of the capsule polysaccharide. The expression of tst, which encodes toxic shock syndrome toxin 1 (TSST-1), was also significantly reduced; and an assay performed to quantify TSST-1 showed that the level of toxin production by cells treated with tigecycline decreased by 10-fold (P < 0.001) compared to the level of production by untreated control cells. This study suggests that tigecycline may reduce the expression of important virulence factors in S. aureus and supports further investigation to determine whether it could be a useful adjunct to therapy for the treatment of biofilm-mediated infections.

Effect of sub-MIC concentrations of metronidazole, vancomycin, clindamycin and linezolid on toxin gene transcription and production in Clostridium difficile

Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5x MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.

Production of toxins by Alternaria alternata and A. radicina and their effects on germination of carrot seeds

A profile of toxin production by Alternaria alternata and A. radicina isolated from carrot seeds was determined. Ten isolates of each fungus were used for the study. All isolates of A. alternate produced alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TeA), and eight produced altertoxin I (ATX I). The isolates varied in level of toxin production. The highest toxigenicity was observed for TeA. In isolates of A. radicina, only radicinin (RAD) was detected. As compared with single isolate cultures of A. alternata and A. radicina, a higher productivity of RAD and, in general, lower productivity of AOH, AME, ALT and TeA was observed in dual cultures of the above-mentioned fungi. Treatment with solutions of extracts containing Alternaria toxins resulted in a lower percentage of normal seedlings and increased the percentage of necrotic seedlings in most cases. TeA at high concentrations as well as the joint presence of RAD and TeA had the greatest negative effects on seed germination.

101 Effect of Karlodinium Micrum Toxin on Success of the Parasitic Dinoflagellate Amoebophyra SP.

Parasitic dinoflagellates of the genus Amoebophrya infect and kill bloom‐forming dinoflagellates, including the toxic species Karlodinium micrum. Unlike non‐toxic hosts, K. micrum is partially resistant to infection, a trait that may be related to toxin production. Here we tested the hypothesis that parasitism of K. micrum is inversely related to toxin concentration in the culture medium. Time‐course studies were conducted to determine the influence of extracted toxin and toxin carrier (methanol) on host growth, parasite prevalence, and parasite load. Results indicate that methanol concentrations below 0.1% have no effect on these variables. When methanol concentration was maintained below 0.1%, extracted toxin equivalent to 100 to 10,000 K. micrum per ml had no effect on host abundance. We are currently analyzing sample to assess the fate of Amoebophrya dinospores when exposed to K. micrum toxin. We will also consider the effect of intracellular host toxin on parasite success, by examining the fate of Amoebophrya dinospores when inoculated to K. micrum cultures that express different levels of toxin production. Understanding the effect of toxins on parasite success will contribute to our knowledge of host‐parasite biology and provide insight into the role of dinoflagellate toxins as a defense against parasitism.

Genotypic and phenotypic characteristics of Corynebacterium diphtheriae strains isolated from patients in belarus during an epidemic period.

One hundred two Corynebacterium diphtheriae strains (93 of the gravis biotype and nine of the mitis biotype) isolated from clinical cases during the Belarus diphtheria epidemic were characterized by biotyping, toxigenicity testing by the Elek test and an indirect hemagglutination assay, phage typing, and ribotyping. The gravis biotype strains were characterized as high and medium toxin producers, and strains of biotype mitis were characterized as low and medium toxin producers. Most strains (82 of 102) were distributed among five phage types. Seventy-two strains (64 of the gravis biotype and 8 of the mitis biotype) belonged to phage type VI ls5,34add. Hybridization of genomic DNA digested with BstEII and PvuII revealed five ribotype patterns, namely, D1, D4, D6, D7, and D13. The majority of gravis biotype strains belonged to ribotypes D1 (49 of 93) and D4 (33 of 93) and included one clonal group of C. diphtheriae. This clone predominated in all regions in Belarus. There was a statistical association between ribotypes and phage types but not between ribotypes and levels of toxin production.

Bacillus cereus produces most emetic toxin at lower temperatures.

Seven emetic toxin-producing strains of Bacillus cereus were examined for toxin production in Skim Milk Medium at incubation temperatures ranging from 10 to 50 degrees C. Minimum and maximum growth temperatures were found to be 12 and 46 degrees C, respectively. At 12 and 15 degrees C, levels of toxin production were significantly higher (P < 0.01) than that observed at 30 degrees C, while no toxin was produced above 37 degrees C. Increased levels of sporulation were observed at increased temperatures, and no correlation was found between levels of sporulation and toxin production (R(2) = 0.086).

Detection of the etx gene (ϵ-toxin inducer) in plasmids of high molecular weight in Clostridium perfringens type D

The purpose of this work is to correlate the production of ϵ-toxin in a set of strains of Clostridium perfringens type D with the presence of the etx gene, either genomic or in plasmids. Total DNA obtained from strains with a different level of toxin production was explored by PCR and all the strains showed the amplification signal. Different methods were used to obtain plasmid profiles and all of the bands were assayed by PCR. The detection of the etx gene was only shown in several high molecular plasmids. These results were confirmed by a Southern blot. We suggest that the localization of the etx gene in different plasmids could be associated with the ϵ-toxin production level.

Detection of theetxgene (ε-toxin inducer) in plasmids of high molecular weight inClostridium perfringenstype D

The purpose of this work is to correlate the production of epsilon-toxin in a set of strains of Clostridium perfringens type D with the presence of the etx gene, either genomic or in plasmids. Total DNA obtained from strains with a different level of toxin production was explored by PCR and all the strains showed the amplification signal. Different methods were used to obtain plasmid profiles and all of the bands were assayed by PCR. The detection of the etx gene was only shown in several high molecular plasmids. These results were confirmed by a Southern blot. We suggest that the localization of the etx gene in different plasmids could be associated with the epsilon-toxin production level.

Trichothecene biosynthesis inGibberella pulicaris: Inheritance of C−8 hydroxylation

Naturally occurring strains ofGibberella pulicaris (Fusarium sambucinum) produce different kinds and levels of trichothecene toxins. Progeny from crosses between strains which produce trichothecenes with an oxygen-containing group at C−8 (C8+) and those that do not (C8−) can segregate in a 1∶1 ratio for this trait. These results define a genetic locus, which we have designatedTox1. The segregation patterns observed for progeny obtained from crosses between high-toxin producers and low-toxin producers indicate that the level of toxin production is determined by several loci. One gene which controls quantitative aspects of toxin production segregates independently of both theTox1 locus and another locus which controls toxin levels. These results suggest that multiple, unlinked nuclear loci are involved in the control of trichothecene biosynthesis.

Isolation and partial characterization of a corynebacteriophage beta, tox operator constitutive-like mutant lysogen of Corynebacterium diphtheriae.

We have isolated and partially characterized a beta-phage mutant lysogen of Corynebacterium diphtheriae, C7(betatoxct1+), which is partially insensitive to iron inhibition of diphtheria toxin production. tox expression by C7(betatoxct1+) was found to be partially constitutive. In the presence of concentrations of iron that almost completely inhibit the expression of diphtheria toxin by the wild type, C7(beta), the level of toxin production by C7(betatoxct1+) was found to be at least 25 times that of the parent. The purified tox gene product of C7(betatoxct1+) was immunologically and electrophoretically identical to, and equally as toxic as, diphtheria toxin purified from C7(beta). In addition, the partial N-terminal amino acid sequence was found to be identical to diphtheria toxin. This data strongly suggests that the mutation allowing for the constitutive expression of tox in C7(betatoxct1+) is outside of the structural gene. Furthermore, the constitutive expression of diphtheria toxin was found to be cis dominant in the double lysogen C7(betacrm45+/betatoxct1+). The data presented is consistent with the existence of a tox operator locus.