Echinococcus multilocularis: developmental stage-specific expression of Antigen B 8-kDa-subunits

Antigen B (AgB) initially found in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa. Four genes encoding these 8-kDa-subunits have been identified from E. granulosus. In this study we isolated five genes encoding 8-kDa-subunits of AgB from Echinococcus multilocularis. Sequence comparison of isolated cDNA clones demonstrated that one of these five clones was completely identical to EmAgB8/1 which had been isolated previously by our group, and three of them were 94.5, 90.8, and 91.9% homologous to E. granulosus antigen B 8-kDa subunit genes, EgAgB8/2, EgAgB8/3, and EgAgB8/4, respectively. The remaining clone shared 51-58% homology with the nucleotide sequences of AgB genes. Gene-specific RT-PCR and Western blot analyses revealed that these genes were expressed in a developmentally regulated manner in E. multilocularis vesicles, protoscoleces, and immature adult worms. Possible functions of different expression manners are also discussed.     

Using mitochondrial and nuclear markers to evaluate the degree of genetic cohesion among Echinococcus populations

Based on the distinctiveness of their mitochondrial haplotypes and other biological features, several recent publications have proposed that some Echinococcus granulosus strains should be regarded as separate species. However, the genetic cohesion of these species has not been extensively evaluated using nuclear markers. We assess the degree of polymorphism of the partial mitochondrial cox1 (366 bp), the nuclear mdh (214 bp) and EgAgB4 (281-283 bp) genes of E. granulosus sensu lato isolates collected from areas where different strains occur sympatrically. Five distinct mitochondrial haplotypes were determined by direct sequencing (G1, G2, G5, G6 and G7). The mdh genotypes were first screened by SSCP: three alleles were identified (Md1-Md3), which were further confirmed by nucleotide sequencing. For EgAgB4, which was analysed by direct sequencing the PCR products, two groups of sequences were found: EgAgB4-1 and EgAgB4-2. No haplotype-specific mdh or EgAgB4 sequences occur. Nevertheless, alleles Md1 and Md2 and type 1 sequences of EgAgB4 showed a higher frequency within the group of haplotypes G1-G2, while allele Md3 and EgAgB4-2 are most frequent in the G5-G7 cluster. By AMOVA it is shown that 79% of the total genetic variability is found among haplotype groups. These findings are compatible with two not mutually exclusive evolutionary hypotheses: (a) that haplotypes share an ancestral polymorphism, or (b) that the reproductive isolation between parasites with distinct haplotypes is not complete, leading to gene introgression. The biologic and epidemiologic consequences of our findings are discussed.     

Analysis on the reactivity of five subunits of antigen B family in serodiagnosis of echinococcosis

In this study, the reactivity and differences of five subunits of echinococcus antigen B (AgB) family, recognizing specific antibodies in echinococcosis patient serum, were analyzed. Eight recombinant subunit antigens from Echinococcus granulosus (EgAgB1-EgAgB4) and Echinococcus multilocularis (EmAgB1-EmAgB3 and EmAgB5) were tested by ELISA using a panel of 243 serum samples collected from cystic echinococcosis (CE), alveolar echinococcosis (AE), cysticercosis (CC) patients and clinically normal individuals (NH). The results showed that the diagnostic sensitivity of the subunits for CE sera were 83.06%, 62.90%, 29.03%, 75.81% and 41.13%, and the specificities were 73.95%, 72.27%, 76.47%, 73.11% and 85.71%, respectively. The reactivity of three paralogous subunits, EgAgB1, EgAgB2 and EgAgB3 from E. granulosus and EmAgB1, EmAgB2 and EmAgB3 from E. multilocularis were compared by serological assay. All of the orthologous subunits showed no statistical difference (P>0.05) in detecting CE and AE sera; it revealed that the reactive epitopes may be similar between the orthologous subunits. In a total of 124 CE sera, the positive recognition rate by EgAgB1 was the highest (103/124), yet cocktail subunit antigens may detect even more positives from 100/124 to 112/124 using different subunit combinations. IgG4 subclass was the predominant antibody in reacting with subunit antigens. To conclude, the epitopes of orthologous AgB subunits from E. granulosus and E. multilocularis that recognize specific antibodies may be similar. The paralogous subunits EgAgB1, EgAgB2 and EgAgB4 were the main reactive subunit in sera detection and may have utility as echinococcosis diagnostics, with EgAgB1 possessing the greatest potential. Cocktail subunits may improve the positive detection rate.     

Induction of T helper 1 response by immunization of BALB/c mice with the gene encoding the second subunit of Echinococcus granulosus antigen B (EgAgB8/2)

A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2) was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/2 or pDRIVE empty cassette using the intramuscular (i.m.), intranasal (i.n.) or the epidermal gene gun (g.g.) routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1) immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I.) ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.     

A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa

We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.     

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.     

Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues

The aim of our study was to establish a new PCR protocol for the detection and discrimination of Echinococcus granulosus complex on one hand and Echinococcus multilocularis in formalin-fixed, paraffin-embedded tissues (FFPTs) on the other. The target sequences for all PCRs are located on a 471bp segment of the mitochondrial ND1 gene, the fragment sizes of the amplification products are 295bp (for the sheep strain of E. granulosus), 204bp (for the pig strain of E. granulosus) and 252bp (for E. multilocularis), respectively. In total, 80 FFPTs from patients with histologically confirmed echinococcosis (76 with E. granulosus and four with E. multilocularis) operated on in Austrian hospitals between 1978 and 2005 were examined. In 68 (85%) samples, we were able to detect specific DNA fragments with our newly established PCR protocols. Thirty-eight (47.5%) of 80 clinical samples were identified as the G1 strain, 26 (32.5%) as the G5, 6 or 7 strains and four (5%) as E. multilocularis. The specificity of all three PCRs was 100%; for the discrimination between G6 and G7 strains, sequencing of an additional 234bp PCR fragment was necessary and showed that three out of 26 G5, 6 or 7 PCR-positive patients were infected with E. granulosus genotype G6 (the camel strain).     

Further molecular discrimination of Spanish strains of Echinococcus granulosus

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.     

First report of Echinococcus granulosus G8 in Eurasia and a reappraisal of the phylogenetic relationships of 'genotypes' G5-G10

In this study, we investigated the presence of the larval stage of the tapeworm Echinococcus granulosus in wild ungulates in Estonia, genetically characterized E. granulosus isolates using mitochondrial gene sequences and used the sequence data, together with those available in a public database, to infer the phylogenic relationships of E. granulosus 'genotypes' G5-G10. While 0.8% of the 2038 moose (Alces alces) examined were found to be infected with E. granulosus, the parasite was not detected in other wild ungulates, such as roe deer (Capreolus capreolus: 1044 specimens examined) and wild boar (Sus scrofa: 442 specimens). Genetic analyses of concatenated atp6, nad1 and cox1 gene (1028 bp) sequences revealed that 2 novel E. granulosus haplotypes, namely E8 (11 samples: 69%) and E10 (5 samples: 31%), grouped with E. granulosus G8 and G10, respectively, are present in Estonia. This is the first record of an E. granulosus G8 in Eurasia. Phylogenetic analyses, using 4 different methods, demonstrated with considerable statistical support that E. granulosus G6/7 forms a subgroup together with G10, whereas G8 is a sister taxon to G6/7-G10.     

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.     

细粒棘球蚴AgB8/2基因的原核表达及免疫学鉴定

旨在克隆细粒棘球蚴 AgB8/2基因,优化原核表达体系,鉴定纯化蛋白并初步确定其诊断价值.采用 RT-PCR扩增 AgB8/2基因 cDNA,构建原核表达载体pET-AgB8/2并在大肠杆菌 BL21(DE3)中表达,优化表达条件.纯化的AgB8/2融合蛋白经 Western blot鉴定正确后,采用斑点免疫金渗滤法初步验证其血清学诊断价值.结果显示,克隆的 AgB8/2基因包含了 273 bp的完整ORF,序列分析表明与其它已报道的AgB8/2 cDNA序列的同源性在 99%～100%之间.优化的表达条件为,当菌液 OD600值为0.6时,加入终浓度为0.05 mmol/L的 IPTG,37℃,振荡培养5 h.纯化的蛋白经 Western blot鉴定为目的蛋白.克隆的 AgB8/2基因高度保守,原核表达载体pET-AgB8/2构建正确并高效表达可溶性融合蛋白,可作为特异性抗原应用于斑点免疫金渗滤法检测血清抗体.     

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.     

The Echinococcus granulosus antigen B shows a high degree of genetic variability

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.     

Echinococcus granulosus strain differentiation in Iran based on sequence heterogeneity in the mitochondrial 12S rRNA gene

Parasite strain characterization is essential for the establishment of a prevention and control strategy in any endemic area. The aim of this study was to characterize different Echinococcus granulosus isolates from Iran by using DNA sequences of the mitochondrial 12S rRNA gene. Thirty livers and lungs of cattle, sheep and goats naturally infected with E. granulosus were collected from abattoirs in northern and western Iran between June and October 2007. These samples yielded 18 fertile cysts which we used for the genetic work. We designed and tested two new primer pairs which specifically amplify portions of the mitochondrial 12S rRNA gene of the two strains (G1 and G6) of E. granulosus known to occur in Iran. One primer pair amplified a fragment of 259 base pairs (bp) from only the G1 strain. The second pair amplified a fragment of 676 bp from the G6 strain. The G1 genotype was identified in all fertile cyst samples, in agreement with previous studies in Iran. Ten of our samples and a single reference sample of the G6 strain were sequenced and compared with the G1 and G6 sequences deposited in GenBank.     

Characterisation of the native lipid moiety of Echinococcus granulosus antigen B

Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40-50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.     

The Echinococcus granulosus Antigen B Gene Family Comprises at Least 10 Unique Genes in Five Subclasses Which Are Differentially Expressed

Background

Antigen B (EgAgB) is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. This protein has been shown to play an important role in modulating host immune responses, although its precise biological function still remains unknown. It is generally accepted that EgAgB is comprised of a gene family of five subfamilies which are highly polymorphic, but the actual number of genes present is unknown.

Methodology/Principal Findings

Based on published sequences for the family, we designed specific primers for each subfamily and used PCR to amplify them from genomic DNA isolated from individual mature adult worms (MAW) taken from an experimentally infected dog in China and individual larval protoscoleces (PSC) excised from a single hydatid cyst taken from an Australian kangaroo. We then used real-time PCR to measure expression of each of the genes comprising the five EgAgB subfamilies in all life-cycle stages including the oncosphere (ONC).

Conclusions/Significance

Based on sequence alignment analysis, we found that the EgAgB gene family comprises at least ten unique genes. Each of the genes was identical in both larval and adult E. granulosus isolates collected from two geographical areas (different continents). DNA alignment comparisons with EgAgB sequences deposited in GenBank databases showed that each gene in the gene family is highly conserved within E. granulosus, which contradicts previous studies claiming significant variation and polymorphism in EgAgB. Quantitative PCR analysis revealed that the genes were differentially expressed in different life-cycle stages of E. granulosus with EgAgB3 expressed predominantly in all stages. These findings are fundamental for determining the expression and the biological function of antigen B.     

Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes

Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains.     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.     

Intraspecific variability among NADH dehydrogenase subunit 1 sequences of Taenia hydatigena.

This paper describes intraspecific variability of the partial sequences of the mitochondrial ND1 gene among isolates of Taenia hydatigena from pigs in Poland, Ukraine and Wales. The differences between studied isolates ranged from 0.4 to 5.5%, which exceeds the variability within the same fragment between the different genetic variants of Echinococcus multilocularis and is comparable with the variability between the most closely related strains (G5/G6/G7) of E. granulosus. The biggest difference (5.5%) was found between the geographically most distant Ukrainian and Welsh samples of T. hydatigena while the samples collected from the neighbouring locations in Poland, were most similar to each other.     

Hydrogen cyanide synthesis and antifungal activity of the biocontrol strain Pseudomonas fluorescens In5 from Greenland is highly dependent on growth medium

Hydrogen cyanide (HCN) is a secondary metabolite produced by many antagonistic Pseudomonas species. In the present study, the gene cluster encoding HCN synthesis in a newly isolated Pseudomonas fluorescens strain, In5, from South Greenland was investigated. Sequence analysis showed that the Greenlandic hcn gene cluster comprises a novel hcn cluster. Transposon mutagenesis of strain In5 resulted in mutants In5-2E1 and In5-1H7 with no production of HCN, and mutant In5-6B9 with reduced HCN synthesis. In mutant In5-2E1, the transposon was inserted into the hcnC gene; in mutant In5-1H7, the Tn5 insertion was found in a region upstream of a putative malate:quinone oxidoreductase gene (mqo); and in mutant In5-6B9, the transposon disrupted a probable enoyl-CoA hydratase/isomerase gene. In vitro inhibition experiments with In5 (wild type) and In5-2E1 (mutant) showed that in nitrogen-rich Luria-Bertani medium, strain In5 but not the hcn mutant In5-2E1 produced HCN and inhibited the growth of hyphae of Rhizoctonia solani and Pythium aphanidermatum . In contrast, when cultivating the strains in the carbohydrate-rich potato dextrose medium, neither of the strains produced any HCN, and thus, they were unable to inhibit hyphal growth of fungi. These experiments strongly indicate that the synthesis of HCN is highly dependent on the growth medium used.