A New Genotype of Trichophyton quinckeanum with Point Mutations in Erg11A Encoding Sterol 14-α Demethylase Exhibits Increased Itraconazole Resistance.

Trichophyton quinckeanum, the causative agent of mouse favus, has been responsible for several infections of animal owners in recent years and showed an infection peak around 2020 in Jena, Thuringia. The isolated T. quinckeanum strains from Thuringia differ in some positions of the ITS region compared to strains from the IHEM collection as well as to Trichophyton schoenleinii. All T. quinckeanum strains of the new genotype show up to a 100-fold increased itraconazole resistance as measured by microplate laser nephelometry (MLN) assays. Analysis of genes involved in Trichophyton indotineae azole resistance, such as Erg1, which encodes squalene epoxidase, and Erg11B, one of two copies of the sterol 14-α demethylase gene, show a 100% identity between the two T. quinckeanum genotypes. In contrast, Erg11A fragments differ in 15-nucleotide positions between both T. quinckeanum genotypes, resulting in the unique amino acid substitution Ala256Ser in resistant strains. The new T. quinckeanum genotype may have evolved through interspecies mating. Mating type analysis showed a nearly 100% identity of the minus type MAT1-1-1 fragment for all T. quinckeanum isolates. The closely related Trichophyton schoenleinii belongs to the plus mating type and has 100% identical fragments of Erg1 and Erg11B. Erg11A protein sequences of T. schoenleinii and T. quinckeanum showed increased diversity.

[Recurrent tinea corporis generalisata due to Terbinafine-resistant Trichophyton rubrum strain : Long-term treatment with super bioavailability itraconazole]. / Rezidivierende Tinea corporis generalisata durch einen Terbinafin-resistenten Trichophyton-rubrum-Stamm : Langzeitbehandlung mit Super-Bioavailability-Itraconazol.

For more than 30 years, an 82-year-old man has been suffering from tinea corporis generalisata in the sense of Trichophyton rubrum syndrome. The patient received long-term treatment with terbinafine. Fluconazole had no effect. There was an increase in liver enzymes with itraconazole. Super bioavailability (SUBA) itraconazole was initially not tolerated. A therapy attempt with voriconazole was successful, but was stopped due to side effects. The Trichophyton (T.) rubrum strain isolated from skin scales was tested for terbinafine resistance using the breakpoint method and found to be (still) sensitive. Sequencing of the squalene epoxidase (SQLE) gene revealed a previously unknown point mutation of the codon for isoleucine ATC→ACC with amino acid substitution I479T (isoleucine479 threonine). Long-term therapy with terbinafine 250 mg had been given every 3 days since 2018. In addition, bifonazole cream, ciclopirox solution, and occasionally terbinafine cream were used. The skin condition was stable until an exacerbation of the dermatophytosis in 2021. There were erythematosquamous, partly atrophic, centrifugal, scaly, confluent plaques on the integument and the extremities. Fingernails and toenails had white to yellow-brown discoloration, and were hyperkeratotic and totally dystrophic. T. rubrum was cultured from skin scales from the integument, from the feet, from nail shavings from the fingernails and also toenails and detected by PCR. In the breakpoint test, the T. rubrum isolates from tinea corporis and nail samples showed a minimum inhibitory concentration (MIC) of 0.5 µg ml-1 (terbinafine resistance in vitro). Sequencing of the SQLE gene of the T. rubrum isolate revealed evidence of a further point mutation that led to amino acid substitution I479V (isoleucine 479 valine). Long-term therapy was started with SUBA itraconazole: 14 days 2â€¯× 1 capsule daily, then twice weekly administration of 2â€¯× 50 mg. During breaks in therapy, the mycosis regularly flared up again. Finally, 50 mg SUBA itraconazole was given 5 days a week, which completely suppressed the dermatophytosis. Topically, ciclopirox and miconazole cream were used alternately. In conclusion, in the case of recurrent and therapy-refractory dermatophytoses caused by T. rubrum, terbinafine resistance must also be considered in individual cases. An in vitro resistance test and point mutation analysis of the squalene epoxidase gene confirms the diagnosis. Itraconazole, also in the form of SUBA itraconazole, is the drug of choice for the oral antifungal treatment of these patients.

Efficacy of antifungal agents against fungal spores: An in vitro study using microplate laser nephelometry and an artificially infected 3D skin model.

Dermal fungal infections seem to have increased over recent years. There is further a shift from anthropophilic dermatophytes to a growing prevalence of zoophilic species and the emergence of resistant strains. New antifungals are needed to combat these fungi and their resting spores. This study aimed to investigate the sporicidal effects of sertaconazole nitrate using microplate laser nephelometry against the microconidia of Trichophyton, chlamydospores of Epidermophyton, blastospores of Candida, and conidia of the mold Scopulariopsis brevicaulis. The results obtained were compared with those from ciclopirox olamine and terbinafine. The sporicidal activity was further determined using infected three-dimensional full skin models to determine the antifungal effects in the presence of human cells. Sertaconazole nitrate inhibited the growth of dermatophytes, molds, and yeasts. Ciclopirox olamine also had good antifungal activity, although higher concentrations were needed compared to sertaconazole nitrate. Terbinafine was highly effective against most dermatophytes, but higher concentrations were required to kill the resistant strain Trichophyton indotineae. Sertaconazole nitrate, ciclopirox olamine, and terbinafine had no negative effects on full skin models. Sertaconazole nitrate reduced the growth of fungal and yeast spores over 72 h. Ciclopirox olamine and terbinafine also inhibited the growth of dermatophytes and molds but had significantly lower effects on the yeast. Sertaconazole nitrate might have advantages over the commonly used antifungals ciclopirox olamine and terbinafine in combating resting spores, which persist in the tissues, and thus in the therapy of recurring dermatomycoses.

Point mutations in the squalene epoxidase erg1 and sterol 14-α demethylase erg11 gene of T indotineae isolates indicate that the resistant mutant strains evolved independently.

BACKGROUND: The T indotineae population shows a high amount of terbinafine resistant isolates based on different point mutations of squalene epoxidase erg1 (ergosterol) gene. A significant proportion of these isolates also show azole resistance. OBJECTIVES: Elucidation of the molecular mechanism for azole resistance, especially the identification of mutations in the sterol 14-α demethylase Erg11 genes, which encode for enzymes interacting with azoles. METHODS: Sequencing of putative Erg11 genes and analysis of phenotypic resistance pattern using a microplate-laser-nephelometry-based growth assay. RESULTS: Four different types of Erg11B mutants were detected; two double mutants with Ala230Thr/Asp441Gly, respectively, Ala230/Tyr444His and single mutants with Gly443Glu, Tyr444Cys and Tyr444His. All isolates featured the wild type genotype of Erg11A. All strains demonstrated different combinations of Erg1 and Erg11 genotypes. CONCLUSION: Resistance against terbinafine and azoles developed several times independently within the T indotineae population. The challenge for fungal treatment is, therefore, that species identification is not enough for prediction of therapeutic efficacy of antifungals. In the future, it will also become important to analyse genes involved in resistance mechanisms.

Spread of Terbinafine-Resistant Trichophyton mentagrophytes Type VIII (India) in Germany-"The Tip of the Iceberg?"

Chronic recalcitrant dermatophytoses, due to Trichophyton (T.) mentagrophytes Type VIII are on the rise in India and are noteworthy for their predominance. It would not be wrong to assume that travel and migration would be responsible for the spread of T. mentagrophytes Type VIII from India, with many strains resistant to terbinafine, to other parts of the world. From September 2016 until March 2020, a total of 29 strains of T. mentagrophytes Type VIII (India) were isolated. All patients were residents of Germany: 12 females, 15 males and the gender of the remaining two was not assignable. Patients originated from India (11), Pakistan (two), Bangladesh (one), Iraq (two), Bahrain (one), Libya (one) and other unspecified countries (10). At least two patients were German-born residents. Most samples (21) were collected in 2019 and 2020. All 29 T. mentagrophytes isolates were sequenced (internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1-α)). All were identified as genotype VIII (India) of T. mentagrophytes. In vitro resistance testing revealed 13/29 strains (45%) to be terbinafine-resistant with minimum inhibitory concentration (MIC) breakpoints ≥0.2 µg/mL. The remaining 16 strains (55%) were terbinafine-sensitive. Point mutation analysis revealed that 10/13 resistant strains exhibited Phe397Leu amino acid substitution of squalene epoxidase (SQLE), indicative for in vitro resistance to terbinafine. Two resistant strains showed combined Phe397Leu and Ala448Thr amino acid substitutions, and one strain a single Leu393Phe amino acid substitution. Out of 16 terbinafine-sensitive strains, in eight Ala448Thr, and in one Ala448Thr +, new Val444 Ile amino acid substitutions were detected. Resistance to both itraconazole and voriconazole was observed in three out of 13 analyzed strains. Treatment included topical ciclopirox olamine plus topical miconazole or sertaconazole. Oral itraconazole 200 mg twice daily for four to eight weeks was found to be adequate. Terbinafine-resistant T. mentagrophytes Type VIII are being increasingly isolated. In Germany, transmission of T. mentagrophytes Type VIII from the Indian subcontinent to Europe should be viewed as a significant public health issue.

Indian Trichophyton mentagrophytes squalene epoxidase erg1 double mutants show high proportion of combined fluconazole and terbinafine resistance.

BACKGROUND: The Indian ITS genotype VIII Trichophyton mentagrophytes population shows a high amount of different erg1 (ergosterol) mutants encoding for squalene epoxidase, which catalyses the first step of ergosterol biosynthesis. OBJECTIVES: Illumination of the implication of point mutations at position Ala448Thr in single and double erg1 T mentagrophytes mutants because mutants of this type were abundantly found within the Indian fungal population. METHODS: Growth in fluconazole or terbinafine containing medium was analysed using a microplate-laser-nephelometry (MLN)-based growth assay. RESULTS: Ala 448 Thr erg1 single mutants were terbinafine sensitive, but about 50% of isolates showed an increased fluconazole resistance, whereas 95% of the double mutants (Phe 397 Leu, Ala 448Thr) demonstrated combined terbinafine and increased fluconazole resistance. CONCLUSION: The new Indian T mentagrophytes populations show several point mutations in erg1. Point mutations at position 397 were previously described and cause terbinafine resistance. A large part of the double mutants exhibit resistance to terbinafine and fluconazole, demonstrating a selective advantage of the combination of both mutations.

Alarming India-wide phenomenon of antifungal resistance in dermatophytes: A multicentre study.

BACKGROUND: An alarming increase in recalcitrant dermatophytosis has been witnessed in India over the past decade. Drug resistance may play a major role in this scenario. OBJECTIVES: The aim of the present study was to determine the prevalence of in vitro resistance to terbinafine, itraconazole and voriconazole in dermatophytes, and to identify underlying mutations in the fungal squalene epoxidase (SQLE) gene. PATIENTS/METHODS: We analysed skin samples from 402 patients originating from eight locations in India. Fungi were identified by microbiological and molecular methods, tested for antifungal susceptibility (terbinafine, itraconazole, voriconazole), and investigated for missense mutations in SQLE. RESULTS: Trichophyton (T.) mentagrophytes internal transcribed spacer (ITS) Type VIII was found in 314 (78%) samples. Eighteen (5%) samples harboured species identified up to the T interdigitale/mentagrophytes complex, and T rubrum was detected in 19 (5%) samples. 71% of isolates were resistant to terbinafine. The amino acid substitution Phe397Leu in the squalene epoxidase of resistant T mentagrophytes was highly prevalent (91%). Two novel substitutions in resistant Trichophyton strains, Ser395Pro and Ser443Pro, were discovered. The substitution Ala448Thr was found in terbinafine-sensitive and terbinafine-resistant isolates but was associated with increased MICs of itraconazole and voriconazole. CONCLUSIONS: The high frequencies of terbinafine resistance in dermatophytes are worrisome and demand monitoring and further research. Squalene epoxidase substitutions between Leu393 and Ser443 could serve as markers of resistance in the future.

Point mutations in the squalene epoxidase gene of Indian ITS genotype VIII T. mentagrophytes identified after DNA isolation from infected scales.

Terbinafine resistant Indian ITS genotype VIII T. mentagrophytes strain was identified by partial sequencing of the squalene epoxidase gene using DNA isolated from infected scales. This method allowed the rapid identification of single point mutations within the squalene epoxidase gene, even before a fungal culture was obtained. Terbinafine resistance was indicated by the amino acid position switch Phe397Leu based on a single point mutation of the codon changing it from TTC to CTC.

A clarion call for preventing taxonomical errors of dermatophytes using the example of the novel Trichophyton mentagrophytes genotype VIII uniformly isolated in the Indian epidemic of superficial dermatophytosis.

An alarming pan Indian increase in the incidence of superficial dermatophytosis has been noticed over the past 5-6 years. Recent studies have demonstrated emerging predominance of Trichophyton (T.) mentagrophytes as the causative organism in such cases. Interestingly, a distinct Indian genotype of T. mentagrophytes has been identified and recognised with the help of sequencing of the ITS region of the rDNA. That has, however, led to a basic confusion owing to the newly introduced taxonomy of dermatophytes in 2017. According to this most recently suggested classification and new taxonomy of dermatophytes, the former "T. mentagrophytes complex" is differentiated into T. mentagrophytes (zoophilic strains) and T. interdigitale (anthropophilic strains). We have noticed that in some recent studies the causative agent of the chronic, relapsing dermatophytosis outbreak in India has been described as T. interdigitale. In our opinion, it is very likely that these T. interdigitale strains isolated in Delhi and Chennai in India are indeed strains more closely related to the neotype of T. mentagrophytes and not strains of T. interdigitale. We therefore want to underscore the importance of a common nomenclature of species in accordance with the new taxonomy of dermatophytes. This would most likely facilitate better understanding of the issue amongst dermatologists and microbiologists in general. Mistaken identification of Trichophyton isolates not limited to India is very likely to occur due to the lack of appropriate molecular diagnosis which in turn is based on the already published data that presumably wrongly identify one species instead of the other.

The current Indian epidemic of superficial dermatophytosis due to Trichophyton mentagrophytes-A molecular study.

The disease burden of chronic-relapsing and therapy-refractory superficial dermatophytosis dramatically increased in India within the past 5-6 years. In order to evaluate the prevalence of this trend, 201 skin scrapings were collected from patients from all parts of India and were tested for dermatophytes using both fungal culture and a PCR-ELISA directly performed with native skin scrapings. Fungal culture material was identified by genomic Sanger sequencing of the internal transcribed spacer (ITS) region and the translation elongation factor (TEF)-1α gene. In total, 149 (74.13%) out of the 201 samples showed a dermatophyte-positive culture result. Out of this, 138 (92.62%) samples were identified as Trichophyton (T.) mentagrophytes and 11 (7.38%) as Trichophyton rubrum. The PCR-ELISA revealed similar results: 162 out of 201 (80.56%) samples were dermatophyte-positive showing 151 (93.21%) T mentagrophytes- and 11 (6.79%) T rubrum-positive samples. In this study, we show for the first time a dramatic Indian-wide switch from T rubrum to T mentagrophytes. Additionally, sequencing revealed a solely occurring T mentagrophytes "Indian ITS genotype" that might be disseminated Indian-wide due to the widespread abuse of topical clobetasol and other steroid molecules mixed with antifungal and antibacterial agents.

The fate of mitochondria after infection of the Mucoralean fungus Absidia glauca by the fusion parasite Parasitella parasitica: comparison of mitochondrial genomes in zygomycetes.

Absidia glauca and Parasitella parasitica constitute a versatile experimental system for studying horizontal gene transfer between a mucoralean host and its fusion parasite. The A. glauca chondriome has a length of approximately 63 kb and a GC content of 28%. The chondriome of P. parasitica is larger, 83 kb, and contains 31% GC base pairs. These mtDNAs contain the standard fungal mitochondrial gene set, small and large subunit rRNAs, plus ribonuclease P RNA. Comparing zygomycete chondriomes reveals an unusually high number of homing endonuclease genes in P. parasitica, substantiating the mobility of intron elements independent of host-parasite interactions.

Post-translational regulation by structural changes of 4-dihydromethyltrisporate dehydrogenase, a key enzyme in sexual and parasitic communication mediated by the trisporic acid pheromone system, of the fungal fusion parasite Parasitella parasitica.

Sexual communication between complementary mating partners in the fungal group of zygomycetes is mediated by the trisporoid pheromone system. A key enzyme towards biosynthesis of hormonally active trisporoids is 4-dihydromethyltrisporate dehydrogenase (TSP1), an enzyme occurring in all zygomycetous fungi. Trisporic acid and some of its precursor molecules serve as pheromones for recognizing complementary mating partners and for induction of the differentiation program towards sexual spore formation. In the parasitic zygomycete Parasitella parasitica, a biotrophic fusion parasite infecting many other zygomycetes, these substances have an additional function: They are also responsible for host-parasite recognition and the formation of the characteristic infection structures. Parasitic interactions are mating type dependent as well. In the Mucor-related mycoparasite P. parasitica we can study both types of communication in parallel. We were interested in protein structures of TSP1 from P. parasitica, the genome of which was recently sequenced by us, and especially in the mechanisms involved in the switch from sexual to parasitic communication. P. parasitica contains at least six genes coding for TSP1-like proteins. We created models of tertiary structures and performed protein-protein docking with the resulting protein structures to simulate dimerization and to provide support for probable regulatory mechanisms at the protein level. The resulting structure models show differences in putative activity and binding preferences between the different TSP1-like proteins. Two of them seem to be able to form solid binding pockets for substrate and cosubstrate after dimerization. The other four TSP1-like proteins are more likely to represent regulating subunits for the two active isoforms. The ability to form homodimers with enzymatic activity could be the crucial difference between sexual and parasitic communication pathways. TSP1 PARPA_07791 forms enzymatically inactive homodimers. The second TSP1, PARPA_04105, forms active homodimers and could be responsible for the parasitic pathway of communication. Both TSP1 proteins can form more or less active heterodimers with the additional TSP1-like proteins. TSP1 PARPA_07791 mediates the sexual pathway probably as in other zygomycetous fungi like Mucor mucedo. High sequence identities between this TSP1 isomer and TSP1 proteins from other zygomycetes substantiate its function. This bioinformatic study supports previous experimental findings of post-translational regulation of 4-dihydromethyltrisporate dehydrogenases in zygomycetes and, for the first time, provides a substantiated hypothesis of the underlying mechanism.

Complete Mitochondrial DNA Sequence of the Mucoralean Fungus Absidia glauca, a Model for Studying Host-Parasite Interactions.

The mitochondrial DNA (mtDNA) ofAbsidia glaucahas been completely sequenced. It is 63,080 bp long, has a G+C content of 28%, and contains the standard fungal gene set.A. glaucais the recipient in a laboratory model for horizontal gene transfer withParasitella parasiticaas a donor of nuclei and mitochondria.

Ingestion and digestion studies in Tetrahymena pyriformis based on chemically modified microparticles.

Recognition of food and, in consequence, ingestion of digestible particles is a prerequisite for energy metabolism in Tetrahymena pyriformis. Understanding why some particles are ingested and digested, whereas others are not, is important for many fields of research, e.g. survival of pathogens in single-celled organisms or establishment of endosymbiotic relationships. We offered T. pyriformis synthetical bovine-serum-albumin (BSA)-methacrylate microparticles of approximately 5.5 µm diameter and studied the ciliates' ingestion and digestion behaviour. Different staining techniques as well as co-feeding with a transformant strain of Escherichia coli revealed that T. pyriformis considers these particles as natural food source and shows no feeding preference. Further, they are ingested at normal rates and may serve as sole food source. A pivotal advantage of these particles is the convenient modification of their surface by binding different ligands resulting in defined surface properties. Ingestion rate of modified microparticles either increased (additional BSA, enzymes) or decreased (amino acids). Furthermore, we investigated glycosylation patterns by lectin binding. By binding different substances to the surface in combination with various staining techniques, we provide a versatile experimental tool for elucidating details on food recognition and digestion that may allow to study evading digestion by pathogens or potential endosymbionts, too.

Complete Mitochondrial DNA Sequence of the Mucoralean Fusion Parasite Parasitella parasitica.

The complete mitochondrial DNA sequence of the Mucor-related fungus Parasitella parasitica has been sequenced. It has a G+C content of 30% and a total length of 83,361 bp. All protein-coding genes normally found in fungi are present in the sequence. A special feature is the remarkably high number of 27 homing endonucleases.

A model for endosymbiosis: interaction between Tetrahymena pyriformis and Escherichia coli.

Endosymbiosis in ciliates is a common and highly diverse phenomenon in nature, but its development at the mechanistic level and the origins are not easy to understand, since these associations may have arisen at any time during evolution. Therefore a laboratory model is helpful. It could be provided by the interaction of Tetrahymena pyriformis and Escherichia coli. Microscopic analyses with a genetically manipulated fluorescent strain of E. coli show single bacteria leaving food vacuoles and escaping digestion, an important prerequisite for further experiments. Under selective conditions, beneficial for T. pyriformis, the ciliate was shown to internalize E. coli cells. After feeding, bacteria, transformed with the plasmids pBS-neoTet or pNeo4, provide T. pyriformis with the ability to handle toxic conditions, caused by the aminoglykoside antibiotic paromomycin. Axenic cultures or cocultures with untransformed bacteria show lower cell numbers and survival rates compared to cocultures with transformed bacteria after transfer to paromomycin containing media. PCR detects bacterial DNA inside T. pyriformis cells. Additionally, microscopical analysis of selectively grown cocultures reveals fluorescing particles in the cytoplasm of T. pyriformis containing DNA and lipids, corresponding in size to E. coli. This system could be a reasonable model for understanding mechanisms of endosymbiosis establishment in ciliates.

Complementation of a stable Met2-1 mutant of the zygomycete Absidia glauca by the corresponding wild-type allele of the mycoparasite Parasitella parasitica, transferred during infection.

Compared with prokaryotes, where horizontal gene transfer events are frequently found and can be studied in the laboratory at the mechanistic level, few systems are known that allow direct experimental access to parasexual phenomena in eukaryotes. In zygomycetes, a basal lineage of fungi, several mycoparasitic fungi are known that inevitably form a cytoplasmic continuum with their hosts during infection. We provide evidence that, corresponding to the expectation suggested by the morphology of the infection process, gene transfer occurs from the parasite to the host. For analysing this parasexual system at the DNA level, we characterized interspecific recombinants obtained by infecting a stable methionine-auxotrophic Absidia glauca mutant with heavy rearrangements at the Met2-1 locus, which encodes homoserine acetyltransferase. Recipients were shown to be complemented by part of the corresponding gene from Parasitella parasitica. This foreign DNA is neither integrated at the putative Met2-2 locus in the recipient strain nor integrated at Met2-1, a locus encoding a hypothetical protein with amino acid similarity but with unknown function. Based on hybridization studies and on the phenotype of recipients that bear some mitotic instability of the acquired prototrophy, we propose that P. parasitica DNA is established in A. glauca recipients as extrachromosomally located replicons.

The mating-related loci sexM and sexP of the zygomycetous fungus Mucor mucedo and their transcriptional regulation by trisporoid pheromones.

The putative mating type locus of mucoralean fungi consists of a single high mobility group (HMG)-domain transcription factor gene, sexM or sexP, flanked by genes for an RNA helicase and a triosephosphate transporter. We used degenerate primers derived from the amino acid sequence of the RNA helicase to sequence a fragment of this gene from Mucor mucedo. This fragment was extended by inverse PCR to obtain the complete sequences of the sex loci from both mating types of M. mucedo. The sex loci in M. mucedo reflect the general picture obtained previously for Phycomyces blakesleeanus, presenting a single HMG-domain transcription factor gene, sexM and sexP in the minus and plus mating types, respectively. These are located next to a gene for RNA helicase. Transcriptional analysis by quantitative real-time PCR showed that only transcription of sexM is considerably stimulated by adding trisporoid pheromones, thus mimicking sexual stimulation, whereas sexP is only slightly affected. These differences in regulation between sexM and sexP are supported by the observation that the promoter sequences controlling these genes show no similarities. The protein structures themselves are considerably different. The SexM, but not the SexP protein harbours a nuclear localization sequence. The SexM protein is indeed transported to nuclei. This was shown by means of a GFP fusion construct that was used to study the localization of SexM in the yeast Saccharomyces cerevisiae. The fusion protein is highly enriched in nuclei.

Transformation of the fungus Absidia glauca by complementation of a methionine-auxotrophic strain affected in the homoserine-acetyltransferase gene.

Transformation of fungi by complementation of auxotrophs is generally much more reliable than usage of antibiotic resistance markers. In order to establish such a system for the model zygomycete Absidia glauca, a stable methionine auxotrophic mutant was isolated after X-ray mutagenesis of the minus mating type and characterized at the molecular level. The mutant is disrupted in the coding region of the Met2-1 gene, encoding homoserine O-acetyltransferase. The corresponding wild type gene was cloned, sequenced and inserted into appropriate vector plasmids. Transformants are prototrophs and show restored methionine-independent growth, based on complementation by the autonomously replicating plasmids.