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Summary: We present a case report of a young female patient with complicated course of inflammatory bowel disease with severe luminal, as well as perianal symptoms. The patient was initially dia­gnosed with ulcerative colitis and underwent proctocolectomy with ileopouch-anal anastomosis. One year after oostomy closure she got pregnant and, at the same time, she developed perianal abscess and fistula. The dia­gnosis was thus changed to Crohn’s disease. During pregnancy, she underwent repeated perianal fistula drainages, surgical revision of ileus and treatment with ustekinumab was started. The patient delivered a healthy boy in November 2022 at 38th gestational week. Key words: Crohn’s disease – perianal Crohn’s disease – ustekinumab – pregnancy

Ecosystems worldwide are exposed to pollutants connected to the industrial production of pharmaceuticals. The objective of this study was to study the composition and characteristics of the soil microbial communities that had been exposed to long-term selection pressure caused by the industrial production of penicillin G. Soil samples from four sites among the penicillin G production plant were analysed using 16S rRNA profiling via Illumina MiSeq platform and were compared with the control samples from four sites outside the plant. Total metagenomic DNA from the impacted soil was also used for the preparation of E. coli T1R-based fosmid library which was consequently qualitatively tested for the presence of penicillin G acylase (PGA)-encoding genes using the method of sequence homology. Analyses of alpha diversity revealed that the long-term antibiotic presence in the soil significantly increased the microbial diversity and richness in terms of Shannon diversity index (p = 0.002) and Chao estimates (p = 0.004). Principal component analysis showed that the two types of communities (on-site and control) could be separated at the phylum, class and genus level. The on-site soil was enriched in Betaproteobacteria, Deltaproteobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetia, while a significant decrease in Actinobacteria was observed. Metagenomic fosmid library revealed high hit rates in identifying PGAs (14 different genes identified) and confirmed the biotechnological potential of soils impacted by anthropogenic activity. This study offers new insights into the changes in microbial communities of soils exposed to anthropogenic activity as well as indicates that those soils may represent a hotspot for biotechnologically interesting targets.

Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors.

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed. © The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.

The sigma H (σΗ) and sigma E (σE) subunits of Corynebacterium glutamicum RNA polymerase belong to Group 4 of sigma factors, also called extracytoplasmic function (ECF) sigma factors. Genes of the C. glutamicum σΗ regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the σE regulon, which is involved in cell surface stress response, have not been explored until now. Using the C. glutamicum RES167 strain and its derivative C. glutamicum ΔcseE with a deletion in the anti-σΕ gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum ΔcseE compared to C. glutamicum RES167. To confirm the functional link between σΕ and the corresponding promoters, we tested selected promoters using the in vivo two-plasmid system with gfpuv as a reporter gene and by in vitro transcription. Analyses with RNAP+σΗ and RNAP+σΕ, which were previously shown to recognize similar promoters, proved that the σΗ and σE regulons significantly overlap. The σE-controlled genes were found to be involved for example in protein quality control (dnaK, dnaJ2, clpB, and clpC), the regulation of Clp proteases (clgR), and membrane integrity maintenance. The single-promoter analyses with σΗ and σΕ revealed that there are two groups of promoters: those which are exclusively σΗ-specific, and the other group of promoters, which are σΗ/σE-dependent. No exclusively σE-dependent promoter was detected. We defined the consensus sequences of exclusively σΗ-regulated promotors to be −35 GGAAt and − 10 GTT and σΗ/σE-regulated promoters to be −35 GGAAC and − 10 cGTT. Fifteen genes were found to belong to the σΗ/σΕ regulon. Homology modeling showed that there is a specific interaction between Met170 in σΗ and the nucleotides −31 and − 30 within the non-coding strand (AT or CT) of the σΗ-dependent promoters. In σE, Arg185 was found to interact with the nucleotides GA at the same positions in the σE-dependent promoters.

Natonodosa is introduced as new genus based on morphology and multigene phylogenetic data, and the monotypic genus Natonodosa, typified by N. speciosa, is characterized by mononematous conidiophores, polyblastic, nodose conidiogenous cells and unicellular, fusiform, hyaline conidia. The other freshly collected material was identified as Poroisariopsis inornata, a species that since its original description had not been reported; it is characterized by synnematous conidiophores with polytretic, cylindrical and uncinate conidiogenous cells with percurrent extensions, and distoseptate, obclavate, brown conidia. A lectotype and an epitype for the species are established and referred since the original specimen of this microfungus has not been found in any herbarium. Placement of both taxa in Xylariales was ascertained by BLASTn searches. Maximum likelihood analysis of the combined sequence data of the ribosomal rDNA (ITS and partial LSU region), as well as partial gene regions coding for the second largest subunit of RNA polymerase II (RPB2) and beta-tubulin (TUB2), was performed. In addition, a new name, Guarroa, is proposed for replacing the illegitimate genus name Phaeobotrys M. Calduch, Gene & Guarro. A dichotomous key to the new genus and morphologically similar taxa is provided.

The Pleuroascaceae (Leotiomycetes) is introduced for Phialophora hyalina (section Catenulatae) and its closest relatives based on analyses of DNA sequences of five gene regions and the comparison of cultural and micromorphological characters. The family is resolved as a strongly supported clade that encompasses Pleuroascus and the new anamorph genera Entimomentora and Venustampulla. The latter includes V. parva, a species placed formerly in Scopulariopsis, and V. echinocandica, which is established for the echinocandin-producing isolate BP-5553. Entimomentora includes E. hyalina, a species based on the ex-type strain of Ph. hyalina. Additional isolates identified as Ph. hyalina are distantly related to the Pleuroacaceae and include Psychrophila antarctica (Arachnopezizaceae) and Cryonesomyces dreyfussii, the sole member of the new genus Cryonesomyces (incertae sedis). Isolates identified or deposited as Ph. alba are also not closely related; they include a species for which we propose the name Neobulgaria koningiana (Gelatinodiscaceae) and a second psychrophilic species that we describe as Psychrophila lagodekhiensis. Of the 13 isolates assessed for in vitro antifungal activity, only V. echinocandica inhibited the growth of Candida albicans.

Ecosystems worldwide are exposed to pollutants connected to the industrial production of pharmaceuticals. The objective of this study was to study the composition and characteristics of the soil microbial communities that had been exposed to long-term selection pressure caused by the industrial production of penicillin G. Soil samples from four sites among the penicillin G production plant were analysed using 16S rRNA profiling via Illumina MiSeq platform and were compared with the control samples from four sites outside the plant. Total metagenomic DNA from the impacted soil was also used for the preparation of E. coli T1R-based fosmid library which was consequently qualitatively tested for the presence of penicillin G acylase (PGA)-encoding genes using the method of sequence homology. Analyses of alpha diversity revealed that the long-term antibiotic presence in the soil significantly increased the microbial diversity and richness in terms of Shannon diversity index (p = 0.002) and Chao estimates (p = 0.004). Principal component analysis showed that the two types of communities (on-site and control) could be separated at the phylum, class and genus level. The on-site soil was enriched in Betaproteobacteria, Deltaproteobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetia, while a significant decrease in Actinobacteria was observed. Metagenomic fosmid library revealed high hit rates in identifying PGAs (14 different genes identified) and confirmed the biotechnological potential of soils impacted by anthropogenic activity. This study offers new insights into the changes in microbial communities of soils exposed to anthropogenic activity as well as indicates that those soils may represent a hotspot for biotechnologically interesting targets.

Two new genera, Rhamphoriopsis and Xylolentia, are described for lignicolous perithecial ascomycetes occurring in terrestrial habitats. Fresh material, living cultures, morphology, and DNA sequence data (nuc rDNA internal transcribed spacers [ITS1-5.8S-ITS2 = ITS], 18S and 28S genes, and second largest subunit of RNA polymerase II = RPB2) of these taxa and morphologically similar fungi were studied to determine their relationships. A monophyletic clade including Rhamphoria, Rhodoveronaea, a dematiaceous hyphomycete Linkosia multiseptum, and the two new genera was recovered in the Sordariomycetes based on the 18S-28S-RPB2 data set. It is introduced as the family Rhamphoriaceae and strongly supported by Bayesian and maximum likelihood methods. Its members are characterized by perithecial ascomata with a cylindrical or rostrate neck, the absence of stromatic tissue or clypeus, similar anatomy of two-layered ascomatal walls, cylindrical paraphyses, unitunicate asci with a distinct, nonamyloid apical annulus, and dictyoseptate or transversely septate, hyaline or brown ascospores. The mode of conidiogenesis is holoblastic, predominantly on polyblastic-denticulate conidiogenous cells. The Phaeoisaria-like anamorph has been linked to Rhamphoria and Rhamphoriopsis, whereas conidia and conidiophores of Idriella-like synanamorph were formed in vitro in two species of Rhamphoria. The Veronaea-like anamorph is associated with Rhodoveronaea. The anamorph of Xylolentia is a dematiaceous hyphomycete with conidiogenous cells with sympodially extending rachis. A key to members of the family is provided. The classification and nature of species boundaries in Rhamphoria are discussed, and diagnostic characters such as ascospore shape, number of transverse and longitudinal septa, a degree of constriction at the septa, and ability to produce ascoconidia are evaluated.

The genus Ceratostomella has a long history of taxonomic confusion. While species with evanescent asci have been transferred to the Microascales and Ophiostomatales, the taxonomic status of species with persistent asci has not been completely resolved. In previous studies using DNA sequence data, cultures and morphology, several Ceratostomella spp. were allocated in 13 genera in the Eurotiomycetes and Sordariomycetes. In our study, the systematics of the remaining Ceratostomella spp. with persistent asci is revisited with new collection data, cultures and phylogeny based on novel DNA sequences from six nuclear loci. Bayesian inference and Maximum Likelihood analyses support the monophyly of several wood-inhabiting species formerly classified in Ceratostomella and other unknown morphologically similar taxa and their division into four genera, i.e. Lentomitella, Spadicoides, Torrentispora and the newly described Calyptosphaeria. This robust clade represents the order Xenospadicoidales in the Sordariomycetidae. Comparative analysis of the ITS2 secondary structure revealed a genetic variation among Lentomitella isolates; 11 species were recognised, of which five are newly introduced and two are new combinations. Other taxonomic novelties include four new species and eight new combinations in Calyptosphaeria, Spadicoides, and Torrentispora. Molecular data suggest that Spadicoides is polyphyletic. The core of the genus is positioned in the Xenospadicoidales; Spadicoides s. str. is experimentally linked with sexual morphs for the first time. Based on DNA sequence data, the monotypic genera Xenospadicoides and Pseudodiplococcium are reduced to synonymy under Spadicoides, while Fusoidispora and Pseudoannulatascus are synonymised with Torrentispora. Members of the Xenospadicoidales inhabit decaying wood in terrestrial and freshwater environments and share a few morphological characters such as the absence of stromatic tissue, ascomata with a cylindrical or rostrate neck, similar anatomies of the ascomatal walls, thin-walled unitunicate asci with a non-amyloid apical annulus, disintegrating paraphyses, usually ellipsoidal to fusiform ascospores and holoblastic-denticulate or tretic conidiogenesis. Revised Ceratostomella spp. with persistent asci are listed and the taxonomic status of each species is re-evaluated based on revision of the holotype and other representative material, published details and available phylogenetic data. Key words: Ceratostomella, Conidiogenesis, Holoblastic-denticulate, Molecular systematics, New taxa, Phaeoisaria-like, Selenosporella-like, Tretic, Taxonomy, Xenospadicoidales, Taxonomic novelties: New genus: Calyptosphaeria Réblová & A.N. Mill, New species: Calyptosphaeria collapsa Réblová & A.N. Mill., C. tenebrosa Réblová & A.N. Mill., Lentomitella magna Réblová, L. obscura Réblová, L. striatella Réblová, L. sulcata Réblová, L. tenuirostris Réblová, Torrentispora calembola Réblová & A.N. Mill., T. novae-zelandiae Réblová & A.N. Mill, New combinations: Calyptosphaeria subdenudata (Peck) Réblová & A.N. Mill., C. tropica (Huhndorf et al.) Réblová & A.N. Mill., Lentomitella conoidea (Feltg.) Réblová, L. investita (Schw.) Réblová, Spadicoides fuscolutea (Rehm) Réblová, S. hyalostoma (Munk) Réblová, Spadicoides iberica (Hern.-Restr. et al.) Réblová & A.N. Mill., Torrentispora aquatica (Vijaykr. et al.) Réblová & A.N. Mill., T. biatriispora (K.D. Hyde) Réblová & A.N. Mill., T. dubia (Sacc.) Réblová & A.N. Mill

A new genus Rhopalophora is described for Phialophora clavispora, a lignicolous species formerly placed in Phialophora section Catenulatae that possesses pigmented conidiophores, phialides with a single conidiogenous locus that occasionally appear as schizophialides, and clavate, aseptate conidia arranged in chains or sometimes in heads. Sexual morphs are not known for this taxon. Phylogenetic analysis of DNA sequences from five loci (nucSSU, ITS, nucLSU, mitSSU, rpb1 and rpb2) of this and related fungi supports the introduction of a new family, Sclerococcaceae, for which we establish the order Sclerococcales. This order belongs to the new subclass Sclerococcomycetidae, a strongly supported clade within the Eurotiomycetes that is basal to a lineage containing the Chaetothyriomycetidae, Coryneliomycetidae and Eurotiomycetidae. Rhopalophora clavispora fits in this new family and is closely related to an isolate of Fusichalara minuta. The Sclerococcales also encompass marine, lignicolous species of Dactylospora, two species of the lichenicolous genus Sclerococcum, and a lineage comprised of strains from the digestive tracts of Neotropical wood-inhabiting beetles. We confirm that Dactylospora is polyphyletic; the phylogenetic placement of D. parasitica, the generic type, remains unknown.

Penicillin G acylase is one of the most employed enzymes in the pharmaceutical industry due to its role in the biotransformation of semi-synthetic β-lactam antibiotics. Recently, the enantioselectivity of the penicillin G acylase markedly broadened its application potential. In this study, we have evaluated effects of in-silico replacements of acyl-binding subsite residue Phe24β of the enzyme from Achromobacter sp. CCM 4824 to seven markedly smaller amino acids on its enantioselectivity towards industrially relevant compounds. Models of the two most promising mutants bearing substitutions Pheβ24Ala and Pheβ24Cys were investigated using molecular docking calculations. The Cys substitution revealed much better enantioselectivity traits with a set of seven substrates. To verify the relevance of in-silico predictions, we constructed a PGA A + Phe24βCys mutant and determined its enantioselectivity in biocatalytic reactions. Since we experimentally confirmed all these predictions, we expanded our in-silico analysis to another set of seven compounds: the prediction suggested increased enantioselectivity for N -phenylacetyl- p- F-α-phenylalanine. The ( R )-enantiomer of this substrate is used as a building block in synthesis of important anti-cancer agent Abarelix. The enantioselectivity of PGA A + Phe24βCys mutant towards this substrate was improved by 75% reaching E -value of about 70. Our results suggest the rapid identification of interesting replacements altering enantioselectivity using in-silico approach as the way for further expanding biotechnological application of penicillin G acylase.

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) sigma factors. These factors sigma(C), sigma(D), sigma(E), sigma(H), and sigma(M) are mostly involved in stress responses. The role of sigma(D) consists in the control of cell wall integrity. The sigma(D) regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential sigma(D)-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly sigma(D)-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAAC(A)/G and -10 GAT. Surprisingly, nearly all of these sigma(D)-dependent promoters were also active to a much lower extent with sigma(H) in vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the sigma(H)-dependent promoters is different (-35 GGAA(T)/C and -10 GTT). In addition to the activity of sigma(H) at the sigma(D)-controlled promoters, we discovered separated or overlapping sigma(A) - or sigma(B)-regulated or sigma(H)-regulated promoters within the upstream region of 8 genes of the sigma(D)-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some sigma(D)-dependent genes. Computer modeling revealed that sigma(H) binds to the promoter DNA in a similar manner as sigma(D) to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in sigma(D) and Lys 53 in sigma(H), bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the sigma(D)-controlled genes also belong to the sigma(H)-regulon and are also transcribed from the overlapping or closely located housekeeping (sigma(A)-regulated) and/or general stress (sigma(B)-regulated) promoters.

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σC, σD, σE, σH, and σM are mostly involved in stress responses. The role of σD consists in the control of cell wall integrity. The σD regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential σD-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σD-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAACA/G and -10 GAT. Surprisingly, nearly all of these σD-dependent promoters were also active to a much lower extent with σH in vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the σH-dependent promoters is different (-35 GGAAT/C and -10 GTT). In addition to the activity of σH at the σD-controlled promoters, we discovered separated or overlapping σA- or σB-regulated or σH-regulated promoters within the upstream region of 8 genes of the σD-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σD-dependent genes. Computer modeling revealed that σH binds to the promoter DNA in a similar manner as σD to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σD and Lys 53 in σH, bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the σD-controlled genes also belong to the σH-regulon and are also transcribed from the overlapping or closely located housekeeping (σA-regulated) and/or general stress (σB-regulated) promoters.

Prace se zamýsli nad aktualnimi trendy slavistickeho badani v balkanistických oborech. Jako významne tema v poslednich dvaceti letech vidi badani o ceských mensinach na Balkanskem poloostrově, zejmena pak o původni zemědělske kolonizaci, ktera vytvořila dodnes životaschopne mensinove ostrovy v Charvatsku (Slavonie), Srbsku (Vojvodina – Banat), Rumunsku (Banat) a castecně i v Bosně a Hercegovině. Přibližuje historii ceskeho badani o mensinach na Balkaně, urcuje nove trendy v mensinovem badani a přinasi prakticky kompletni bibliografii zasadnich praci s touto tematikou v poslednich dvou desetiletich a v zavěru pak urcuje nove směry, ktere pro slavistiku badani o mensinach nabizi.

Thirteen morphologically similar strains of barbatosphaeria- and tectonidula-like fungi were studied based on the comparison of cultural and morphological features of sexual and asexual morphs and phylogenetic analyses of five nuclear loci, i.e. internal transcribed spacer rDNA operon (ITS), large and small subunit nuclear ribosomal DNA, β-tubulin, and second largest subunit of RNA polymerase II. Phylogenetic results were supported by in-depth comparative analyses of common core secondary structure of ITS1 and ITS2 in all strains and the identification of non-conserved, co-evolving nucleotides that maintain base pairing in the RNA transcript. Barbatosphaeria is defined as a well-supported monophyletic clade comprising several lineages and is placed in the Sordariomycetes incertae sedis. The genus is expanded to encompass nine species with both septate and non-septate ascospores in clavate, stipitate asci with a non-amyloid apical annulus and non-stromatic ascomata with a long decumbent neck and carbonised wall often covered by pubescence. The asexual morphs are dematiaceous hyphomycetes with holoblastic conidiogenesis belonging to Ramichloridium and Sporothrix types. The morphologically similar Tectonidula, represented by the type species T. hippocrepida, grouped with members of Barbatosphaeria and is transferred to that genus. Four new species are introduced and three new combinations in Barbatosphaeria are proposed. A dichotomous key to species accepted in the genus is provided.

Four morphologically similar specimens of an unidentified perithecial ascomycete were collected on decaying wood submerged in fresh water. Phylogenetic analysis of DNA sequences from protein-coding and ribosomal nuclear loci supports the placement of the unidentified fungus together with Achroceratosphaeria in a strongly supported monophyletic clade. The four collections are described as two new species of the new genus Pisorisporium characterised by non-stromatic, black, immersed to superficial perithecial ascomata, persistent paraphyses, unitunicate, persistent asci with an amyloid apical annulus and hyaline, fusiform, cymbiform to cylindrical, transversely multiseptate ascospores with conspicuous guttules. The asexual morph is unknown and no conidia were formed in vitro or on the natural substratum. The clade containing Achroceratosphaeria and Pisorisporium is introduced as the new order Pisorisporiales, family Pisorisporiaceae in the class Sordariomycetes. It represents a new lineage of aquatic fungi. A sister relationship for Pisorisporiales with the Lulworthiales and Koralionastetales is weakly supported by Bayesian inference and maximum likelihood analyses. The systematic position of Pisorisporium among morphologically similar perithecial ascomycetes is discussed.

The bacterial strain KDF8 capable of growth in the presence of diclofenac and codeine analgesics was obtained after chemical mutagenesis of nature isolates from polluted soils. The strain KDF8 was identified as Raoultella sp. based on its morphology, biochemical properties, and 16S rRNA gene sequence. It was deposited in the Czech Collection of Microorganisms under the number CCM 8678. A growing culture efficiently removed diclofenac (92% removal) and partially also codeine (about 30% degradation) from culture supernatants within 72 h at 28 °C. The degradation of six analgesics by the whole cell catalyst was investigated in detail. The maximum degradation of diclofenac (91%) by the catalyst was achieved at pHINI of 7 (1 g/L diclofenac). The specific removal rate at high concentrations of diclofenac and codeine increased up to 16.5 mg/gCDW per h and 5.1 mg/gCDW per h, respectively. HPLC analysis identified 4′-hydroxydiclofenac as a major metabolite of diclofenac transformation and 14-hydroxycodeinone as codeine transformation product. The analgesics ibuprofen and ketoprofen were also removed, albeit to a lower extent of 3.2 and 2.0 mg/gCDW per h, respectively. Naproxen and mefenamic acid were not degraded.

A Gram-negative bacterium transforming codeine 1a to 14-hydroxycodeine 1c was isolated from the sample of composted wastes of Papaver somniferum deposited in a factory producing poppy seed. Based on 16S ribosomal RNA gene phylogeny, biochemical and morphological properties, the strain was designated as Rhizobium radiobacter R89-1. The bacterium is able to transform 1a into four C-14-hydroxy derivatives of codeine. When used as a whole-cell catalyst, 100% conversion was achieved at concentration of 1a up to 6.0 g/L to 14-hydroxycodeine 1c as a single product. Biohydroxylation of codeine to C-14-hydroxy derivatives, a high tolerance of the strain to 1a and activity with morphine 2a make the biocatalyst promising for pharmaceutical applications.

This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.

Two new genera, Atractospora and Rubellisphaeria, are described for perithecial ascomycetes occurring on decaying wood submerged in freshwater habitats. Their relationships with other morphologically similar fungi were investigated utilising four nuclear ribosomal and protein-coding loci. They are nested together with members of five orders and families and numerous genera incertae sedis in the Sordariomycetidae in a clade supported by Maximum Likelihood and Bayesian inference methods. Their closest relative is Lentomitella. Atractospora includes species characterised by dark, immersed to semi-immersed beaked ascomata lying horizontally to the host, stipitate asci with a pronounced non-amyloid apical annulus and hyaline, fusiform, septate, thick-walled ascospores with smooth or ornamented wall. Three novel species are introduced (A. decumbens, A. reticulata and A. verruculosa). A new combination for Aquaticola ellipsoidea is proposed in Atractospora based on molecular data and morphological characters. Rubellisphaeria, with a new species R. abscondita, is distinct by subhyaline to reddish brown, immersed ascomata with a lateral neck, short-stipitate asci and ellipsoidal smooth ascospores with a delayed formation of a middle septum. The sexual morph of the dematiaceous hyphomycete genus Myrmecridium was encountered for the first time on decaying wood submerged in a stream. It is nested in a strongly supported monophyletic clade of the Myrmecridiales. The novel species M. montsegurinum is described; it possesses papillate ascomata immersed beneath a clypeus with subhyaline to weakly pigmented ascomatal wall, long-stipitate asci with a non-amyloid apical annulus and three-septate, hyaline, ellipsoidal, delicately verruculose ascospores. A morphology-based key is provided to facilitate identification of species of Atractospora.

Optimised culture conditions for the recombinant prototrophic strain Escherichia coli RE3(pSEKau2) were established to obtain sufficient amounts of biomass with high specific epoxide hydrolase (EH) activity. Both batch and fed-batch cultivations resulted in high EH production after induction. The recombinant enzyme was extracted from three different crude extracts using an optimised polyethylene glycol-based aqueous two-phase system (ATPS) with yields of ≥95% and selectivity factors of ≥50. The purification factors of the optimised ATPS were dependent on the cultivation conditions used for biomass production and were determined to be 2.0, 2.6, and 8.2 for the three crude extracts. Several oxirane-activated supports were evaluated for immobilizing the enriched EH directly in the recovered polyethylene glycol-containing top phase of the ATPS. Eupergit C 250L proved to be the most interesting, resulting in an immobilized biocatalyst with an EH activity of 44 μmol min−1 (g of dry support)−1 for the fastest substrate tested. The immobilized EH exhibited a significantly higher thermal stability, with 50% of its initial activity maintained after 90 min at an incubation temperature of 40 °C, compared to 19 min for the free enzyme. Immobilization did not alter, to a large extent, the enantioselectivity and regioselectivity of the enzyme.

Metagenomic DNA is a rich source of genes encoding novel epoxide hydrolases (EHs). We retrieved two genes encoding functional EHs from total DNA isolated from biofilter-derived biomass, using PCR with EH-specific degenerate primers followed by genome-walking PCR. The degenerate primers were based on two EH-specific consensus sequences: HGWP and GHDWG. The resulting recombinant EHs, Kau2 and Kau8, were expressed in Escherichia coli , and their enantioselectivity and regioselectivity were determined using 13 different epoxide substrates. The EH Kau2 had broad substrate specificity and preferentially hydrolyzed epoxides with S -configuration. It showed high enantioselectivity towards aromatic epoxides such as styrene oxide, p -nitrostyrene oxide, and trans -1-phenyl-1,2-epoxypropane. In addition, Kau2 showed enantioconvergent hydrolysis activity. The EH Kau8 also showed broad substrate specificity and preferentially hydrolyzed epoxides with R -configuration. High enantioselectivity was observed for p -nitrostyrene oxide, and the hydrolysis activity of Kau8 was less enantioconvergent than that of Kau2. To determine the usefulness of Kau2 for synthetic applications, preparative-scale biohydrolysis reactions were performed. Specifically, two kinetic resolutions were carried out with 80 g/L of racemic trans -1-phenyl-1,2-epoxypropane, affording both (1 R ,2 R )-epoxide and the corresponding (1 R ,2 S )-diol in high enantiomeric excess (>99%) and good yield (>45%). In addition, a process based on enantioconvergent hydrolysis by the EH Kau2 was established for racemic cis -1-phenyl-1,2-epoxypropane at a concentration of 13 g/L, resulting in the formation of the corresponding (1 R ,2 R )-diol with a 97% yield and an enantiomeric excess exceeding 98%.

We describe a convenient method for amplification of novel epoxide hydrolase-encoding genes directly from the metagenome. In a first step, small specific regions of putative epoxide hydrolase genes were amplified by using PCR with degenerate consensus primers specific for prokaryotic epoxide hydrolases, and environmental DNA as template. In a second step, the sequence obtained from one randomly selected epoxide hydrolase gene fragment served as the starting point for genome-walking PCR. This technique enabled us to recover a complete novel epoxide hydrolase gene with a GC content of 64.7%. A database search revealed that this novel gene was 44% and 43% identical to two putative epoxide hydrolases from Ralstonia metallidurans and Ralstonia eutropha, respectively, at the amino acid level, the highest among all orthologs searched. The gene, which encodes a polypeptide with a molecular mass of 34 kDa, was cloned and overexpressed in Escherichia coli . The recombinant enzyme showed hydrolyzing activity toward aliphatic terminal epoxides with chain lengths ranging from 6 to 10 carbon atoms. In all cases, the enantioselectivity of the enzyme was low. Determination of the regioselectivity coefficients α R and α S revealed that the oxirane ring was attacked almost exclusively at the non-substituted carbon of the R -epoxide. The preference for attack at the non-substituted ring carbon of the S -epoxide was dependent on the chain length of the substrate and ranged from 55% to 78%, resulting in a partially enantioconvergent reaction.

Phylogenetic analyses of DNA sequences from nuclear ribosomal and protein-coding loci support the placement of several perithecial ascomycetes and dematiaceous hyphomycetes from freshwater and terrestrial environments in two monophyletic clades closely related to the Savoryellales. One clade formed by five species of Conioscypha and a second clade containing several genera of uncertain taxonomic status centred on Pleurothecium, represent two distinct taxonomic groups at the ordinal systematic rank. They are proposed as new orders, the Conioscyphales and Pleurotheciales. Several taxonomic novelties are introduced in the Pleurotheciales, i.e. two new genera (Adelosphaeria and Melanotrigonum), three novel species (A. catenata, M. ovale, Phaeoisaria fasciculata) and a new combination (Pleurotheciella uniseptata). A new combination is proposed for Savoryella limnetica in Ascotaiwania s.str. based on molecular data and culture characters. A strongly supported lineage containing a new genus Plagiascoma, species of Bactrodesmiastrum and Ascotaiwania persoonii, was identified as a sister to the Conioscyphales/Pleurotheciales/Savoryellales clade in our multilocus phylogeny. Together, they are nested in a monophyly in the Hypocreomycetidae, significantly supported by Bayesian inference and Maximum Likelihood analyses. Members of this clade share a few morphological characters, such as the absence of stromatic tissue or clypeus, similar anatomies of the 2-layered ascomatal walls, thin-walled unitunicate asci with a distinct, non-amyloid apical annulus, symmetrical, transversely septate ascospores and holoblastic conidiogenesis. They represent the only fungi in the Hypocreomycetidae with apically free, filiform to cylindrical, persistent or partially disintegrating paraphyses. The systematic placement of two other dematiaceous hyphomycetes was resolved based on DNA sequences; Phragmocephala stemphylioides is a member of the Pleurotheciales and Triadelphia uniseptata is within the Savoryellales.

The Calosphaeriales is revisited with new collection data, living cultures, morphological studies of ascoma centrum, secondary structures of the internal transcribed spacer (ITS) rDNA and phylogeny based on novel DNA sequences of five nuclear ribosomal and protein-coding loci. Morphological features, molecular evidence and information from predicted RNA secondary structures of ITS converged upon robust phylogenies of the Calosphaeriales and Togniniales. The current concept of the Calosphaeriales includes the Calosphaeriaceae and Pleurostomataceae encompassing five monophyletic genera, Calosphaeria, Flabellascus gen. nov., Jattaea, Pleurostoma and Togniniella, strongly supported by Bayesian and Maximum Likelihood methods. The structural elements of ITS1 form characteristic patterns that are phylogenetically conserved, corroborate observations based on morphology and have a high predictive value at the generic level. Three major clades containing 44 species of Phaeoacremonium were recovered in the closely related Togniniales based on ITS, actin and β-tubulin sequences. They are newly characterized by sexual and RNA structural characters and ecology. This approach is a first step towards understanding of the molecular systematics of Phaeoacremonium and possibly its new classification. In the Calosphaeriales, Jattaea aphanospora sp. nov. and J. ribicola sp. nov. are introduced, Calosphaeria taediosa is combined in Jattaea and epitypified. The sexual morph of Phaeoacremonium cinereum was encountered for the first time on decaying wood and obtained in vitro. In order to achieve a single nomenclature, the genera of asexual morphs linked with the Calosphaeriales are transferred to synonymy of their sexual morphs following the principle of priority, i.e. Calosphaeriophora to Calosphaeria, Phaeocrella to Togniniella and Pleurostomophora to Pleurostoma. Three new combinations are proposed, i.e. Pleurostoma ochraceum comb. nov., P. repens comb. nov. and P. richardsiae comb. nov. The morphology-based key is provided to facilitate identification of genera accepted in the Calosphaeriales.

Cryptic plasmid pRK2 of the strain Escherichia coli W (ATCC 9637), an ancestor of production strains for penicillin G acylase, was sequenced and characterized. Based on the data on replication region and origin (ori sequence AAC, 924–926 nt), the plasmid was classified as ColE1-like plasmid. DNA sequence analysis revealed five orfs hypothetical products of which shared a significant sequence similarity with putative proteins encoded by DNA of plasmid pColE1. orf1 codes for protein Rom involved in the control of plasmid replication, orfs 2–5 code for putative mobilization proteins (Mob A–D) that show a high level of similarity with the ones encoded by DNA of plasmids pColE1 and pLG13 (E. coli), pECL18 and pEC01 (Enterobacter cloacae), pSFD10 (Salmonella choleraesuis), and pScol7 (Shigella sonnei). Recombinant plasmids pRS11 (4.91 kbp), pRS12 (4.91 kbp), pRS2 (2.996 kbp), and pRS3 (2.623 kbp) that bear the Spectinomycin resistance determinant (SpcR) were prepared on the basis of nucleotide sequence of pRK2. These constructs are stably maintained in the population of E. coli cells grown in the absence of the selection pressure for 63 generations. The copy number of SpcR constructs in E. coli host grown in antibiotic-free LB medium ranges from 25 to 40 molecules per chromosomal equivalent.

Segregational stability of the recombinant plasmid pKA18 bearing the pga gene encoding penicillin G acylase (PGA) and the production stability for PGA was studied in Escherichia coli strain W and its descendants. The hosts differ in the number of indigenous plasmids (pRK1–3) and the specific activity of PGA. The production of PGA by the recombinant strain RE3(pKA18) is the highest among the hosts and the increase of activity is proportional to the increase of copy number (CN) of the plasmid pKA18. The plasmid is segregationally stable in spite of the presence of the indigenous plasmid pRK2 that belong to the same ColE1 plasmid family. Both plasmids (total sum of CN equals 271) are maintained without selection pressure during 102 generations. Subculture-based selection experiments taking advantage of a very high segregational and production stability of plasmid pKA18 in the host RE3 were used to study the optimization of the host–plasmid interaction. The clones with increased synthesis of the enzyme average value of 833 U/g DM were isolated: the expression of PGA per plasmid molecule increased in these strains from 3 to 4 U. Once this expression is reached, the population of segregationally stable, high-producing strains becomes heterogeneous: segregationally stable, low-producing clones and segregationally unstable, high-producing clones appear in population.

The monotypic genus Xylochrysis is introduced for a lignicolous perithecial ascomycete that possesses golden yellow ascomata with black glabrous necks, a three-layered ascomatal wall, persistent paraphyses, and cylindrical, long-stipitate unitunicate asci with an inamyloid apical annulus, and hyaline, ellipsoidal, unicellular ascospores. In culture it produces hyaline conidiophores with terminally arranged branches bearing metulae, conidiogenous cells and holoblastic conidia. Phylogenetic analysis of two ribosomal (nc18S and nc28S rDNA) and one protein-coding (RPB2) gene position this species within the Sordariomycetidae but without close ordinal or familial affiliation. Morphological and molecular DNA data support the recognition of this new genus and suggest that Xylochrysis is most closely related to the genera Ceratolenta, Cyanoannulus and Woswasia.

Three indigenous plasmids designated pRK1, pRK2 and pRK3 were identified among producers of penicillin G acylase (PGA) derived from the strain Escherichia coli W ATCC 9637. Their size and copy number (CN) in E. coli W were determined (kb; CN): pRK1 (80; 3.4), pRK2 (5.1; 71), and pRK3 (4.8; 13.7). Strain E. coli RE2 harboring these plasmids was used for selection of strains with reduced number of plasmids: the strain RE3 without plasmid pRK1 and the plasmid-less strain cERE3 were isolated. Indigenous plasmids did not code for the resistance determinants against 23 antibiotics and 10 heavy metals.

Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of β-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160 mM 6-APA, 350 mM acyl donor methylester[Symbol: see text]HCl, pH 6.3, 25 °C, reaction time of 200 min) with immobilized NPGA equaled 96.9 % and 91.1 %, respectively. The enzyme was highly thermostable with maximum activity at 60 °C (pH 8.0) and 65 °C (pH 6.0). Activity half-life at 60 °C (pH 8.0) and at 60 °C (pH 6.0) was 24 min and 6.9 h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230 mM 6-APA, 340 mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5 °C, pH 6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.

The systematics of the ascomycete genera Apiorhynchostoma, Endoxyla and Pseudovalsaria are reevaluated based on the comparison of cultural characteristics, teleomorph morphology and DNA sequence data. Analyses of sequences of the internal transcribed spacer (ITS) of the ribosomal DNA operon and the large subunit (LSU) of the nuclear ribosomal DNA gene resolve Boliniales as a robustly supported lineage comprising Apiorhynchostoma, Camarops, Camaropella, Cornipulvina, Endoxyla and Pseudovalsaria. Within Boliniales, species of Endoxyla form a strongly supported lineage. Apiorhynchostoma curreyi and Pseudovalsaria ferruginea group with Cornipulvina ellipsoides. Species of Camarops are paraphyletic and comprise two clades, one of which includes Camaropella. Boliniaceae is emended, Endoxyla mallochii is described as new and Apiorhynchostoma trabicola is considered a synonym of Apiorhynchostoma altipetum. We also propose the combinations Endoxyla occulta, Endoxylina luteobasis and Jobellisia peckii. Keys to genera included in the Boliniaceae and to species of Apiocamarops, Apiorhynchostoma and Endoxyla are provided.

A high-expression system for penicillin G acylase (PGA) was developed using the high PGA-producing strain Escherichia coli RE3 as a host and a recombinant plasmid pKA18 which was constructed by cloning the chromosomal pga gene coding for PGA in the strain RE3 on multicopy vector pK19. One particular objective for the elaboration of this expression system was the selection of a convenient host strain and modification of the growth conditions. From a total of 15 hosts, the strain RE3 cultured in a mineral medium exhibited the highest expression of a pga -encoding gene recombinant plasmid and high segregational plasmid stability. The high-expression system led to an increase in the specific activity of PGA up to 1,000 U g −1 cell dry weight and a total activity of about 4,500 U l −1 of the culture in a laboratory fermentor.

Pga gene from the industrial strain of E. coli RE3 hyperproducing penicillin G acylase (PGA) was cloned using a simple and rapid NIPAB-based chromogenic method for the detection of PGA-positive recombinant clones. Heterogeneous genetical material was prepared by subcloning pga in vectors pACYC184, pBR322-2 and pK19. The highest constitutive expression of pga was observed in a strain bearing the recombinant plasmid pKA18 synthesizing 2.5 times more of enzyme than the parent strain.

Penicillin G acylases (PGAs) are robust industrial catalysts used for biotransformation of β-lactams into key intermediates for chemical production of semi-synthetic β-lactam antibiotics by hydrolysis of natural penicillins. They are used also in reverse, kinetically controlled synthetic reactions for large-scale productions of these antibiotics from corresponding beta-lactam nuclei and activated acyl donors. Further biocatalytic applications of PGAs have recently been described: catalysis of peptide syntheses and the resolutions of racemic mixtures for the production of enantiopure active pharmaceutical ingredients that are based on enantioselective acylation or chiral hydrolysis. Moreover, PGAs rank among promiscuous enzymes because they also catalyze reactions such as trans-esterification, Markovnikov addition or Henry reaction. This particular biocatalytic versatility represents a driving force for the discovery of novel members of this enzyme family and further research into the catalytic potential of PGAs. This review deals with biocatalytic applications exploiting enantioselectivity and promiscuity of prokaryotic PGAs that have been recently reported. Biocatalytic applications are discussed and presented with reaction substrates converted into active compounds useful for the pharmaceutical industry.

The ferric uptake regulator gene (fur), its promoter region and Fur box of pvdS gene involved in siderophore-mediated iron uptake system were sequenced in the parent strain Pseudomonas aeruginosa PAO1 and in the fur mutant FPA121 derived from the strain PAO1. We identified the gene fur 179 bearing a novel, single-point mutation that changed the amino acid residue Gln60Pro in the DNA-binding domain of the Fur protein. The synthesis of pyoverdine was studied in cultures of the strains PAO1 and FPA121 grown in iron-deplete and iron-replete (60 μmol/L FeIII) medium. The amino acid replacement in the regulatory Fur protein is responsible for the overproduction of pyoverdine in iron-deplete and iron-replete medium. No mutation was identified in the Fur box of the gene pvdS.

Production of enantiopure esomeprazole by biocatalysis is of great demand by pharmaceutical industry. A Gram-positive bacterium oxidizing omeprazole sulfide 1a (5-methoxy-2-[((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)thio]-1H-benzoimidazole) to (S)-sulfoxide esomeprazole 2a (S)-5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl) methylsulfinyl]-3H-benzoimidazole was isolated from soil polluted with elemental sulfur. The strain exhibited the highest identity with the genus Lysinibacillus and catalyzed oxidation of 1a into enantiopure esomeprazole with conversion of 77% in a stirred bioreactor, fed-batch culture. No consecutive oxidation of (S)-sulfoxide to sulfone was observed during whole-cell catalysis. The unique characteristics of the catalyst provide a solid basis for further improvement and development of sustainable green bioprocess.

Recombinant plasmid pKA18 of the high expression bacterial system for penicillin amidase ('penicillin G acylase') bears the 3' end region of IS2 element. The IS2 sequence replaces the -35 region of promoter of pga and extends up to TAGTAT box at position -10 of the promoter region. It therefore forms a hybrid promoter of pga ppgaHT. A natural promoter ppgaWT was not detected on any recombinant plasmid isolated from recombinant strains of Escherichia coli constitutively producing penicillin amidase. PCR fragments carrying both types of promoters were cloned into the promoter-probe vector pET2 to compare their transcriptional activity: the activity of ppgaWT was 5x higher than that of ppgaHT. The same nucleotide "G" localized 28 nucleotides upstream of the translation start point was identified as the respective transcription start point of both mRNAs. An attempt was made to place the pga gene cloned on a plasmid under the control of the natural promoter: not a single clone expressing penicillin amidase was found among 150 transformants. High transcriptional activity of the natural promoter together with high pga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme.