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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

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.

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.

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

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.

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.

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.

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.