New crustose Teloschistaceae in Central Europe

Central Europe in general is poor in Teloschistaceae lichen crusts (Caloplaca s. lat.). Diversity of these lichens is increased by the occurrence of some Arctic, Mediterranean and continental species, which are here close to the limits of their range. Examples include: 1) Caloplaca interfulgens, previously known from arid territories of northern Africa and western Asia, is recorded, surprisingly, from Austria, Czech Republic, Germany, Slovakia and southern Russia. In Central Europe, it is restricted to scattered xerothermic limestone outcrops. 2) Caloplaca scabrosa, previously known only from Svalbard, is recorded from the Sudetes in the Czech Republic. It is similar to, but not conspecific with, C. furfuracea. Its diagnostic characters include a blastidiate thallus and the presence of atranorin. Our results show that atranorin is absent in the majority of taxa related to C. furfuracea with only two exceptions: the sample from Eastern Carpathians, here called C. aff. scabrosa, and in one Sudetan sample identified as C. crenularia. 3) Caloplaca emilii, newly described below, is closely related to the Mediterranean C. areolata. We consider C. emilii a Mediterranean species rarely occurring in higher latitudes in Austria, the Czech Republic and Germany. It is distinguished from C. areolata mainly by the presence of vegetative diaspores (blastidia); a possible role of blastidia in the distribution pattern of C. emilii is discussed below. Status of the names Caloplaca areolata, C. isidiigera and C. spalatensisis, formerly used for the new taxon, is clarified. 4) Caloplaca molariformis, newly described below, belongs to the Pyrenodesmia group (a lineage of Caloplaca without anthraquinones). It is a continental species, frequently collected on limestone or lime-rich tuffs in steppes or deserts in Turkey, Iran, western Kazakhstan and southern Russia, and is also known from eastern Ukraine and southern Slovakia. Caloplaca molariformis is characterized by its thick thallus with fungal and algal tissues arranged in high stacks. 5) Caloplaca substerilis, newly described below, is distinguished from the closely related C. ulcerosa by its endophloeodal or minutely squamulose thallus with soralia formed in bark crevices or on margins of squamules. While C. ulcerosa has a maritime distribution in Europe, C. substerilis is typically a continental species. North American continental lichens called C. ulcerosa are phylogenetically closer and more similar to C. substerilis. The positions within Teloschistaceae of the taxa considered are demonstrated by ITS phylogenies. The distributions of C. areolata, C. emilii and C. interfulgens are mapped. The new species are fully described using more than a hundred phenotype characters, and diagnostic characters are indicated separately. Copyright © British Lichen Society 2013

Chemical diversity and cellular effects of antifungal cyclic lipopeptides from cyanobacteria

Cyanobacteria produce a variety of chemically diverse cyclic lipopeptides with potent antifungal activities. These cyclic lipopeptides have an amphipathic structure comprised of a polar peptide cycle and hydrophobic fatty acid side chain. Many have antibiotic activity against a range of human and plant fungal pathogens. This review article aims to summarize the present knowledge on the chemical diversity and cellular effects of cyanobacterial cyclic lipopeptides that display antifungal activity. Cyclic antifungal lipopeptides from cyanobacteria commonly fall into four structural classes; hassallidins, puwainaphycins, laxaphycins, and anabaenolysins. Many of these antifungal cyclic lipopeptides act through cholesterol and ergosterol-dependent disruption of membranes. In many cases, the cyclic lipopeptides also exert cytotoxicity in human cells, and a more extensive examination of their biological activity and structure-activity relationship is warranted. The hassallidin, puwainaphycin, laxaphycin, and anabaenolysin structural classes are unified through shared complex biosynthetic pathways that encode a variety of unusual lipoinitiation mechanisms and branched biosynthesis that promote their chemical diversity. However, the biosynthetic origins of some cyanobacterial cyclic lipopeptides and the mechanisms, which drive their structural diversification in general, remain poorly understood. The strong functional convergence of differently organized chemical structures suggests that the production of lipopeptide confers benefits for their producer. Whether these benefits originate from their antifungal activity or some other physiological function remains to be answered in the future. However, it is clear that cyanobacteria encode a wealth of new cyclic lipopeptides with novel biotechnological and therapeutic applications.Peer reviewe

Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins

Low-molecular weight natural products display vast structural diversity and have played a key role in the development of novel therapeutics. Here we report the discovery of novel members of the aeruginosin family of natural products, which we named varlaxins. The chemical structures of varlaxins 1046A and 1022A were determined using a combination of mass spectrometry, analysis of one- and two-dimensional NMR spectra, and HPLC analysis of Marfey's derivatives. These analyses revealed that varlaxins 1046A and 1022A are composed of the following moieties: 2-O-methylglyceric acid 3-O-sulfate, isoleucine, 2-carboxy-6-hydroxyoctahydroindole (Choi), and a terminal arginine derivative. Varlaxins 1046A and 1022A differ in the cyclization of this arginine moiety. Interestingly, an unusual alpha-d-glucopyranose moiety derivatized with two 4-hydroxyphenylacetic acid residues was bound to Choi, a structure not previously reported for other members of the aeruginosin family. We sequenced the complete genome of Nostoc sp. UHCC 0870 and identified the putative 36 kb varlaxin biosynthetic gene cluster. Bioinformatics analysis confirmed that varlaxins belong to the aeruginosin family of natural products. Varlaxins 1046A and 1022A strongly inhibited the three human trypsin isoenzymes with IC50 of 0.62-3.6 nM and 97-230 nM, respectively, including a prometastatic trypsin-3, which is a therapeutically relevant target in several types of cancer. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and provide evidence that the aeruginosin family is a source of strong inhibitors of human serine proteases.Peer reviewe

Discovery of a Pederin Family Compound in a Nonsymbiotic Bloom-Forming Cyanobacterium

The pederin family includes a number of bioactive compounds isolated from symbiotic organisms of diverse evolutionary origin. Pederin is linked to beetle-induced dermatitis in humans, and pederin family members possess potent antitumor activity caused by selective inhibition of the eukaryotic ribosome. Their biosynthesis is accomplished by a polyketide/nonribosomal peptide synthetase machinery employing an unusual trans-acyltransferase mechanism. Here, we report a novel pederin type compound, cusperin, from the free-living cyanobacterium Cuspidothrix issatschenkoi (earlier Aphanizomenon). The chemical structure of cusperin is similar to that of nosperin recently isolated from the lichen cyanobiont Nostoc sharing the tehrahydropyran moiety and major part of the linear backbone. However, the cusperin molecule is extended by a glycine residue and lacks one hydroxyl substituent. Pederins were previously thought to be exclusive to symbiotic relationships. However, C. issatschenkoi is a nonsymbiotic planktonic organism and a frequent component of toxic water blooms. Cusperin is devoid of the cytotoxic activity reported for other pederin family members. Hence, our findings raise questions about the role of pederin analogues in cyanobacteria and broaden the knowledge of ecological distribution of this group of polyketides.Peer reviewe

Fatty Acid Substitutions Modulate the Cytotoxicity of Puwainaphycins/Minutissamides Isolated from the Baltic Sea Cyanobacterium Nodularia harveyana UHCC-0300

Puwainaphycins (PUW) and minutissamides (MIN) are structurally homologous cyclic lipopeptides that exhibit high structural variability and possess antifungal and cytotoxic activities. While only a minor variation can be found in the amino acid composition of the peptide cycle, the fatty acid (FA) moiety varies largely. The effect of FA functionalization on the bioactivity of PUW/MIN chemical variants is poorly understood. A rapid and selective liquid chromatography-mass spectrometry-based method led us to identify 13 PUW/MIN (1-13) chemical variants from the benthic cyanobacterium Nodularia harveyana strain UHCC-0300 from the Baltic Sea. Five new variants identified were designated as PUW H (1), PUW I (2), PUW J (4), PUW K (10), and PUW L (13) and varied slightly in the peptidic core composition, but a larger variation was observed in the oxo-, chloro-, and hydroxy-substitutions on the FA moiety. To address the effect of FA substitution on the cytotoxic effect, the major variants (3 and 5-11) together with four other PUW/MIN variants (14-17) previously isolated were included in the study. The data obtained showed that hydroxylation of the FA moiety abolishes the cytotoxicity or significantly reduces it when compared with the oxo-substituted C-18-FA (compounds 5-8). The oxo-substitution had only a minor effect on the cytotoxicity of the compound when compared to variants bearing no substitution. The activity of PUW/ MIN variants with chlorinated FA moieties varied depending on the position of the chlorine atom on the FA chain. This study also shows that variation in the amino acids distant from the FA moiety (position 4-8 of the peptide cycle) does not play an important role in determining the cytotoxicity of the compound. These findings confirmed that the lipophilicity of FA is essential to maintain the cytotoxicity of PUW/MIN lipopeptides. Further, a 63 kb puwainaphycin biosynthetic gene cluster from a draft genome of the N. harveyana strain UHCC-0300 was identified. This pathway encoded two specific lipoinitiation mechanisms as well as enzymes needed for the modification of the FA moiety. Examination on biosynthetic gene clusters and the structural variability of the produced PUW/MIN suggested different mechanisms of fatty-aryl-AMP ligase cooperation with accessory enzymes leading to a new set of PUW/MIN variants bearing differently substituted FA.Peer reviewe

Semi-synthetic puwainaphycin/minutissamide cyclic lipopeptides with improved antifungal activity and limited cytotoxicity

Microbial cyclic lipopeptides are an important class of antifungal compounds with applications in pharmacology and biotechnology. However, the cytotoxicity of many cyclic lipopeptides limits their potential as antifungal drugs. Here we present a structure-activity relationship study on the puwainaphycin/minutissamide (PUW/MIN) family of cyclic lipopeptides isolated from cyanobacteria. PUWs/MINs with variable fatty acid chain lengths differed in the dynamic of their cytotoxic effect despite their similar IC50 after 48 hours (2.8 mu M for MIN A and 3.2 mu M for PUW F). Furthermore, they exhibited different antifungal potency with the lowest MIC values obtained for MIN A and PUW F against the facultative human pathogen Aspergillus fumigatus (37 mu M) and the plant pathogen Alternaria alternata (0.6 mu M), respectively. We used a Grignard-reaction with alkylmagnesium halides to lengthen the lipopeptide FA moiety as well as the Steglich esterification on the free hydroxyl substituents to prepare semi-synthetic lipopeptide variants possessing multiple fatty acid tails. Cyclic lipopeptides with extended and branched FA tails showed improved strain-specific antifungal activity against A. fumigatus (MIC = 0.5-3.8 mu M) and A. alternata (MIC = 0.1-0.5 mu M), but with partial retention of the cytotoxic effect (similar to 10-20 mu M). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 mu M), and greatly reduced or abolished cytotoxic activity (>20 mu M). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.Peer reviewe

Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata. Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola. We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production

Accumulation of cyanobacterial oxadiazine nocuolin A is enhanced by temperature shift during cultivation and is promoted by bacterial co-habitants in the culture

© 2019 Elsevier B.V. Proper setting of cultivation conditions is essential for production of high-value compounds in microbial biotechnology. The present study characterizes photoautotrophic growth and capacity to accumulate the antiproliferative secondary metabolite Nocuolin A (NoA) in cyanobacterium Nostoc sp. CCAP 1453/38. As the cyanobacterial culture was found to be non-axenic, the bacteria accompanying the culture were characterized, then the growth demands and NoA production in the Nostoc-bacterial consortium were determined, and finally an axenic strain was prepared. For the purposes of growth characterization, the culture was maintained in a quasi-continuous regime under various light intensities, temperatures, and inorganic carbon concentrations in a small-scale laboratory photobioreactor. The maximum biomass growth rate obtained was 0.10 h−1 (doubling time Dt = 6.93 h). Following optimal growth conditions were identified: temperature of 35 °C, light intensity 600 μmol(photons) m−2 s−1, and 2500 ppm CO2 in the sparging gas. As the temperature optima for the biomass production and for NoA accumulation differed, biphasic cultivation for maximal NoA yield was designed, leading to a three times more effective cultivation procedure compared to batch culture maintained at a temperature optimal for NoA production. The increased NoA accumulation at reduced temperature that correlated with enhanced expression of NoA biosynthetic genes after the temperature shift suggested its regulation occurs at the expression level. It has further been shown that NoA production is reduced in axenic culture, which indicates that it is also triggered by presence of bacteria. This study shows an example of how a biphasic cultivation mode with different temperatures can be used in high-value compound production processes. It also brings direct evidence that cyanobacterial strain axenization can lead to a rapid decrease in production of valuable compounds and that non-axenic strains may be considered more suitable for retrieval and initial production of novel pharmaceutical leads

Taxonomy and phylogeny of the <em>Caloplaca cerina</em> group in Europe

Using ITS nrDNA sequence data, the Caloplaca cerina group (Teloschistaceae) is defined here as a monophyletic, but internally richly branched lineage. The group is also characterized by a combination of morphological and anatomical characters. Its internal lineages are supported by phenotypic characters in addition to ecology and distribution. Within the large C. cerina group, we have found at least 20 phylospecies in the temperate zone of the Northern Hemisphere. Two species complexes do not produce any vegetative diaspores: the polyphyletic, corticolous Caloplaca cerina s. lat. (six separated cryptic or semi-cryptic species) and the monophyletic C. stillicidiorum s. lat. that grows mainly on plant debris, small shrubs and bryophytes and consists of at least four internal lineages. All lineages producing vegetative diaspores (soredia, blastidia, isidia or lobules) are phenotypically characteristic and represent fairly easily distinguishable species: C. chlorina, C. isidiigera, C. monacensis, C. subalpina, C. thracopontica, C. turkuensis and C. ulmorum. Only the North American sorediate C. pinicola possibly represents an aggregate of species. Caloplaca sterilis is described as a new species. A key to the phenotypically distinguishable species is provided. Lectotypes are designated for C. albolutea, Caloplaca cerina f. coronulata and for C. monacensis. The Australian C. hanneshertelii belongs to this group, but this and other possible species from the Southern Hemisphere are not treated here in detail. Some species traditionally placed in the C. cerina group due to their similar morphology are excluded here on the basis of our phenotype examinations and molecular data. Caloplaca albolutea, C. mydalaea and C. virescens are uncertain taxa and their identities still remain unclear