Membrane Lipids and Osmolytes in the Response of the Acidophilic Basidiomycete Phlebiopsis gigantea to Heat, Cold, and Osmotic Shocks.

Previously, we found for the first time the participation of osmolytes in adaptation to acidic conditions in three acidophilic fungi. Because trehalose can protect membranes, we hypothesized a relationship between osmolyte and membrane systems in adaptation to stressors. In the mycelium of Phlebiopsis gigantea, the level of osmolytes reaches 8% of the dry mass, while trehalose and arabitol make up 60% and 33% of the sum, respectively. Cold shock does not change the composition of osmolytes, heat shock causes a twofold increase in the trehalose level, and osmotic shock leads to a marked increase in the amount of trehalose and arabitol. Predominance of phospholipids (89% of the sum) and low proportions of sterols and sphingolipids are characteristic features of the membrane lipids' composition. Phosphatidic acids, along with phosphatidylethanolamines and phosphatidylcholines, are the main membrane lipids. The composition of the membrane lipids remains constant under all shocks. The predominance of linoleic (75% of the sum) and palmitic (20%) acids in phospholipids results in a high degree of unsaturation (1.5). Minor fluctuations in the fatty acid composition are observed under all shocks. The results demonstrate that maintaining or increasing the trehalose level provides stability in the membrane lipid composition during adaptation.

Genome Analysis and Potential Ecological Functions of Members of the Genus Ensifer from Subsurface Environments and Description of Ensifer oleiphilus sp. nov.

The current work deals with genomic analysis, possible ecological functions, and biotechnological potential of two bacterial strains, HO-A22T and SHC 2-14, isolated from unique subsurface environments, the Cheremukhovskoe oil field (Tatarstan, Russia) and nitrate- and radionuclide-contaminated groundwater (Tomsk region, Russia), respectively. New isolates were characterized using polyphasic taxonomy approaches and genomic analysis. The genomes of the strains HO-A22T and SHC 2-14 contain the genes involved in nitrate reduction, hydrocarbon degradation, extracellular polysaccharide synthesis, and heavy metal detoxification, confirming the potential for their application in various environmental biotechnologies. Genomic data were confirmed by cultivation studies. Both strains were found to be neutrophilic, chemoorganotrophic, facultatively anaerobic bacteria, growing at 15-33 °C and 0-1.6% NaCl (w/v). The 16S rRNA gene sequences of the strains were similar to those of the type strains of the genus Ensifer (99.0-100.0%). Nevertheless, genomic characteristics of strain HO-A22T were below the thresholds for species delineation: the calculated average nucleotide identity (ANI) values were 83.7-92.4% (<95%), and digital DNA-DNA hybridization (dDDH) values were within the range of 25.4-45.9% (<70%), which supported our conclusion that HO-A22T (=VKM B-3646T = KCTC 92427T) represented a novel species of the genus Ensifer, with the proposed name Ensifer oleiphilus sp. nov. Strain SHC 2-14 was assigned to the species 'Ensifer canadensis', which has not been validly published. This study expanded the knowledge about the phenotypic diversity among members of the genus Ensifer and its potential for the biotechnologies of oil recovery and radionuclide pollution treatment.

The Role of Osmolytes and Membrane Lipids in the Adaptation of Acidophilic Fungi.

Acidophiles maintain near-neutral intracellular pH using proton pumps. We have suggested the protective role of osmolytes and membrane lipids in the adaptation to an acidic environment. Previously we have observed, for the first time, high levels of trehalose in acidophilic basidiomycete Sistotrema brinkmannii. Here, we have studied the composition of both osmolytes and membrane lipids of two more acidophilic fungi. Trehalose and polyols were among the main osmolytes during growth under optimal conditions (pH 4.0) in basidiomycete Phlebiopsis gigantea and ascomycete Mollisia sp. Phosphatidic acids, phosphatidylethanolamines, phosphatidylcholines, and sterols, were predominant membrane lipids in both fungi. P. gigantea had a narrow optimum of growth at pH 4.0, resulting in a sharp decline of growth rate at pH 2.6 and 5.0, accompanied by a decrease in the number of osmolytes and significant changes in the composition of membrane lipids. In contrast, Mollisia sp. had a broad optimal growth range (pH 3.0-5.0), and the number of osmolytes either stayed the same (at pH 6.0) or increased (at pH 2.6), while membrane lipids composition remained unchanged. Thus, the data obtained indicate the participation of osmolytes and membrane lipids in the adaptation of acidophilic fungi.

A novel bacteriobiont of the Arctic lichen Flavocetraria nivalis, Lichenifustis flavocetrariae gen. nov, sp. nov. demonstrating hydrolytic properties and containing a full set of the Calvin-Benson-Bassham cycle genes.

A Gram-negative, strictly aerobic, chemoorganotrophic, bacteriochlorophyll a-containing, slow-growing bacterium was isolated from the lichen Flavocetraria nivalis and designated strain BP6-180914 T. Cells of this strain were large nonmotile rods, which reproduced by binary fission. Cells grew under oxic conditions and were able to utilize sugars and several polysaccharides, including starch and pectin. Strain BP6-180914 T was psychrotolerant and moderately acidophilic growing at 4-35 °C (optimum 20-28 °C) and between pH 4.0 and 7.5 (optimum 4.5-5.5). The major fatty acids were C18:1ω7c, C19:0 cyclo, C16:0 and C18:0. The polar lipids were diphosphatidylglycerols, phosphatidylglycerols, phosphatidylethanolamines, phosphatidylcholines, unidentified aminolipids, and a number of glycolipids, the major one being an unidentified glycolipid. The quinone was Q-10. The DNA G + C content was 63.65%. Comparative 16S rRNA gene sequence analysis revealed that strain BP6-180914 T was a member of the order Hyphomicrobiales and belonged to the family Lichenihabitantaceae defined by the lichen-dwelling facultative aerobic chemo-organotroph Lichenihabitans psoromatis (92.7% sequence similarity). The results of phylogenomic and genomic relatedness analyses showed that strain BP6-180914 T could clearly be distinguished from other species in the order Hyphomicrobiales with average nucleotide identity values of < 74.05% and genome-to-genome distance values of < 21.1%. The AAI value of 65.9% between strain BP6-180914 T and L. psoromatis allowed us to assign this strain to the novel genus of the family Lichenihabitantaceae. Therefore, it is proposed that strain BP6-180914 T represents a novel species in a new genus, Lichenifustis flavocetrariae gen. nov., sp. nov.; strain BP6-180914 T (= KCTC 92872 T = VKM B-3641 T = UQM 41506 T) is the type strain.

Acquired thermotolerance, membrane lipids and osmolytes profiles of xerohalophilic fungus Aspergillus penicillioides under heat shock.

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides, growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose.

Epinephrine extensively changes the biofilm matrix composition in Micrococcus luteus C01 isolated from human skin.

The importance of the impact of human hormones on commensal microbiota and microbial biofilms is established in lots of studies. In the present investigation, we continued and extended the research of epinephrine effects on the skin commensal Micrococcus luteus C01 and its biofilms, and also the matrix changes during the biofilm growth. Epinephrine in concentration 4.9 × 10-9 M which is close to normal blood plasma level increased the amount of polysaccharides and extracellular DNA in the matrix, changed extensively its protein, lipid and polysaccharide composition. The Ef-Tu factor was one of the most abundant proteins in the matrix and its amount increased in the presence of the hormone. One of the glucose-mannose polysaccharide was absent in the matrix in presence of epinephrine after 24 h of incubation. The matrix phospholipids were also eradicated by the addition of the hormone. Hence, epinephrine has a great impact on the M. luteus biofilms and their matrix composition, and this fact opens wide perspectives for the future research.

Correction: Semenova et al. Physiological and Genomic Characterization of Actinotalea subterranea sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family Actinotaleaceae. Microorganisms 2022, 10, 378.

The authors wish to make the following correction to this paper [...].

Physiological and Genomic Characterization of Actinotalea subterranea sp. nov. from Oil-Degrading Methanogenic Enrichment and Reclassification of the Family Actinotaleaceae.

The goal of the present work was to determine the diversity of prokaryotes involved in anaerobic oil degradation in oil fields. The composition of the anaerobic oil-degrading methanogenic enrichment obtained from an oil reservoir was determined by 16S rRNA-based survey, and the facultatively anaerobic chemoorganotrophic bacterial strain HO-Ch2T was isolated and studied using polyphasic taxonomy approach and genome sequencing. The strain HO-Ch2T grew optimally at 28 °C, pH 8.0, and 1-2% (w/v) NaCl. The 16S rRNA gene sequence of the strain HO-Ch2T had 98.8% similarity with the sequence of Actinotalea ferrariae CF5-4T. The genomic DNA G + C content of strain HO-Ch2T was 73.4%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the genome of strain HO-Ch2T and Actinotalea genomes were 79.8-82.0% and 20.5-22.2%, respectively, i.e., below the thresholds for species delineation. Based on the phylogenomic, phenotypic, and chemotaxonomic characterization, we propose strain HO-Ch2T (= VKM Ac-2850T = KCTC 49656T) as the type strain of a new species within the genus Actinotalea, with the name Actinotalea subterranea sp. nov. Based on the phylogenomic analysis of 187 genomes of Actinobacteria we propose the taxonomic revision of the genera Actinotalea and Pseudactinotalea and of the family Actinotaleaceae. We also propose the reclassification of Cellulomonas carbonis as Actinotalea carbonis comb. nov., Cellulomonas bogoriensis as Actinotalea bogoriensis comb. nov., Actinotalea caeni as Pseudactinotalea caeni comb. nov., and the transfer of the genus Pseudactinotalea to the family Ruaniaceae of the order Ruaniales.

Membrane lipid and osmolyte readjustment in the alkaliphilic micromycete Sodiomyces tronii under cold, heat and osmotic shocks.

Previously, we showed for the first time that alkaliphilic fungi, in contrast to alkalitolerant fungi, accumulated trehalose under extremely alkaline conditions, and we have proposed its key role in alkaliphilia. We propose that high levels of trehalose in the mycelium of alkaliphiles may promote adaptation not only to alkaline conditions, but also to other stressors. Therefore, we studied changes in the composition of osmolytes, and storage and membrane lipids under the action of cold (CS), heat (HS) and osmotic (OS) shocks in the obligate alkaliphilic micromycete Sodiomyces tronii. During adaptation to CS, an increase in the degree of unsaturation of phospholipids was observed while the composition of osmolytes, membrane and storage lipids remained the same. Under HS conditions, a twofold increase in the level of trehalose and an increase in the proportion of phosphatidylethanolamines were observed against the background of a decrease in the proportion of phosphatidic acids. OS was accompanied by a decrease in the amount of membrane lipids, while their ratio remained unchanged, and an increase in the level of polyols (arabitol and mannitol) in the fungal mycelium, which suggests their role for adaptation to OS. Thus, the observed consistency of the composition of membrane lipids suggests that trehalose can participate in adaptation not only to extremely alkaline conditions, but also to other stressors - HS, CS and OS. Taken together, the data obtained indicate the adaptability of the fungus to the action of various stressors, which can point to polyextremotolerance.

Osmolytes and membrane lipids in the adaptation of micromycete Emericellopsis alkalina to ambient pH and sodium chloride.

The accumulation of low molecular weight cytoprotective compounds (osmolytes) and changes in the membrane lipids composition are of key importance for the adaptation to stress impacts. However, the reason behind the wide variety of osmolytes present in the cell remains unclear. We suggest that specific functions of osmolytes can be revealed by studying the adaptation mechanisms of the mycelial fungus Emericellopsis alkalina (Hypocreales, Ascomycota) that is resistant to both alkaline pH values and high sodium chloride concentrations. It has been established that the fungus uses different osmolytes to adapt to ambient pH and NaCl concentration. Arabitol was predominant osmolyte in alkaline conditions, while mannitol prevailed in acidic conditions. On the salt-free medium mannitol was the main osmolyte; under optimal conditions (pH 10.2; 0.4 M NaCl) arabitol and mannitol were both predominant. Higher NaCl concentrations (1.0-1.5 M) resulted in the accumulation of low molecular weight polyol - erythritol, which amounted up to 12-14%, w/w. On the contrary, changes in the composition of membrane lipids were limited under pH and NaCl impacts; only higher NaCl concentrations led to the increase in the degree of unsaturation of membrane lipids. Results obtained indicated the key role of the osmolytes in the adaptation to the ambient pH and osmotic impacts.

Osmolytes and membrane lipids in adaptive response of thermophilic fungus Rhizomucor miehei to cold, osmotic and oxidative shocks.

In contrast to mesophiles, in which levels of trehalose and phosphatidic acids (PA) increased only under heat shock (HS), in thermophiles trehalose and PA were predominant under optimal growth conditions. To study the role of trehalose protection in the adaptation of thermophiles to various stressors, the composition of osmolytes and membrane lipids in the thermophilic fungus Rhizomucor miehei was studied under cold (CS), osmotic (OS) and oxidative (OxS) shocks. CS resulted in no accumulation of glycerol in the mycelium, while the amount of trehalose decreased. The main lipid changes were the increase in the PA proportion with simultaneous decrease of sterols (St), the increase of the unsaturation degree of polar lipids and the decrease of the ergosterol proportion in total St. OS did not cause changes in the lipid composition, but led to the decrease of ergosterol proportion too. Despite the low ability of Mucorales to produce polyols, increase in the level of arabitol and glycerol was observed under OS. OxS led to the decrease of trehalose level and had no effect on the lipid composition. Thus, our results show the similarity (OS) and the difference (CS and OxS) between adaptation mechanisms of thermophiles and mesophiles.

Lipids and soluble carbohydrates in the mycelium and ascomata of alkaliphilic fungus Sodiomyces alkalinus.

Alkaliphilic fungi are fundamentally different from alkalitolerant ones in terms of mechanisms of adaptation. They accumulate trehalose in cytosol and phosphatidic acids (PA) in the membrane lipids, whereas alkalitolerants contain these compounds in low amounts. But it is unclear how the composition of osmolytes and lipids changes during cytodifferentiation. In this article the composition of lipids and soluble cytosol carbohydrates in the mycelium and fruit bodies of the alkaliphilic fungus Sodiomyces alkalinus was studied. In the mycelium, mannitol and trehalose dominated, while in fruit bodies only trehalose was predominant. Phosphatidylcholines (PC), PA and sterols were major membrane lipids of the mycelium, while PC and sterols were predominant in fruit bodies. The degree of fatty acids unsaturation of the main mycelium phospholipids (PC and PA) increased with age, while that of PC did not change regardless of the developmental stage. In young mycelium, storage lipids were represented mainly by free fatty acids, and in mature mycelium and fruit bodies-by triacylglycerols. Fruit bodies contained three times less membrane lipids and twice as many storage lipids as mycelium. Trehalose was the main cytosol carbohydrate in the mycelium and fruit bodies, which confirms its key value for alkaliphily.

Combinatorial impact of osmotic and heat shocks on the composition of membrane lipids and osmolytes in Aspergillus niger.

The combinatorial action of osmotic (OS) and heat (HS) shocks on the composition of soluble cytosol carbohydrates and membrane lipids was studied. For the first time it was demonstrated that the combinatorial effect of these shocks led to the non-additive response - an increase in the trehalose level, characteristic for HS, but at the same time suppression of glycerol production, uncharacteristic of the OS response. In addition, combinatorial action resulted in a new effect - increase in the mannitol level, which was not typical for the individual HS or OS responses. On the contrary, a general pattern of change was observed in the composition of membrane lipids in response to both individual HS and OS, and their combinations, which was a twofold increase in the proportion of phosphatidic acids. At the same time, the mechanism of alteration in the degree of unsaturation of membrane phospholipids was not involved in adaptation. The response to combinatorial shocks includes the accumulation of trehalose and mannitol, and increase in the proportion of phosphatidic acids in membrane lipids.

Membrane lipids and soluble sugars dynamics of the alkaliphilic fungus Sodiomyces tronii in response to ambient pH.

Alkaliphily, the ability of an organism to thrive optimally at high ambient pH, has been well-documented in several lineages: archaea, bacteria and fungi. The molecular mechanics of such adaptation has been extensively addressed in alkaliphilic bacteria and alkalitolerant fungi. In this study, we consider an additional property that may have enabled fungi to prosper at alkaline pH: altered contents of membrane lipids and cytoprotectant molecules. In the alkaliphilic Sodiomyces tronii, we showed that at its optimal growth pH 9.2, the fungus accumulates abundant cytosolic trehalose (4-10% dry weight) and phosphatidic acids in the membrane lipids, properties not normally observed in neutrophilic species. At a very high pH 10.2, the major carbohydrate, glucose, was rapidly substituted by mannitol and arabitol. Conversely, lowering the pH to 5.4-7.0 had major implications both on the content of carbohydrates and membrane lipids. It was shown that trehalose dominated at pH 5.4. Fractions of sphingolipids and sterols of plasma membranes rapidly elevated possibly indicating the formation of membrane structures called rafts. Overall, our results reveals complex dynamics of the contents of membrane lipids and cytoplasmic sugars in alkaliphilic S. tronii, suggesting their adaptive functionality against pH stress.

Heat shock response of thermophilic fungi: membrane lipids and soluble carbohydrates under elevated temperatures.

The heat shock (HS) response is an adaptation of organisms to elevated temperature. It includes substantial changes in the composition of cellular membranes, proteins and soluble carbohydrates. To protect the cellular macromolecules, thermophilic organisms have evolved mechanisms of persistent thermotolerance. Many of those mechanisms are common for thermotolerance and the HS response. However, it remains unknown whether thermophilic species respond to HS by further elevating concentrations of protective components. We investigated the composition of the soluble cytosol carbohydrates and membrane lipids of the thermophilic fungi Rhizomucor tauricus and Myceliophthora thermophilaat optimum temperature conditions (41-43 °Ð¡), and under HS (51-53 °Ð¡). At optimum temperatures, the membrane lipid composition was characterized by a high proportion of phosphatidic acids (PA) (20-35 % of the total), which were the main components of the membrane lipids, together with phosphatidylcholines (PC), phosphatidylethanolamines (PE) and sterols (St). In response to HS, the proportion of PA and St increased, and the amount of PC and PE decreased. No decrease in the degree of fatty acid desaturation in the major phospholipids under HS was detected. The mycelium of all fungi at optimum temperatures contained high levels of trehalose (8-10 %, w/w; 60-95 % of the total carbohydrates), which is a hallmark of thermophilia. In contrast to mesophilic fungi, heat exposure decreased the trehalose level and the fungi did not acquire thermotolerance to lethal HS, indicating that trehalose plays a key role in this process. This pattern of changes appears to be conserved in the studied filamentous thermophilic fungi.