Filamentous Strains Research Articles

New 6,19-oxidoandrostan derivatives obtained by biotransformation in environmental filamentous fungi cultures

BackgroundSteroid compounds with a 6,19-oxirane bridge possess interesting biological activities including anticonvulsant and analgesic properties, bacteriostatic activity against Gram-positive bacteria and selective anti-glucocorticoid action, while lacking mineralocorticoid and progestagen activity.ResultsThe study aimed to obtain new derivatives of 3β-acetyloxy-5α-chloro-6,19-oxidoandrostan-17-one by microbial transformation. Twelve filamentous fungal strains were used as catalysts, including entomopathogenic strains with specific activity in the transformation of steroid compounds. All selected strains were characterised by high biotransformation capacity for steroid compounds. However, high substrate conversions were obtained in the cultures of 8 strains: Beauveria bassiana KCh BBT, Beauveria caledonica KCh J3.4, Penicillium commune KCh W7, Penicillium chrysogenum KCh S4, Mucor hiemalis KCh W2, Fusarium acuminatum KCh S1, Trichoderma atroviride KCh TRW and Isaria farinosa KCh KW1.1. Based on gas chromatography (GC) and nuclear magnetic resonance (NMR) analyses, it was found that almost all strains hydrolysed the ester bond of the acetyl group. The strain M. hiemalis KCh W2 reduced the carbonyl group additionally. From the P. commune KCh W7 and P. chrysogenum KCh S4 strain cultures a product of D-ring Baeyer–Villiger oxidation was isolated, whereas from the culture of B. bassiana KCh BBT a product of hydroxylation at the 11α position and oxidation of the D ring was obtained. Three 11α-hydroxy derivatives were obtained in the culture of I. farinosa KCh KW1.1: 3β,11α-dihydroxy-5α-chloro-6,19-oxidoandrostan-17-one, 3β,11α,19-trihydroxy-5α-chloro-6,19-oxidoandrostan-17-one and 3β,11α-dihydroxy-5α-chloro-6,19-oxidoandrostan-17,19-dione. They are a result of consecutive reactions of hydrolysis of the acetyl group at C-3, 11α- hydroxylation, then hydroxylation at C-19 and its further oxidation to lactone.ConclusionsAs a result of the biotransformations, seven steroid derivatives, not previously described in the literature, were obtained: 3β-hydroxy-5α-chloro-6,19-oxidoandrostan-17-one, 3β,17α-dihydroxy-5α-chloro-6,19-oxidoandrostane, 3β-hydroxy-5α-chloro-17α-oxa-D-homo-6,19-oxidoandrostan-17-one, 3β,11α-dihydroxy-5α-chloro-17α-oxa-D-homo-6,19-oxidoandrostan-17-one and the three above–mentioned 11α-hydroxy derivatives. This study will allow a better understanding and characterisation of the catalytic abilities of individual microorganisms, which is crucial for more accurate planning of experiments and achieving more predictable results.

Ecogenomics of the Marine Benthic Filamentous Cyanobacterium Adonisia.

Turfs are among the major benthic components of reef systems worldwide. The nearly complete genome sequences, basic physiological characteristics, and phylogenomic reconstruction of two phycobiliprotein-rich filamentous cyanobacteria strains isolated from turf assemblages from the Abrolhos Bank (Brazil) are investigated. Both Adonisia turfae CCMR0081T (= CBAS 745T) and CCMR0082 contain approximately 8Mbp in genome size and experiments identified that both strains exhibit chromatic acclimation. Whereas CCMR0081T exhibits chromatic acclimation type 3 (CA3) regulating both phycocyanin (PC) and phycoerythrin (PE), CCMR0082 strain exhibits chromatic acclimation type 2 (CA2), in correspondence with genes encoding specific photosensors and regulators for PC and PE. Furthermore, a high number and diversity of secondary metabolite synthesis gene clusters were identified in both genomes, and they were able to grow at high temperatures (28°C, with scant growth at 30°C). These characteristics provide insights into their widespread distribution in reef systems.

The first crenarchaeon capable of growth by anaerobic carbon monoxide oxidation coupled with H2 production

The ability to grow by anaerobic CO oxidation with production of H2 from water is known for some thermophilic bacteria, most of which belong to Firmicutes, as well as for a few hyperthermophilic Euryarchaeota isolated from deep-sea hydrothermal habitats. A hyperthermophilic, neutrophilic, anaerobic filamentous archaeon strain 1505=VKM B-3180=KCTC 15798 was isolated from a terrestrial hot spring in Kamchatka (Russia) in the presence of 30% CO in the gas phase. Strain 1505 could grow lithotrophically using carbon monoxide as the energy source with the production of hydrogen according to the equation CO+H2O→CO2+H2; mixotrophically on CO plus glucose; and organotrophically on peptone, yeast extract, glucose, sucrose, or Avicel. The genome of strain 1505 was sequenced and assembled into a single chromosome. Based on 16S rRNA gene sequence analysis and in silico genome-genome hybridization, this organism was shown to be closely related to the Thermofilum adornatum species. In the genome of Thermofilum sp. strain 1505, a gene cluster (TCARB_0867-TCARB_0879) was found that included genes of anaerobic (Ni,Fe-containing) carbon monoxide dehydrogenase and genes of energy-converting hydrogenase ([Ni,Fe]-CODH–ECH gene cluster). Compared to the [Ni,Fe]-CODH–ECH gene clusters occurring in the sequenced genomes of other H2-producing carboxydotrophs, the [Ni,Fe]-CODH–ECH gene cluster of Thermofilum sp. strain 1505 presented a novel type of gene organization. The results of the study provided the first evidence of anaerobic CO oxidation coupled with H2 production performed by a crenarchaeon, as well as the first documented case of lithotrophic growth of a Thermofilaceae representative.

The Pkn22 Kinase of Nostoc PCC 7120 Is Required for Cell Differentiation via the Phosphorylation of HetR on a Residue Highly Conserved in Genomes of Heterocyst-Forming Cyanobacteria

Hanks-type kinases encoding genes are present in most cyanobacterial genomes. Despite their widespread pattern of conservation, little is known so far about their role because their substrates and the conditions triggering their activation are poorly known. Here we report that under diazotrophic conditions, normal heterocyst differentiation and growth of the filamentous cyanobacterium Nostoc PCC 7120 require the presence of the Pkn22 kinase, which is induced under combined nitrogen starvation conditions. By analyzing the phenotype of pkn22 mutant overexpressing genes belonging to the regulatory cascade initiating the development program, an epistatic relationship was found to exist between this kinase and the master regulator of differentiation, HetR. The results obtained using a bacterial two hybrid approach indicated that Pkn22 and HetR interact, and the use of a genetic screen inducing the loss of this interaction showed that residues of HetR which are essential for this interaction to occur are also crucial to HetR activity both in vitro and in vivo. Mass spectrometry showed that HetR co-produced with the Pkn22 kinase in Escherichia coli is phosphorylated on Serine 130 residue. Phosphoablative substitution of this residue impaired the ability of the strain to undergo cell differentiation, while its phosphomimetic substitution increased the number of heterocysts formed. The Serine 130 residue is part of a highly conserved sequence in filamentous cyanobacterial strains differentiating heterocysts. Heterologous complementation assays showed that the presence of this domain is necessary for heterocyst induction. We propose that the phosphorylation of HetR might have been acquired to control heterocyst differentiation.

Functions for Cdc42p BEM adaptors in regulating a differentiation-type MAP kinase pathway.

Ras homology (Rho) GTPases regulate cell polarity and signal transduction pathways to control morphogenetic responses in different settings. In yeast, the Rho GTPase Cdc42p regulates cell polarity, and through the p21-activated kinase Ste20p, Cdc42p also regulates mitogen-activated protein kinase (MAPK) pathways (mating, filamentous growth or fMAPK, and HOG). Although much is known about how Cdc42p regulates cell polarity and the mating pathway, how Cdc42p regulates the fMAPK pathway is not clear. To address this question, Cdc42p-dependent MAPK pathways were compared in the filamentous (Σ1278b) strain background. Each MAPK pathway showed a unique activation profile, with the fMAPK pathway exhibiting slow activation kinetics compared with the mating and HOG pathways. A previously characterized version of Cdc42p, Cdc42pE100A, that is specifically defective for fMAPK pathway signaling, was defective for interaction with Bem4p, the pathway-specific adaptor for the fMAPK pathway. Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK pathway signaling. The polarity adaptor Bem1p also regulated the fMAPK pathway. Versions of Bem1p defective for recruitment of Ste20p to the plasma membrane, intramolecular interactions, and interaction with the GEF, Cdc24p, were defective for fMAPK pathway signaling. Bem1p also regulated effector pathways in different ways. In some pathways, multiple domains of the protein were required for its function, whereas in other pathways, a single domain or function was needed. Genetic suppression tests showed that Bem4p and Bem1p regulate the fMAPK pathway in an ordered sequence. Collectively, the study demonstrates unique and sequential functions for Rho GTPase adaptors in regulating MAPK pathways.

Synthesis of sulfonamides bearing 1,3,5-triarylpyrazoline and 4-thiazolidinone moieties as novel antimicrobial agents

Two series of sulfonamides were synthesized from 4-hydrazinylbenzenesulfonamide as the key starting material. 1,3,5-Triarylpyrazoline sulfonamides (2a?i) were obtained by cyclocondensation of various chalcones in 53? ?64 % yields, while 4-thiazolidinone derivatives (4a?e) were synthesized by cyclocondensation between mercaptoacetic acid and different phenylhydrazones in 43?62 % yields. The synthesized compounds were characterized based on FTIR, 1H-NMR, 13C-NMR and HRMS data. The sulfonamides were evaluated for their in vitro antimicrobial activities against four bacterial strains (E. coli, P. aeruginosa, B. subtillis and S aureus), two filamentous fungal strains (A. niger and F. oxysporum) and two yeast strains (C. albicans and S. cerevisiae). Seven pyrazolines, 2a?c and 2e?h, exhibited significant inhibition of different microbial strains. Among them, compound 2b displayed good antifungal activity against A. niger (MIC value at 12.5 ?g mL-1) over the reference drug.

SCREENING OF FILAMENTOUS FUNGI FROM THE ATLANTIC FOREST BIOME PRODUCING ENZYMES OF THE PECTINOLYTIC COMPLEX

Fungi are the major producers of enzymes such as cellulases, pectinases and xylanases, so these microorganisms were selected in the present work to analyze the ideal conditions to improve the production of the activity of the pectinase enzyme. A total of 36 fungi previously collected from the Atlantic Forest of Parana were cultivated in a test tube containing 10 to 15 mL of BDA medium for 7 days at 40 °C. Among all analyzed, the filamentous fungus Aspergillus terreus strain PA3A5T was the best producer of pectinases, whose greatest enzymatic activity occurred at a temperature of 60 °C and at pH 5. The ideal culture medium for inducing production Enzyme was the defined medium Vogel supplemented with passion fruit peel at 1% (w/v) as a carbon source and incubated for 96 hours. To our knowledge, this is the first time that these data are reported in the literature.

Identification of a gene regulated by HetR, a master regulator of heterocyst differentiation, in the non-heterocyst-forming filamentous cyanobacterium Arthrospira platensis NIES-39.

Cyanobacteria are a morphologically and physiologically diverse group of bacteria, which contains unicellular and multicellular filamentous strains. Some filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, form a differentiated cell called a heterocyst. The heterocyst is a specialized cell for nitrogen fixation and is differentiated from a vegetative cell in response to depletion of combined nitrogen in the medium. In Anabaena PCC 7120, it has been demonstrated that hetR, which encodes a transcriptional regulator, is necessary and sufficient for heterocyst differentiation. However, comprehensive genomic analysis of cyanobacteria has shown that hetR is present in non-heterocyst-forming cyanobacteria. Almost all filamentous cyanobacteria have hetR, but unicellular cyanobacteria do not. In this study, we conducted genetic and biochemical analyses of hetR (NIES39_C03480) of the non-heterocyst-forming cyanobacterium Arthrospira platensis NIES-39. HetR of A. platensis was able to complement the hetR mutation in Anabena PCC 7120 and recognized the same DNA sequence as Anabaena HetR. A search of the A. platensis genome revealed the HetR-recognition sequence within the promoter region of NIES39_O04230, which encodes a protein of unknown function. Expression from the NIES39_O04230 promoter could be suppressed by HetR in Anabaena PCC 7120. These data support the conclusion that NIES39_O04230 is regulated by HetR in A. platensis NIES-39.

<p><strong><em>Neowollea manoromense</em> gen. & sp. nov. (Nostocales, Cyanobacteria), a novel geosmin producer isolated from Thailand</strong></p>

In the present study, two new filamentous cyanobacterial strains (NUACC12 and NUACC15) were described based on a comprehensive study of morphological characteristics and molecular analyses. The novel strains were isolated from a shallow freshwater pond in the central region of Thailand. The morphological features of both strains are similar to those of Wollea species, W. ambigua, but are different in the dimensions of heterocyte and akinete cells as well as the absence of a sheath around the trichome. Phylogenetic analyses based on 16S rDNA and 16S–23S rDNA internal transcribed spacer sequences (ITS) revealed that new strains formed a robust supported clade which does not belong to the same clade with the type species of other cyanobacterial genera. Moreover, 16S rDNA sequence similarity between new strains and other putatively related taxa such as Wollea, Amphiheterocytum, Sphaerospermopsis, and Raphidiopsis raciborskii ranged from 96.1–97.4%, a level of similarity low enough for separating species differences. The unique patterns of the ITS secondary structure also clearly support two new strains representing a distinct cyanobacterial species. Considering all the results obtained in the present study, we here name the new genus Neowollea gen. nov., with the type species N. manoromense sp. nov. Based on morphology, ecology, 16S rDNA data and D1–D1′ helix structure, we also propose N. salina comb. nov. (basionym Wollea salina) as an additional species in the novel genus. Furthermore, the results of PCR amplification and GC/MS/MS analysis confirmed that two N. manoromense strains could produce an odorous compound, geosmin.

Polyphasic taxonomy of green algae strains isolated from Mediterranean freshwaters

BackgroundTerrestrial, freshwater and marine green algae constitute the large and morphologically diverse phylum of Chlorophyta, which gave rise to the core chlorophytes. Chlorophyta are abundant and diverse in freshwater environments where sometimes they form nuisance blooms under eutrophication conditions. The phylogenetic relationships among core chlorophyte clades (Chlorodendrophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae), are of particular interest as it is a species-rich phylum with ecological importance worldwide, but are still poorly understood. In the Mediterranean ecoregion, data on molecular characterization of eukaryotic microalgae strains are limited and current knowledge is based on ecological studies of natural populations. In the present study we report the isolation and characterization of 11 green microalgae strains from Greece contributing more information for the taxonomy of Chlorophyta. The study combined morphological and molecular data.ResultsPhylogenetic analysis based on 18S rRNA, internal transcribed spacer (ITS) region and the large subunit of the ribulose-bisphosphate carboxylase (rbcL) gene revealed eight taxa. Eleven green algae strains were classified in four orders (Sphaeropleales, Chlorellales, Chlamydomonadales and Chaetophorales) and were represented by four genera; one strain was not assigned to any genus. Most strains (six) were classified to the genus Desmodesmus, two strains to genus Chlorella, one to genus Spongiosarcinopsis and one filamentous strain to genus Uronema. One strain is placed in a separate independent branch within the Chlamydomonadales and deserves further research.ConclusionsOur study reports, for the first time, the presence of Uronema in an aquatic environment up to 40 °C and reveals new diversity within the Chlamydomonadales. The results from the ITS region and the rbcL gene corroborated those obtained from 18S rRNA without providing further information or resolving the phylogenetic relationships within certain genera, due to the limited number of ITS and rbcL sequences available. The comparison of molecular and morphological data showed that they were congruent. Cosmopolitan genera with high worldwide distribution inhabit Greek freshwaters.

Draft Genome Sequences of Three Filamentous Cyanobacterial Strains, Dolichospermum planctonicum NIES-80, Planktothrix agardhii NIES-905, and Sphaerospermopsis reniformis NIES-1949.

Three freshwater planktonic filamentous cyanobacterial strains, Dolichospermum planctonicum NIES-80, Planktothrix agardhii NIES-905, and Sphaerospermopsis reniformis NIES-1949, were sequenced. The genome sizes of NIES-80, NIES-905, and NIES-1949 were 4,571,002 bp, 5,512,454 bp, and 6,025,023 bp, and the number of protein-coding genes in each genome was 4,009, 4,925, and 5,408, respectively.

Biofilm formation behaviour of marine filamentous cyanobacterial strains in controlled hydrodynamic conditions.

Marine biofouling has severe economic impacts and cyanobacteria play a significant role as early surface colonizers. Despite this fact, cyanobacterial biofilm formation studies in controlled hydrodynamic conditions are scarce. In this work, computational fluid dynamics was used to determine the shear rate field on coupons that were placed inside the wells of agitated 12-well microtiter plates. Biofilm formation by three different cyanobacterial strains was assessed at two different shear rates (4 and 40 s-1 ) which can be found in natural ecosystems and using different surfaces (glass and perspex). Biofilm formation was higher under low shear conditions, and differences obtained between surfaces were not always statistically significant. The hydrodynamic effect was more noticeable during the biofilm maturation phase rather than during initial cell adhesion and optical coherence tomography showed that different shear rates can affect biofilm architecture. This study is particularly relevant given the cosmopolitan distribution of these cyanobacterial strains and the biofouling potential of these organisms.

Solid-state fermentation of pearl millet with Aspergillus oryzae and Rhizopus azygosporus: effects on bioactive profile and DNA damage protection activity

The present scientific work envisaged the effect of fermentation, starter culture, solvent type, concentration of solvent and extraction conditions on pearl millet phenolics. Fermentation of pearl millet grains was performed using two filamentous fungal strains (Aspergillus oryzae and Rhizopus azygosporus) for a specific period (336 h). Pearl millet koji was extracted under optimized conditions and studied for the presence of specific bioactive compounds using high performance liquid chromatography (HPLC). Results confirmed the presence of ascorbic acid (1.55 mg g−1), p-coumaric acid (1.04 mg g−1) and cinnamic acid (0.71 mg g−1 as major compounds in unfermented pearl millet and ascorbic acid (10.23 mg g−1), gallic acid (8.95 mg g−1), resorcinol (2.90 mg g−1, catechol (7.60 mg g−1), vanillin (4.58 mg g−1), p-coumaric acid (3.96 mg g−1), quercetin (2.74 mg g−1), benzoic acid (5.10 mg g−1) and cinnamic acid (5.14 mg g−1) in A. oryzae fermented millet (AoFM). Specific phenolics in R. azygosporus fermented millet (RaFM) were ascorbic acid (8.80 mg g−1), gallic acid (4.70 mg g−1) and p-coumaric acid (1.27 mg g−1) along with other minor phenolics. DNA damage protection activity was observed maximum till 288 h of fermentation for AoFM and 240 h of fermentation for RaFM.

Ancylomarina salipaludis sp. nov., isolated from a salt marsh.

A Gram-stain-negative, aerobic, non-motile and rod-shaped or filamentous bacterial strain, designated SHSM-M15T, was isolated from a salt marsh at Siheung in Republic of Korea and identified by polyphasic taxonomic study. Strain SHSM-M15T grew optimally at 25 °C, at pH 7.0-8.0 and in the presence of 1.0-2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain SHSM-M15T clusters with the type strain of Ancylomarina subtilis, showing 16S rRNA gene sequence similarity of 97.8 %. Strain SHSM-M15T had 16S rRNA gene sequence similarities of less than 93.7 % with the type strains of other recognised species. Strain SHSM-M15T contained MK-7 as the predominant menaquinone and iso-C15 : 0, iso-C15 : 0 3-OH and anteiso-C15 : 0 as the major fatty acids. The major polar lipid detected in strain SHSM-M15T was phosphatidylethanolamine. The DNA G+C content of strain SHSM-M15T from genomic sequence was 36.6 %. Mean DNA-DNA relatedness value between strain SHSM-M15T and the type strain of A. subtilis was 18 % and the average nucleotide identity value between strain SHSM-M15T and the type strain of A. subtilis was 87.98 %. The phylogenetic and genetic data and differential phenotypic properties indicated that strain SHSM-M15T is separated from A. subtilis. On the basis of the polyphasic data presented, strain SHSM-M15T is considered to represent a novel species of the genus Ancylomarina, for which the name Ancylomarina salipaludis sp. nov. is proposed. The type strain is SHSM-M15T (=KACC 19862T=NBRC 113749T).

Unveiling the bioactive potential of the essential oil of a Portuguese endemism, Santolina impressa

Ethnopharmacological relevanceSantolina species are widely used in traditional medicine in the Mediterranean region for their anti-inflammatory, antimicrobial, antispasmodic, digestive, and analgesic properties. S. impressa, a Portuguese endemism, is traditionally recognized for its beneficial anti-inflammatory properties in several gastrointestinal affections and is also used in oropharyngeal infections. Aim of the studyThe present study aims to characterize the essential oil of S. impressa growing in Portugal and validate its traditional uses by assessing the anti-inflammatory potential of its essential oil at concentrations without toxicity. The antifungal properties of the oil are also addressed, as well as, the putative mechanism of action underlying these effects. Material and methodsThe essential oil was obtained in accordance with the European Pharmacopoeia and characterized by GC and GC-MS. The anti-inflammatory potential of the oil was assessed on LPS-stimulated macrophages, through the production of nitric oxide (NO) using the Griess reaction. Putative mechanisms of action included the role of the oil as a NO scavenger, as well as its effect on the expression of two key pro-inflammatory enzymes, iNOS and COX-2 by Western blot analysis. The antifungal effect of the oil was evaluated according to the CLSI guidelines on several yeast and filamentous strains and on two major virulence factors in Candida albicans, namely germ tubes and biofilms. Ultrastructural modifications on dermatophytes were also unveiled by transmission electron microscopy. ResultsS. impressa essential oil was primarily characterized by the presence of monoterpene hydrocarbons and oxygenated monoterpenes, being the main compounds β-pinene (22.5%), 1,8-cineole (10.0%), limonene (9.1%), camphor (8.1%) and β-phellandrene (8.0%). A significant decrease (ca 60.0%) in nitrite levels was observed in LPS-stimulated macrophages treated with the oil without affecting cell viability. This effect could be explained by a great reduction on iNOS expression (85.0% inhibition), thus underpinning the anti-inflammatory potential of the oil.The oil also showed a fungicidal effect, being more active against Cryptococcus neoformans, Epidermophyton floccosum and Trichophytum rubrum. For these dermatophytes, significant ultrastructural modifications in cell wall structure were detected. Strikingly, for C. albicans, the oil showed a significant anti-infective potential (at 0.07 mg/mL for germ tube inhibition and 0.02 mg/mL for biofilm disruption) before fungal growth inhibition occurred. ConclusionsOur results validate the main traditional use ascribed to S. impressa, namely its anti-inflammatory effect. In addition, an antifungal potential is pointed out, thus corroborating the antimicrobial uses and adding new value to an endemic species poorly recognized by the industry.

Arsenic-contaminated sediment from mining areas as source of morphological and phylogenetic distinct cyanobacterial lineages

The prospection of cyanobacteria from tropical/subtropical ecosystems and from man-made environments has attracted attention as source of novel strains favoring studies on systematics, molecular/chemical prospection, physiology and biotechnology. Here, a deep characterization of abiotic variables of surface water and sediment samples collected from an Arsenic (As) contaminated region in a gold mining area was provided coupled with a complete morphological and phylogenetic evaluation of isolated cyanobacterial strains. Sediment samples presented higher As content than water samples, indicating As contamination as a result of mining activities. From the sediment samples, eight filamentous strains were isolated. These novel strains were placed into six distinct phylogenetic lineages, considering 16S rRNA sequences, from which two may correspond to novel genera. This is the first report of an Anagnostidinema strain from a brazilian environment as well as Desmonostoc and Kamptonema strains from As-impacted environment. In addition, Pseudanabaena strain displaying microcystin biosynthetic genes was also documented. The isolation and culturing of these novel cyanobacterial strains are not solely relevant for systematics, but it also represent a step further to better understand the role of cyanobacteria in the circulation of As in aquatic environments as well as for their application in As bioremediation.

Environmental characteristics and taxonomy of microscopic fungi isolated from washing machines

Washing machines (WMs) are convenient places for fungal colonization. This study is focused on fungal diversity of WMs, and investigates relationships between habits of WM users and colonising species. Housekeeping conditions and habits were assessed in Hungary with a questionnaire. Several fungal species were identified by microscopy and sequence analysis of diagnostic loci. Based on the results, 32 % of the sampled WMs were highly polluted with various species of fungi. Forty six percent of them were colonised also by opportunistically human pathogenic species. In total, 32 yeast and 39 filamentous fungal strains were isolated. Growth tests were conducted with five selected taxa (Cutaneotrichosporon dermatis, Cystobasidium slooffiae, Meyerozyma guilliermondii, Candida parapsilosis and the Fusarium oxysporum species complex (FOSC)) to ascertain their tolerance ranges. None of the examined isolates were able to grow >50 °C, 4.10 < pH < 10.88. FOSC could grow at high salinity. More species were detected in WMs operated in rooms without heating systems (p = 0.0025). The number of species was higher in WMs located in the kitchen than the ones kept in bathroom or in other rooms (p = 0.0205). WMs may serve as a reservoir of pathogenic fungi, the presence of which may depend on the usage of these devices.

Regulation by FurC in Anabaena Links the Oxidative Stress Response to Photosynthetic Metabolism.

The FUR (Ferric Uptake Regulator) family in Anabaena sp. PCC 7120 consists of three paralogs named FurA (Fur), FurB (Zur) and FurC (PerR). furC seems to be an essential gene in the filamentous nitrogen-fixing strain Anabaena sp. PCC 7120, suggesting that it plays a fundamental role in this organism. In order to better understand the functions of FurC in Anabaena, the phenotype of a derivative strain that overexpresses this regulator (EB2770FurC) has been characterized. The furC-overexpressing variant presented alterations in growth rate, morphology and ultrastructure, as well as higher sensitivity to peroxide than Anabaena sp. PCC 7120. Interestingly, the overexpression of furC led to reduced photosynthetic O2 evolution, increased respiratory activity, and had a significant influence in the composition and efficiency of both photosystems. Comparative transcriptional analyses, together with electrophoretic mobility shift assays allowed the identification of different genes directly controlled by FurC, and involved in processes not previously related to PerR proteins, such as the cell division gene ftsZ and the major thylakoid membrane protease ftsH. The rise in the transcription of ftsH in EB2770FurC cells correlated with reduced levels of the D1 protein, which is involved in the PSII repair cycle. Deregulation of the oxidative stress response in EB2770FurC cells led to the identification of novel FurC targets involved in the response to H2O2 through different mechanisms. These results, together with the effect of furC overexpression on the composition, stability and efficiency of the photosynthetic machinery of Anabaena, disclose novel links between PerR proteins, cell division and photosynthesis in filamentous cyanobacteria.

Near-Infrared Confocal Imaging of Single-Walled Carbon Nanotube Uptake in Bacterial Cells

The distinctive properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of innovative applications in the field of cell nanobiotechnology. Most studies to date have focused on eukaryotic cells capable of internalizing SWCNTs functionalized with a variety of non-covalent wrappings. However, the effect of SWCNT functionalization on transport across the thick cell wall of prokaryotes remains unexplored. In this study, we explore the uptake of SWCNTs in Gram-negative cyanobacteria and demonstrate selective internalization of SWCNTs decorated with charged protein wrappings. The functionalized SWCNTs are shown to traverse the outer cell wall of both filamentous and unicellular strains of cyanobacteria, independent of whether the strain is naturally competent for DNA uptake, with adsorption and internalization rate constants of kads = (9.08 ± 0.16) x 10-8 s-1 and kin = (1.466 ± 0.011) x 10-4 s-1, respectively. A custom-built, spinning disc confocal microscope was used for the first time to directly image near-infrared (NIR) SWCNT fluorescence within cells, revealing a highly inhomogeneous distribution of SWCNTs that is otherwise overlooked using conventional NIR widefield imaging. The nanobionic cells show sustained photosynthetic activity and growth, offering a powerful avenue for engineering photosynthetic organisms with augmented and even inherited nanobionic capabilities.

Differential transcriptomic profiling of filamentous fungus during solid-state and submerged fermentation and identification of an essential regulatory gene PoxMBF1 that directly regulated cellulase and xylanase gene expression

BackgroundSolid-state fermentation (SSF) mimics the natural decay environment of soil fungi and can be employed to investigate the production of plant biomass-degrading enzymes. However, knowledge on the transcriptional regulation of fungal genes during SSF remains limited. Herein, transcriptional profiling was performed on the filamentous fungus Penicillium oxalicum strain HP7-1 cultivated in medium containing wheat bran plus rice straw (WR) under SSF (WR_SSF) and submerged fermentation (WR_SmF; control) conditions. Novel key transcription factors (TFs) regulating fungal cellulase and xylanase gene expression during SSF were identified via comparative transcriptomic and genetic analyses.ResultsExpression of major cellulase genes was higher under WR_SSF condition than that under WR_SmF, but the expression of genes involved in the citric acid cycle was repressed under WR_SSF condition. Fifty-six candidate regulatory genes for cellulase production were screened out from transcriptomic profiling of P. oxalicum HP7-1 for knockout experiments in the parental strain ∆PoxKu70, resulting in 43 deletion mutants including 18 constructed in the previous studies. Enzyme activity assays revealed 14 novel regulatory genes involved in cellulase production in P. oxalicum during SSF. Remarkably, deletion of the essential regulatory gene PoxMBF1, encoding Multiprotein Bridging Factor 1, resulted in doubled cellulase and xylanase production at 2 days after induction during both SSF and SmF. PoxMBF1 dynamically and differentially regulated transcription of a subset of cellulase and xylanase genes during SSF and SmF, and conferred stress resistance. Importantly, PoxMBF1 bound specifically to the putative promoters of major cellulase and xylanase genes in vitro.ConclusionsWe revealed differential transcriptional regulation of P. oxalicum during SSF and SmF, and identified PoxMBF1, a novel TF that directly regulates cellulase and xylanase gene expression during SSF and SmF. These findings expand our understanding of regulatory mechanisms of cellulase and xylanase gene expression during fungal fermentation.