Influence of carbon source complexity on porosity, water retention and extracellular matrix composition of Neurospora discreta biofilms.

AIMS: To evaluate carbon source complexity as a process lever to impact the microstructure, chemical composition and water retention capacity of biofilms produced by Neurospora discreta. METHODS AND RESULTS: Biofilms were produced by nonpathogenic fungus N. discreta, using sucrose, cellulose or lignin as carbon source. The increase in complexity of carbon source from sucrose to lignin resulted in decreased water retention values (WRV) and wet weights of harvested biofilms. Confocal laser scanning microscopy was used to calculate porosity from bright-field images, and relative stained areas of cells and carbohydrates from fluorescence imaging of samples stained with Trypan blue and Alexa Fluor 488. Porosity and relative quantity of cells increased with increase in carbon source complexity while the amount of carbohydrates decreased. The chemical analysis of the extracted extracellular matrix (ECM) showed that biofilms grown on more complex carbon sources had lower carbohydrate and protein content, which also explains the lower WRV trend, as carbohydrates are hydrophilic. CONCLUSIONS: The nature of carbon source impacts the metabolic pathway of cells, thereby influencing the relative proportions of ECM and cells. This in turn impacts the microstructure, composition and water content of biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that carbon source can be used as process lever to control the properties of biofilms and presents a novel view of biofilms as potentially useful biomaterials.

Arrested fungal biofilms as low-modulus structural bio-composites: Water holds the key.

Biofilms are self-assembling structures consisting of rigid microbial cells embedded in a soft biopolymeric extracellular matrix (ECM), and have been commonly viewed as being detrimental to health and equipment. In this work, we show that biofilms formed by a non-pathogenic fungus Neurospora discreta, are fungal bio-composites (FBCs) that can be directed to self-organize through active stresses to achieve specific properties. We induced active stresses by systematically varying the agitation rate during the growth of FBCs. By growing FBCs that are strong enough to be conventionally tensile loaded, we find that as agitation rate increases, the elongation strain at which the FBCs break, increases linearly, and their elastic modulus correspondingly decreases. Using results from microstructural imaging and thermogravimetry, we rationalize that agitation increases the production of ECM, which concomitantly increases the water content of agitated FBCs up to 250% more than un-agitated FBCs. Water held in the nanopores of the ECM acts a plasticizer and controls the ductility of FBCs in close analogy with polyelectrolyte complexes. This paradigm shift in viewing biofilms as bio-composites opens up the possibility for their use as sustainable, biodegradable, low-modulus structural materials.

Abundant Perithecial Protein (APP) from Neurospora is a primitive functional analog of ocular crystallins.

The eye arose during the Cambrian explosion from pre-existing proteins that would have been recruited for the formation of the specialized components of this organ, such as the transparent lens. Proteins suitable for the role of lens crystallins would need to possess unusual physical properties and the study of such earliest analogs of ocular crystallins would add to our understanding of the nature of recruitment of proteins as lens/corneal crystallins. We show that the Abundant Perithecial Protein (APP) of the fungi Neurospora and Sordaria fulfils the criteria for an early crystallin analog. The perithecia in these fungal species are phototropic, and APP accumulates at a high concentration in the neck of the pitcher-shaped perithecium. Spores are formed at the base of the perithecium, and light contributes to their maturation. The hydrodynamic properties of APP appear to exclude dimer formation or aggregation at high protein concentrations. APP is also deficient in Ca2+ binding, a property seen in its close homolog, the calcium-binding cell adhesion molecule (DdCAD-1) from Dictyostelium discoidum. Comparable to crystallins, APP occurs in high concentrations and seems to have dispensed with Ca2+ binding in exchange for greater stability. These crystallin-like attributes of APP lead us to demonstrate that it is a primitive form of ocular crystallins.

Evaluation of broiler chickens' digestibility of Neurospora intermedia biomass.

A total of 70 broiler chickens were used to evaluate the apparent ileal digestibility coefficient (AIDC) of protein and amino acids, and apparent digestibility coefficient (ADC) of energy in the protein rich Fungal Biomass of Neurospora intermedia (FBN) obtained from bioethanol production. The chickens were housed in 10 pens with seven chickens per pen and fed one of two experimental diets between day 28 and 35 of age. The experimental diets were wheat-soybean meal-based control diet and a diet composed of 70% control diet and 30% of FBN. There were no difference (P > 0.05) between the control and FBN diet on chick feed intake and body weight at day 35. However, the AIDCs of crude protein and amino acids were significantly (P < 0.01) higher in control diet than in the FBN diet except for proline and phenylalanine. The AIDC of CP (0.74), cysteine (0.68), methionine (0.70), AME (15.6), and threonine (0.69) in FBN were comparable to the corresponding values in other protein-rich feedstuffs such as soybean meal and fish meal. The results from this study show that FBN may be an alternative protein source for poultry.

Mechanism study of silver nanoparticle production using Neurospora intermedia.

Elucidation of the molecular mechanism of silver nanoparticle (AgNP) synthesis is necessary to control nanoparticle size, shape, and monodispersity. In this study, the mechanism of AgNP formation by Neurospora intermedia was investigated. The higher production rate of AgNP formation using a culture supernatant heat-treated at 100° and 121°C relative to that with an un-treated culture supernatant indicated that the native form of the molecular species is not essential. The effect of the protein molecular weight (MW) on the nanoparticle size distribution and average size was studied by means of ultraviolet-visible spectroscopy and dynamic light scattering. Using un-treated and concentrated cell-free filtrate passed through 10 and 20 kDa cut-off filters led to the production of AgNPs with average sizes of 25, 30, and 34 nm, respectively. Also, using the permeate fraction of cell-free filtrate passed through a 100 kDa cut-off filter led to the formation of the smallest nanoparticles with the narrowest size distribution (average size of 16 nm and polydispersity index of 0.18). Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of the fungal extracellular proteins showed two notable bands with the MWs of 15 and 23 kDa that are involved in the reduction and stabilisation of the nanoparticles, respectively.

Insights into RNA structure and dynamics from recent NMR and X-ray studies of the Neurospora Varkud satellite ribozyme.

Despite the large number of noncoding RNAs and their importance in several biological processes, our understanding of RNA structure and dynamics at atomic resolution is still very limited. Like many other RNAs, the Neurospora Varkud satellite (VS) ribozyme performs its functions through dynamic exchange of multiple conformational states. More specifically, the VS ribozyme recognizes and cleaves its stem-loop substrate via a mechanism that involves several structural transitions within its stem-loop substrate. The recent publications of high-resolution structures of the VS ribozyme, obtained by NMR spectroscopy and X-ray crystallography, offer an opportunity to integrate the data and closely examine the structural and dynamic properties of this model RNA system. Notably, these investigations provide a valuable example of the divide-and-conquer strategy for structural and dynamic characterization of a large RNA, based on NMR structures of several individual subdomains. The success of this divide-and-conquer approach reflects the modularity of RNA architecture and the great care taken in identifying the independently-folding modules. Together with previous biochemical and biophysical characterizations, the recent NMR and X-ray studies provide a coherent picture into how the VS ribozyme recognizes its stem-loop substrate. Such in-depth characterization of this RNA enzyme will serve as a model for future structural and engineering studies of dynamic RNAs and may be particularly useful in planning divide-and-conquer investigations. WIREs RNA 2017, 8:e1421. doi: 10.1002/wrna.1421 For further resources related to this article, please visit the WIREs website.

Crystal structure of the Varkud satellite ribozyme.

The Varkud satellite (VS) ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.

The NMR structure of the II-III-VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure.

As part of an effort to structurally characterize the complete Neurospora VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III-IV-V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II-III-VI three-way junction and its adjoining stems. In addition, we localize Mg(2+)-binding sites within this structure using Mn(2+)-induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg(2+) ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge-bulge interaction and Mg(2+) ions are critical for the stable folding of the II-III-VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II-III-VI junction that does not contain the II-VI bulge-bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique ribozyme.

Hydrogen-bonding changes of internal water molecules upon the actions of microbial rhodopsins studied by FTIR spectroscopy.

Microbial rhodopsins are classified into type-I rhodopsins, which utilize light energy to perform wide varieties of function, such as proton pumping, ion pumping, light sensing, cation channels, and so on. The crystal structures of several type-I rhodopsins were solved and the molecular mechanisms have been investigated based on the atomic structures. However, the crystal structures of proteins of interest are not always available and the basic architectures are sometimes quite similar, which obscures how the proteins achieve different functions. Stimulus-induced difference FTIR spectroscopy is a powerful tool to detect minute structural changes providing a clue for elucidating the molecular mechanisms. In this review, the studies on type-I rhodopsins from fungi and marine bacteria, whose crystal structures have not been solved yet, were summarized. Neurospora rhodopsin and Leptosphaeria rhodopsin found from Fungi have sequence similarity. The former has no proton pumping function, while the latter has. Proteorhodopsin is another example, whose proton pumping machinery is altered at alkaline and acidic conditions. We described how the structural changes of protein were different and how water molecules were involved in them. We reviewed the results on dynamics of the internal water molecules in pharaonis halorhodopsin as well. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags.

Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3' homogeneity. Here, we explored strategies to also ensure 5' homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5' heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II 2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5'-sequence heterogeneity in several cases, significant levels of 5' heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5' homogeneity, we tested the suitability of using a small CRISPR RNA stem-loop at the 5' end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5'-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR-SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5'-CRISPR-based strategy can be combined with affinity purification using the 3'-ARiBo tag for quick purification of RNA with both 5' and 3' homogeneity.

Endo-ß-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity.

The crystal structure of wild-type endo-ß-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (ß/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

Arginine changes the conformation of the arginine attenuator peptide relative to the ribosome tunnel.

The fungal arginine attenuator peptide (AAP) is a regulatory peptide that controls ribosome function. As a nascent peptide within the ribosome exit tunnel, it acts to stall ribosomes in response to arginine (Arg). We used three approaches to probe the molecular basis for stalling. First, PEGylation assays revealed that the AAP did not undergo overall compaction in the tunnel in response to Arg. Second, site-specific photocross-linking showed that Arg altered the conformation of the wild-type AAP, but not of nonfunctional mutants, with respect to the tunnel. Third, using time-resolved spectral measurements with a fluorescent probe placed in the nascent AAP, we detected sequence-specific changes in the disposition of the AAP near the peptidyltransferase center in response to Arg. These data provide evidence that an Arg-induced change in AAP conformation and/or environment in the ribosome tunnel is important for stalling.

Gel-based mass spectrometric and computational approaches to the mitochondrial proteome of Neurospora.

We have used gel electrophoretic techniques including isoelectric focusing, blue native, acid-urea, 16-benzyldimethyl-n-hexadecylammonium chloride or SDS first dimensions and SDS Laemmli or tricine second dimensions to separate the proteins from highly-purified Neurospora mitochondria and sub-mitochondrial fractions (membrane, soluble, protein complexes and ribonucleoproteins). The products of 260 genes, many of them in multiple processed or modified forms, were identified by MALDI-TOF mass spectrometry. This work confirms the existence, expression, and mitochondrial localization of the products of 55 Neurospora genes previously annotated only as predicted or hypothetical, and of 101 genes not identified in previous mass spectrometry studies. Combined with previous mass spectrometry studies, and re-evaluation of genome annotations, we have compiled a curated list of 358 proteins identified in proteomic studies that are likely to be bona fide mitochondrial proteins plus 80 other identified proteins that may be mitochondrial. Literature data mining and computational predictions suggest that Neurospora mitochondria also contain another 299 proteins not yet identified in proteomics projects. Taken together, these data suggest that the products of at least 738 genes comprise the Neurospora mitochondrial proteome.

Data variance and statistical significance in 2D-gel electrophoresis and DIGE experiments: comparison of the effects of normalization methods.

Identifying changes in the relative abundance of proteins between different biological samples is often confounded by technical noise. In this work, we compared eight normalization methods commonly used in two-dimensional gel electrophoresis and difference gel electrophoresis (DIGE) experiments for their ability to reduce noise and for their influence on the list of proteins whose difference in abundance between two samples is determined to be statistically significant. With respect to reducing noise we find that, while all methods improve upon unnormalized data, cyclic linear normalization is the least well suited to gel-based proteomics and the performances of the other methods are similar. We also find in DIGE data that the choice of normalization method has less of an impact on the noise than does the decision to use an internal reference in the experimental design and that both normalization and standardization using the internal reference are required to maximally reduce variance. Despite the similar noise reduction achieved by most normalization methods, the list of proteins whose abundance was determined to differ significantly between biological groups differed depending on the choice of normalization method. This work provides a direct comparison of the impact of normalization methods in the context of common experimental designs.

Fatty acid methyl ester from Neurospora intermedia N-1 isolated from Indonesian red peanut cake (oncom merah).

The objective of this study was to identify the Fatty Acid Methyl Ester (FAME) from Neurospora intermedia N-1 that isolated from Indonesian red peanut cake (oncom). FAME profiles have been used as biochemical characters to study many different groups of organisms, such as bacteria and yeasts. FAME from N. intermedia N-1 was obtained by some stages of extraction the orange spores and fractination using a chromatotron. The pure compound (1) was characterized by 500 mHz NMR (1H and 13C), FTIR and LC-MS. Summarized data's of 1H and 13C NMR spectra of compound 1 contained 19 Carbon, 34 Hydrogen and 2 Oxygen (C19H34O2). The position of the double bonds at carbon number 8 and 12 were indicated in the HMBC spectrum (2D-NMR). LC-MS spectrum indicates molecular weight of the compound 1 as 294 which is visible by the presence of protonated molecular ion [M+H] at m/z 295. Methyl esters of long chain fatty acids was presented by a 3 band pattern of IR spectrum with bands near 1249, 1199 and 1172 cm(-1). We suggested that the structure of the pure compound 1 is methyl octadeca-8,12-dienoate. The presence methyl octadeca-8,12-dienoate in N. intermedia is the first report.

Allenyl and alkynyl phenyl ethers from the endolichenic fungus Neurospora terricola.

Terricollenes A-C (1-3), new allenyl phenyl ethers, and terricolynes (4-6), new alkynyl phenyl ethers, have been isolated from the crude extract of the endolichenic fungus Neurospora terricola. The structures of these compounds were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were determined by reversed-phase HPLC analysis on the o-tolylthiocarbamates of the liberated sugars upon acid hydrolysis, whereas that of 4 was assigned using the modified Mosher method. Compound 1 showed modest cytotoxicity against the human tumor cell lines HeLa and MCF-7.

DCL-1 colocalizes with other components of the MSUD machinery and is required for silencing.

In Neurospora, a gene present in an abnormal number of copies is usually a red flag for mischief. One way to deal with these potential intruders is by destroying their transcripts. Widely known as RNA interference (RNAi), this mechanism depends on the "dicing" of a double-stranded RNA intermediate into small-interfering RNA, which in turn guide the degradation of mRNA from the target gene. Quelling is a vegetative silencing system in Neurospora that utilizes such a mechanism. Quelling depends on the redundant activity of two Dicer-like ribonucleases, DCL-1 and DCL-2. Here, we show that Meiotic Silencing by Unpaired DNA (MSUD), a mechanism that silences expression from unpaired DNA during meiosis, requires the dcl-1 (but not the dcl-2) gene for its function. This result suggests that MSUD operates in a similar manner to Quelling and other RNAi systems. DCL-1 colocalizes with SAD-1 (an RdRP), SAD-2, and SMS-2 (an Argonaute) in the perinuclear region.

Circadian clocks and natural antisense RNA.

Eukaryotes regulate gene expression in a number of different ways. On a daily and seasonal timescale, the orchestration of gene expression is to a large extent governed by circadian clocks. These endogenous timekeepers enable organisms to prepare for predictable environmental conditions from one day to the next and thus allow adaptation to a given temporal niche. In general, circadian clocks have been shown to employ the classical transcriptional and posttranscriptional control mechanisms to generate rhythmicity. However, the discovery of antisense clock gene transcripts suggests that mechanisms of gene regulation operating through antisense RNA may also be integral to the circadian clockwork. Following a brief history of the impact of genetic and molecular techniques in aiding our understanding of circadian clocks, this review concentrates on the few examples of antisense clock gene transcripts so far investigated and their effect on circadian timing.

Single fungal kinesin motor molecules move processively along microtubules.

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.