Thermal adaptation strategies of the extremophile bacterium Thermus filiformis based on multi-omics analysis.

Thermus filiformis is an aerobic thermophilic bacterium isolated from a hot spring in New Zealand. The experimental study of the mechanisms of thermal adaptation is important to unveil response strategies of the microorganism to stress. In this study, the main pathways involved on T. filiformis thermoadaptation, as well as, thermozymes with potential biotechnological applications were revealed based on omics approaches. The strategy adopted in this study disclosed that pathways related to the carbohydrate metabolism were affected in response to thermoadaptation. High temperatures triggered oxidative stress, leading to repression of genes involved in glycolysis and the tricarboxylic acid cycle. During heat stress, the glucose metabolism occurred predominantly via the pentose phosphate pathway instead of the glycolysis pathway. Other processes, such as protein degradation, stringent response, and duplication of aminoacyl-tRNA synthetases, were also related to T. filiformis thermoadaptation. The heat-shock response influenced the carotenoid profile of T. filiformis, favoring the synthesis of thermozeaxanthins and thermobiszeaxanthins, which are related to membrane stabilization at high temperatures. Furthermore, antioxidant enzymes correlated with free radical scavenging, including superoxide dismutase, catalase and peroxidase, and metabolites, such as oxaloacetate and α-ketoglutarate, were accumulated at 77 °C.

Functional characterization and synergic action of fungal xylanase and arabinofuranosidase for production of xylooligosaccharides.

Plant cell wall degrading enzymes are key technological components in biomass bioconversion platforms for lignocellulosic materials transformation. Cost effective production of enzymes and identification of efficient degradation routes are two economic bottlenecks that currently limit the use of renewable feedstocks through an environmental friendly pathway. The present study describes the hypersecretion of an endo-xylanase (GH11) and an arabinofuranosidase (GH54) by a fungal expression system with potential biotechnological application, along with comprehensive characterization of both enzymes, including spectrometric analysis of thermal denaturation, biochemical characterization and mode of action description. The synergistic effect of these enzymes on natural substrates such as sugarcane bagasse, demonstrated the biotechnological potential of using GH11 and GH54 for production of probiotic xylooligosaccharides from plant biomass. Our findings shed light on enzymatic mechanisms for xylooligosaccharide production, as well as provide basis for further studies for the development of novel enzymatic routes for use in biomass-to-bioethanol applications.

Inducible RNA Interference of brlAbeta in Aspergillus nidulans.

An inducible RNA interference (RNAi) construct composed of inverted repeating alcA promoters flanking the developmental regulatory gene brlAbeta was tested in Aspergillus nidulans. On inducing medium, the RNAi strains failed to sporulate and lacked brlAalpha and brlAbeta expression. RNAi was specific for brlAbeta, but not brlAalpha, silencing, indicating brlAalpha regulation by brlAbeta.

Accumulation of stress and inducer-dependent plant-cell-wall-degrading enzymes during asexual development in Aspergillus nidulans.

Determination and interpretation of fungal gene expression profiles based on digital reconstruction of expressed sequenced tags (ESTs) are reported. A total of 51,524 DNA sequence files processed with PipeOnline resulted in 9775 single and 5660 contig unique ESTs, 31.2% of a typical fungal transcriptome. Half of the unique ESTs shared homology with genes in public databases, 35.8% of which are functionally defined and 64.2% are unclear or unknown. In Aspergillus nidulans 86% of transcripts associate with intermediate metabolism functions, mainly related to carbohydrate, amino acid, protein, and peptide biosynthesis. During asexual development, A. nidulans unexpectedly accumulates stress response and inducer-dependent transcripts in the absence of an inducer. Stress response genes in A. nidulans ESTs total 1039 transcripts, contrasting with 117 in Neurospora crassa, a 14.3-fold difference. A total of 5.6% of A. nidulans ESTs implicate inducer-dependent cell wall degradation or amino acid acquisition, 3.5-fold higher than in N. crassa. Accumulation of stress response and inducer-dependent transcripts suggests general derepression of cis-regulation during terminal asexual development.

Expression and action pattern of Botryotinia fuckeliana (Botrytis cinerea) rhamnogalacturonan hydrolase in Pichia pastoris.

The cDNA sequence coding for the complete rhamnogalacturonan hydrolase (RGase) of Botryotinia fuckeliana (Botrytis cinerea) was introduced into Pichia pastoris and expressed under the control of the alcohol oxidase promoter. The RGase was secreted into the medium of the yeast driven by the alpha-factor secretion peptide and could be purified using the C-terminal His6-tag fusion. RGase activity was measured using a traditional reducing end assay with linseed rhamnogalacturonan (RG) as the substrate, or with an assay using a fluorescent RG oligomer as the substrate and detection and identification of hydrolysis products by capillary zone electrophoresis (CZE). Both methods showed the recombinant enzyme to have a specific activity of about ten units per milligram of protein. Since the CZE method allows identification of the hydrolysis products, it was used to show that the RGase lacks a multiple attack mechanism and needs at least five GalA-Rha repeating disaccharides to be active. This finding is contrary to the action pattern of the native RGase of Aspergillus aculeatus which has the same substrate length requirement, but exhibits multiple attack, leading to products containing only two and three Rha-GalA repeat units without the appearance of intermediate sized fragments. No plant cell wall degrading enzymes were detected in the culture medium of un-transformed P. pastoris, thus the recombinant enzyme, devoid of extraneous activities, can be applied for fine structural studies on cell walls.

The reliability of the Aspergillus nidulans physical map.

Here we report an evaluation of the Aspergillus nidulans physical map (a cosmid contig map) emphasizing quantification and description of obvious mapping errors. Classification and appraisal of mapping errors should be helpful to researchers working on particular regions of the map. We estimate between 47 (4.1%) and 63 (5.4%) probe/clone-linking errors. The majority of identified false links (38) permit reciprocal exchanges among linking clones located on disconnected mapping regions. The order of adjacent clones or probes on the affected contigs remains unchanged. In addition we describe an Internet-accessible resource in which genetic and physical maps were integrated through a graphic interface. A simple search engine allows retrieval of cosmids from redundant clone lists and provides links to the minimal clone order. Integration of genetic and physical maps provides an additional level of accountability in which mapping discrepancies are visually located.

Multicellular ascomycetous fungal genomes contain more than 8000 genes.

Fungi comprise a large monophyletic group of uni- and multicellular eukaryotic organisms in which many species are of economic or medical importance. Fungal genomes are variable in size (13-42 Mb), and multicellular species support true spatial and temporal cell-type-specific regulation of gene expression. In a 38.8-kb Aspergillus nidulans contiguous genomic DNA region, a transposable element and 12 potential genes were identified, 7 similar to genes in other organisms. This observation is consistent with the prediction that multicellular ascomycetous fungi harbor 8000-9000 genes in a 36-Mb average genome. Thus, the genomic DNA sequence of filamentous fungi will provide substantial amounts of genetic and functional information that is not available in yeast, for the human and other metazoan minimal gene complement.

In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome.

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.

Detection of Toxoplasma gondii in feline and canine biological samples by use of the polymerase chain reaction.

OBJECTIVE: To develop a polymerase chain reaction (PCR) technique to identify Toxoplasma gondii DNA in biological samples from cats and dogs. DESIGN: To artificially create samples that would mimic those acquired in a clinical setting from animals with naturally acquired toxoplasmosis. Using these samples, a PCR test to identify T gondii DNA was developed. SAMPLE POPULATION: Feline and canine aqueous humor, CSF, serum, and blood samples. PROCEDURE: Tachyzoites of several strains of T gondii grown in cell culture were added to feline and canine aqueous humor, CSF, serum, and blood samples. Protocols for identifying T gondii DNA by use of the PCR were developed. RESULTS: The DNA from as few as 10 tachyzoites of T gondii could be identified in feline and canine aqueous humor, CSF, and serum samples. One hundred tachyzoites could be identified in blood samples. CONCLUSIONS: Toxoplasma gondii can be identified in feline and canine biological samples by use of the PCR. CLINICAL RELEVANCE: Correlation of clinical disease to T gondii serum antibodies provides only a presumptive diagnosis of toxoplasmosis. Use of PCR to detect T gondii DNA in biological samples from cats and dogs may provide a sensitive tool for the antemortem diagnosis of toxoplasmosis and may be most beneficial when used in conjunction with serum antibody titers.

On the consistency of a physical mapping method to reconstruct a chromosome in vitro.

During recent years considerable effort has been invested in creating physical maps for a variety of organisms as part of the Human Genome Project and in creating various methods for physical mapping. The statistical consistency of a physical mapping method to reconstruct a chromosome, however, has not been investigated. In this paper, we first establish that a model of physical mapping by binary fingerprinting of DNA fragments is identifiable using the key assumption-for a large randomly generated recombinant DNA library, there exists a staircase of DNA fragments across the chromosomal region of interest. Then we briefly introduce epi-convergence theory of variational analysis and transform the physical mapping problem into a constrained stochastic optimization problem. By doing so, we prove epi-convergence of the physical mapping model and epi-convergence of the physical mapping method. Combining the identifiability of our physical mapping model and the epi-convergence of a physical mapping method, finally we establish strong consistency of a physical mapping method.

Xylanases: from biology to biotechnology.

Xylan is the main carbohydrate found in the hemicellulosic fraction of plant tissues and accounts for one third of all renewable organic carbon available on earth. Xylanase, the major component of an enzymatic consortium, acts in nature by depolymerizing xylan molecules into monomeric pentosan units that are used by bacterial and fungal populations as a primary carbon source. Xylanase producers have been isolated from all ecological niches where plant material is deposited, and microorganisms often contain multiple loci encoding overlapping xylanolytic functions. The numerical excess of genes and the extensive sharing of structural features within beta-glycanase families suggests that extensive gene duplication and conversion events have occurred during xylanase evolution. Hydrolysis of beta-glycosidic linkages is sponsored by a general acid catalytic reaction common to all glycanases, whereas substrate recognition is specified by subsites that interact with adjacent glycosyl units. Under natural conditions xylanases are inducible by the products of their own action and subject to carbon catabolite repression. Bleaching paper pulps with xylanases is the first successful commercial application for these enzymes. The recovery of cellulosic textile fibers is the next logical application and bioconversion of biomass into fuels and chemicals, remains the ultimate target. Recent developments have shown that metabolic pathways can be transferred from one organism to another and proteins can be modified to gain conformational stability, suggesting that naturally occurring systems can be custom engineered to the situation in the fermentation tank. Thus, biotechnologies developed to transform biomass into marketable products that gradually substitute materials derived from non-renewable resources are becoming commercially worthwhile.

ODS_BOOTSTRAP: assessing the statistical reliability of physical maps by bootstrap resampling.

In the program ODS_BOOTSTRAP we provide a methodology for quickly ordering clones in a genomic library into a physical map and for applying a statistical tool known as the bootstrap to assess the statistical reliability of a clonal ordering. Each clone is assigned a binary fingerprint by one of a variety of experimental approaches to physical mapping. For example, the binary fingerprints might be generated by hybridizing a panel of m probes to a library of n clones. The resulting n x m binary data matrix, X, is input to ODS_BOOTSTRAP, which utilizes the similarity in binary fingerprints of clones to construct a physical map. Under this particular implementation of bootstrap resampling, the m probes (or columns of the data matrix) are sampled randomly with replacement in the computer to generate a new n x m data matrix, X*, from which a second physical map is constructed. The resampling process is repeated 100 or more times to generate 100 or more X* matrices. The resulting 100 or more physical maps are compared with the original physical map based on the original data matrix X by counting how often links in the original physical map reappear. Three confidence statistics are introduced for each link in a physical map. The statistic C1 is defined as the percentage of time two neighboring clones on the original map reappear as neighbors under resampling. The statistic C2 is defined as the percentage of time that two neighboring clones i and j on the original map reappear as neighbors or that a clone with an identical binary fingerprint to clone i reappears as a neighbor to clone j. The statistic C3 is defined as the percentage of time that two neighboring clones on the original map reappear in the same contig under resampling.

A fast random cost algorithm for physical mapping.

Ordering clones from a genomic library into physical maps of whole chromosomes presents a central computational/statistical problem in genetics. Here we present a physical mapping algorithm for creating ordered genomic libraries or contig maps by using a random cost approach [Berg, A. (1993) Nature (London) 361, 708-710]. This random cost algorithm is 5-10 times faster than existing physical mapping algorithms and has optimization performance comparable to existing procedures. The speedup in the algorithm makes practical the widespread use of bootstrap resampling to assess the statistical reliability of links in the physical map as well as the use of more elaborate physical mapping criteria to improve map quality. The random cost algorithm is illustrated by its application in assembling a physical map of chromosome IV from the filamentous fungus Aspergillus nidulans.

The Penicillium chrysogenum and Aspergillus nidulans wetA developmental regulatory genes are functionally equivalent.

Aspergillus nidulans and Penicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. In A. nidulans, spore maturation is controlled by the wetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) from P. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. The PwetA and AwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5' flanking regions, including conserved binding sites for the product of the regulatory gene abaA. PwetA fully complemented an A. nidulans wetA deletion mutation, demonstrating that PwetA and its 5' regulatory sequences function normally in A. nidulans. These results indicate that the mechanisms controlling sporulation in A. nidulans and P. chrysogenum are evolutionarily conserved.

The Aspergillus nidulans brlA regulatory locus consists of overlapping transcription units that are individually required for conidiophore development.

The Aspergillus nidulans brlA locus controls conidiophore development in conjunction with the products of several other regulatory loci. In this paper, we show that the brlA locus consists of overlapping transcription units, designated alpha and beta, with alpha transcription initiating within beta intronic sequences. The predicted BrlA polypeptides differ by 23 amino acid residues at their N-termini. Targeted mutations specifically eliminating either the alpha or beta transcript led to developmental abnormalities similar to those produced by previously identified hypomorphic mutants, showing that both transcripts have essential functions for normal development. However, provision of additional doses of alpha in a beta- strain or of beta in an alpha- strain remediated the developmental defects, indicating that the polypeptides have redundant functions. It is likely that differential regulation of alpha and beta expression in the wild type is important for the initiation and temporal regulation of development.

Role of sulfhydryl compounds in the control of tyrosinase activity in Neurospora crassa.

It is known that Neurospora crassa mycelia cultured in standard concentrations (76 to 190 micrograms/ml) of sulfate accumulate a low molecular weight inhibitor of tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase; EC 1.14.1.18.1.). This is not observed in cultures grown under sulfate-limiting conditions. The chemical nature of tyrosinase inhibition was investigated. It was shown to be due to the low molecular weight sulfhydryl fraction of the extracts, in which glutathione is predominant. The concentration of low molecular weight sulfhydryl compounds decreased sharply in mycelia submitted to various treatments which also derepressed tyrosinase, such as (i) starvation in phosphate buffer, (ii) treatment with cycloheximide, and (iii) mating. These results suggest that the concentration of sulfhydryl compounds may be of physiological significance in the control of tyrosinase activity in N. crassa.