Comparative analysis of functional diversity of rumen microbiome in bison and beef heifers.

IMPORTANCE: Ruminants play a key role in the conversion of cellulolytic plant material into high-quality meat and milk protein for humans. The rumen microbiome is the driver of this conversion, yet there is little information on how gene expression within the microbiome impacts the efficiency of this conversion process. The current study investigates gene expression in the rumen microbiome of beef heifers and bison and how transplantation of ruminal contents from bison to heifers alters gene expression. Understanding interactions between the host and the rumen microbiome is the key to developing informed approaches to rumen programming that will enhance production efficiency in ruminants.

Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain.

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.

Genetic Characterization of Mutations Related to Conidiophore Stalk Length Development in Aspergillus niger Laboratory Strain N402.

Aspergillus niger is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the A. niger N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of A. niger, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of A. niger strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed cspA and cspB) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (cspA) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the A. nidulans UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (cspB) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.

Identification of a Conserved Transcriptional Activator-Repressor Module Controlling the Expression of Genes Involved in Tannic Acid Degradation and Gallic Acid Utilization in Aspergillus niger.

Tannic acid, a hydrolysable gallotannin present in plant tissues, consists of a central glucose molecule esterified with gallic acid molecules. Some microorganisms, including several Aspergillus species, can metabolize tannic acid by releasing gallic acid residues from tannic acid by secreting tannic acid specific esterases into the medium. The expression of these so-called tannases is induced by tannic acid or gallic acid. In this study, we identified a conserved transcriptional activator-repressor module involved in the regulation of predicted tannases and other genes involved in gallic acid metabolism. The transcriptional activator-repressor module regulating tannic acid utilization resembles the transcriptional activator-repressor modules regulating galacturonic acid and quinic acid utilization. Like these modules, the Zn(II)2Cys6 transcriptional activator (TanR) and the putative repressor (TanX) are located adjacent to each other. Deletion of the transcriptional activator (ΔtanR) results in inability to grow on gallic acid and severely reduces growth on tannic acid. Deletion of the putative repressor gene (ΔtanX) results in the constitutive expression of tannases as well as other genes with mostly unknown function. Known microbial catabolic pathways for gallic acid utilization involve so-called ring cleavage enzymes, and two of these ring cleavage enzymes show increased expression in the ΔtanX mutant. However, deletion of these two genes, and even deletion of all 17 genes encoding potential ring cleavage enzymes, did not result in a gallic acid non-utilizing phenotype. Therefore, in A. niger gallic acid utilization involves a hitherto unknown pathway. Transcriptome analysis of the ΔtanX mutant identified several genes and gene clusters that were significantly induced compared to the parental strain. The involvement of a selection of these genes and gene clusters in gallic acid utilization was examined by constructing gene deletion mutants and testing their ability to grow on gallic acid. Only the deletion of a gene encoding an FAD-dependent monooxygenase (NRRL3_04659) resulted in a strain that was unable to grow on gallic acid. Metabolomic studies showed accumulation of gallic acid in the ΔNRRL3_04659 mutant suggesting that this predicted monooxygenase is involved in the first step of gallic acid metabolism and is likely responsible for oxidation of the aromatic ring.

Application of Transcriptomics to Compare the Carbohydrate Active Enzymes That Are Expressed by Diverse Genera of Anaerobic Fungi to Degrade Plant Cell Wall Carbohydrates.

The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well-recognized and in recent years has received renewed interest. To further understand the biological mechanisms that are utilized by the rumen anaerobic fungi to break down lignocellulose, we have used a transcriptomic approach to examine carbohydrate digestion by Neocallimastix frontalis, Piromyces rhizinflata, Orpinomyces joyonii, and Anaeromyces mucronatus cultured on several carbon sources. The number of predicted unique transcripts ranged from 6,633 to 12,751. Pfam domains were identified in 62-70% of the fungal proteins and were linked to gene ontology terms to infer the biological function of the transcripts. Most of the predicted functions are consistent across species suggesting a similar overall strategy evolved for successful colonization of the rumen. However, the presence of differential profiles in enzyme classes suggests that there may be also be niche specialization. All fungal species were found to express an extensive array of transcripts encoding carbohydrate active enzymes (CAZymes) ranging from 8.3 to 11.3% of the transcriptome. CAZyme families involved in hemicellulose digestion were the most abundant across all four fungi. This study provides additional insight into how anaerobic fungi have evolved to become specialists at breaking down the plant cell wall in the complex and, strictly anaerobic rumen ecosystem.

Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid-responsive transcription factor gaaR.

The transcription factor GaaR is needed for the expression of genes required for pectin degradation and transport and catabolism of the main degradation product, D-galacturonic acid (GA) in Aspergillus niger. In this study, we used the strong constitutive gpdA promoter of Aspergillus nidulans to overexpress gaaR in A. niger. Overexpression of gaaR resulted in an increased transcription of the genes encoding pectinases, (putative) GA transporters, and catabolic pathway enzymes even under non-inducing conditions, i.e., in the absence of GA. Exoproteome analysis of a strain overexpressing gaaR showed that this strain secretes highly elevated levels of pectinases when grown in fructose. The genes encoding exo-polygalacturonases were found to be subjected to CreA-mediated carbon catabolite repression, even in the presence of fructose. Deletion of creA in the strain overexpressing gaaR resulted in a further increase in pectinase production in fructose. We showed that GaaR localizes mainly in the nucleus regardless of the presence of an inducer, and that overexpression of gaaR leads to an increased concentration of GaaR in the nucleus.

An Evolutionarily Conserved Transcriptional Activator-Repressor Module Controls Expression of Genes for D-Galacturonic Acid Utilization in Aspergillus niger.

The expression of genes encoding extracellular polymer-degrading enzymes and the metabolic pathways required for carbon utilization in fungi are tightly controlled. The control is mediated by transcription factors that are activated by the presence of specific inducers, which are often monomers or monomeric derivatives of the polymers. A D-galacturonic acid-specific transcription factor named GaaR was recently identified and shown to be an activator for the expression of genes involved in galacturonic acid utilization in Botrytis cinerea and Aspergillus niger Using a forward genetic screen, we isolated A. niger mutants that constitutively express GaaR-controlled genes. Reasoning that mutations in the gaaR gene would lead to a constitutively activated transcription factor, the gaaR gene in 11 of the constitutive mutants was sequenced, but no mutations in gaaR were found. Full genome sequencing of five constitutive mutants revealed allelic mutations in one particular gene encoding a previously uncharacterized protein (NRRL3_08194). The protein encoded by NRRL3_08194 shows homology to the repressor of the quinate utilization pathway identified previously in Neurospora crassa (qa-1S) and Aspergillus nidulans (QutR). Deletion of NRRL3_08194 in combination with RNA-seq analysis showed that the NRRL3_08194 deletion mutant constitutively expresses genes involved in galacturonic acid utilization. Interestingly, NRRL3_08194 is located next to gaaR (NRRL3_08195) in the genome. The homology to the quinate repressor, the chromosomal clustering, and the constitutive phenotype of the isolated mutants suggest that NRRL3_08194 is likely to encode a repressor, which we name GaaX. The GaaR-GaaX module and its chromosomal organization is conserved among ascomycetes filamentous fungi, resembling the quinate utilization activator-repressor module in amino acid sequence and chromosomal organization.

Real-Time 3D Tracking and Reconstruction on Mobile Phones.

We present a novel framework for jointly tracking a camera in 3D and reconstructing the 3D model of an observed object. Due to the region based approach, our formulation can handle untextured objects, partial occlusions, motion blur, dynamic backgrounds and imperfect lighting. Our formulation also allows for a very efficient implementation which achieves real-time performance on a mobile phone, by running the pose estimation and the shape optimisation in parallel. We use a level set based pose estimation but completely avoid the, typically required, explicit computation of a global distance. This leads to tracking rates of more than 100 Hz on a desktop PC and 30 Hz on a mobile phone. Further, we incorporate additional orientation information from the phone's inertial sensor which helps us resolve the tracking ambiguities inherent to region based formulations. The reconstruction step first probabilistically integrates 2D image statistics from selected keyframes into a 3D volume, and then imposes coherency and compactness using a total variational regularisation term. The global optimum of the overall energy function is found using a continuous max-flow algorithm and we show that, similar to tracking, the integration of per voxel posteriors instead of likelihoods improves the precision and accuracy of the reconstruction.

A biochemical genomics screen for substrates of Ste20p kinase enables the in silico prediction of novel substrates.

The Ste20/PAK family is involved in many cellular processes, including the regulation of actin-based cytoskeletal dynamics and the activation of MAPK signaling pathways. Despite its numerous roles, few of its substrates have been identified. To better characterize the roles of the yeast Ste20p kinase, we developed an in vitro biochemical genomics screen to identify its substrates. When applied to 539 purified yeast proteins, the screen reported 14 targets of Ste20p phosphorylation. We used the data resulting from our screen to build an in silico predictor to identify Ste20p substrates on a proteome-wide basis. Since kinase-substrate specificity is often mediated by additional binding events at sites distal to the phosphorylation site, the predictor uses the presence/absence of multiple sequence motifs to evaluate potential substrates. Statistical validation estimates a threefold improvement in substrate recovery over random predictions, despite the lack of a single dominant motif that can characterize Ste20p phosphorylation. The set of predicted substrates significantly overrepresents elements of the genetic and physical interaction networks surrounding Ste20p, suggesting that some of the predicted substrates are in vivo targets. We validated this combined experimental and computational approach for identifying kinase substrates by confirming the in vitro phosphorylation of polarisome components Bni1p and Bud6p, thus suggesting a mechanism by which Ste20p effects polarized growth.

MonoSLAM: real-time single camera SLAM.

We present a real-time algorithm which can recover the 3D trajectory of a monocular camera, moving rapidly through a previously unknown scene. Our system, which we dub MonoSLAM, is the first successful application of the SLAM methodology from mobile robotics to the "pure vision" domain of a single uncontrolled camera, achieving real time but drift-free performance inaccessible to Structure from Motion approaches. The core of the approach is the online creation of a sparse but persistent map of natural landmarks within a probabilistic framework. Our key novel contributions include an active approach to mapping and measurement, the use of a general motion model for smooth camera movement, and solutions for monocular feature initialization and feature orientation estimation. Together, these add up to an extremely efficient and robust algorithm which runs at 30 Hz with standard PC and camera hardware. This work extends the range of robotic systems in which SLAM can be usefully applied, but also opens up new areas. We present applications of MonoSLAM to real-time 3D localization and mapping for a high-performance full-size humanoid robot and live augmented reality with a hand-held camera.