MAPLE 2.3.0: an improved system for evaluating the functionomes of genomes and metagenomes

MAPLE is an automated system for inferring the potential comprehensive functions harbored by genomes and metagenomes. To reduce runtime in MAPLE analyzing the massive amino acid datasets of over 1 million sequences, we improved it by adapting the KEGG automatic annotation server to use GHOSTX and verified no substantial difference in the MAPLE results between the original and new implementations.     

Prediction of enzymes and non-enzymes from protein sequences based on sequence derived features and PSSM matrix using artificial neural network

The problem of predicting the enzymes and non-enzymes from the protein sequence information is still an open problem in bioinformatics. It is further becoming more important as the number of sequenced information grows exponentially over time. We describe a novel approach for predicting the enzymes and non-enzymes from its amino-acid sequence using artificial neural network (ANN). Using 61 sequence derived features alone we have been able to achieve 79 percent correct prediction of enzymes/non-enzymes (in the set of 660 proteins). For the complete set of 61 parameters using 5-fold cross-validated classification, ANN model reveal a superior model (accuracy = 78.79 plus or minus 6.86 percent, Q(pred) = 74.734 plus or minus 17.08 percent, sensitivity = 84.48 plus or minus 6.73 percent, specificity = 77.13 plus or minus 13.39 percent). The second module of ANN is based on PSSM matrix. Using the same 5-fold cross-validation set, this ANN model predicts enzymes/non-enzymes with more accuracy (accuracy = 80.37 plus or minus 6.59 percent, Q(pred) = 67.466 plus or minus 12.41 percent, sensitivity = 0.9070 plus or minus 3.37 percent, specificity = 74.66 plus or minus 7.17 percent).     

基于比较基因组学重构细菌的代谢途径和调控网络

微生物基因组学的迅速发展为从功能基因与蛋白、网络及其调控等不同的角度,全面理解与认识微生物的代谢过程、构建细胞工厂,提供了丰富的背景信息。基于基因组序列进行代谢网络重构,有助于发现新的代谢功能基因、调控元件、甚至新的代谢途径,从而优化设计细胞内从原料到产品的生物合成路线。然而,目前公共数据库平台中代谢途径基因的功能注释,许多是错误或不完整的。以下着重介绍了近年来出现的一些用于代谢途径和调控网络重构的新型比较基因组学技术,并以近期的丙酮丁醇梭菌中木糖代谢途径的重构工作为例来说明其应用。     

An automated annotation tool for genomic DNA sequences using GeneScan and BLAST

Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.     

Phylogenetic and metagenomic analysis of Verrucomicrobia in former agricultural grassland soil

The bacterial phylum Verrucomicrobia has a widespread distribution, and is known to be one of the most common and diverse phyla in soil habitats. However, members of this phylum have typically been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we examine the phylogenetic diversity of the Verrucomicrobia in a former agricultural field and gain access to genomic information via a metagenomic approach. We examined Verrucomicrobia -like 16S rRNA gene sequences recovered from general bacterial and phylum-specific libraries, revealing a dominance of subdivisions 1 and 2. A PCR-based screening method was developed to identify inserts containing verrucomicrobial 16S rRNA genes within a large-insert metagenomic library, and on screening of 28 800 clones, four fosmids were identified as containing verrucomicrobial genomic DNA. Full-length sequencing of fosmid inserts and gene annotation identified a total of 98 ORFs, representing a range of functions. No conservation of gene order was observed adjacent to the ribosomal operons. Fosmid inserts were further analyzed for tetranucleotide frequencies to identify remnants of past horizontal gene transfer events. The metagenomic approach utilized proved to be suitable for the recovery of verrucomicrobial genomic DNA, thereby providing a window into the genomes of members of this important, yet poorly characterized, bacterial phylum.     

Genetic innovations: Transposable element recruitment and de novo formation lead to the birth of orphan genes in the rice genome

Orphan genes are genetic innovations that lack homologs in other lineages. Orphan genes can rapidly originate and become substantially functional, yet the mechanisms underlying their origins are still largely unknown in plants. Here, we investigated the origin of orphan genes in the Oryza sativa ssp. japonica “Nipponbare” genome using genome‐wide comparisons with 10 closely related Oryza species. We identified a total of 37 orphan genes in the Nipponbare genome that show short sequence lengths, elevated GC content, and absence of introns. Interestingly, half of the identified orphan genes originated by way of a distinctive mechanism that involved the generation of new coding sequences through independent and rapid divergence within the inserted transposable element. Our results provide valuable insight into genetic innovations in the model rice genome that formed on a very short timescale.     

Computational genome analyses of metabolic enzymes in Mycobacterium leprae for drug target identification

Leprosy is an infectious disease caused by Mycobacterium leprae. M. leprae has undergone a major reductive evolution leaving a minimal set of functional genes for survival. It remains non-cultivable. As M. leprae develops resistance against most of the drugs, novel drug targets are required in order to design new drugs. As most of the essential genes mediate several biosynthetic and metabolic pathways, the pathway predictions can predict essential genes. We used comparative genome analysis of metabolic enzymes in M. leprae and H. sapiens using KEGG pathway database and identified 179 non-homologues enzymes. On further comparison of these 179 non-homologous enzymes to the list of minimal set of 48 essential genes required for cell-wall biosynthesis of M. leprae reveals eight common enzymes. Interestingly, six of these eight common enzymes map to that of peptidoglycan biosynthesis and they all belong to Mur enzymes. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy and thus targeting these enzymes is a step towards facilitating the search for new antibiotics.     

Toward molecular trait‐based ecology through integration of biogeochemical,geographical and metagenomic data

Using metagenomic ‘parts lists’ to infer global patterns on microbial ecology remains a significant challenge. To deduce important ecological indicators such as environmental adaptation, molecular trait dispersal, diversity variation and primary production from the gene pool of an ecosystem, we integrated 25 ocean metagenomes with geographical, meteorological and geophysicochemical data. We find that climatic factors (temperature, sunlight) are the major determinants of the biomolecular repertoire of each sample and the main limiting factor on functional trait dispersal (absence of biogeographic provincialism). Molecular functional richness and diversity show a distinct latitudinal gradient peaking at 20°N and correlate with primary production. The latter can also be predicted from the molecular functional composition of an environmental sample. Together, our results show that the functional community composition derived from metagenomes is an important quantitative readout for molecular trait‐based biogeography and ecology.     

Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems

Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability and pressure. Survival under these conditions must have necessitated the adaptation and the development of unique cellular biochemistry and metabolism by these microbes. Thus, enzymes isolated from these microbes have the potential to possess quite unique physiological and biochemical properties. This review outlines a number of function-based metagenomic approaches which are available to screen metagenomic libraries constructed from marine ecosystems to facilitate the exploitation of some of these potentially novel biocatalysts. Functional screens to isolate novel cellulases, lipases and esterases, proteases, laccases, oxidoreductases and biosurfactants are described, together with approaches which can be employed to help overcome some of the typical problems encountered with functional metagenomic-based screens.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

基因组信息的整合与植物功能基因组学研究的策略

近年来,随着许多植物基因组测序和可利用序列的增加,相继建立了一些基于靶基因诱变的“反向”遗传学研究策略,如T—DNA诱变、基因敲除、基因沉默和超表达分析等。同时,DNA微阵列和基因芯片技术的发展使得快速、定量检测植物发育不同时期和不同组织器官的基因转录时空变化成为现实。作图技术的改进和来自不同物种基因组信息的整合也正在加速图谱克隆程序的简化和发展。因此,随着生物基因组测序工作日益增多,整合不同类群植物基因组的信息和资源,在植物功能基因组学研究中的重要性日趋显著。     

Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis

Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks.     

贵州典型煤矸石堆场微生物群落结构及功能特征

【背景】煤矸石堆场用于堆放煤矿开采过程中产生的一种热值低、含重金属的固体废弃物,会产生大量的酸性废水,对堆场周边的生态环境造成严重影响。【目的】探究煤矸石堆场的微生物群落结构和功能特征。【方法】选择贵州省六枝特区典型煤矸石堆场作为研究对象,采集堆场表层土壤、矸石层土壤、废水浸出口沉积物和堆场下游河道沉积物,通过宏基因组学技术进行分析。【结果】细菌的优势菌门为变形菌门(Proteobacteria)和放线菌门(Actinobacteria),优势菌属为钩端螺菌属(Leptospirillum)和硫化杆菌属(Sulfobacillus);古菌的优势菌门为Candidatus＿Thermoplasmatota和泉古菌门(Crenarchaeota),优势菌属为热原体属(Thermoplasma)和金属球菌属(Metallosphaera)。不同采样点细菌和古菌的优势菌属存在显著差异,矸石层土壤和废水浸出口沉积物中的铁氧化细菌和硫氧化细菌比其他2个样点丰富。煤矸石堆场中微生物的碳、氮、硫代谢基因丰度较高,共检测到6条固碳途径、6条氮代谢途径和3条硫代谢途径。主要的固碳基因为ACAT和E2.2....     

metaxa2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data

The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species‐ or genus‐level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The metaxa software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to metaxa – metaxa 2 – that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short‐read (100 bp) and paired‐end sequences, among other changes. The performance of metaxa 2 was compared to other commonly used taxonomic classifiers, showing that metaxa 2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. metaxa 2 is freely available from http://microbiology.se/software/metaxa2/ .     

Comparison of large-insert,small-insert and pyrosequencing libraries for metagenomic analysis

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation'' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.     

Structure and function of enzymes in heme biosynthesis

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V‐shaped glutamyl‐tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the “Radical SAM enzyme” coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure–function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.     

A combined computational-experimental analyses of selected metabolic enzymes in Pseudomonas species

Comparative genomic analysis has revolutionized our ability to predict the metabolic subsystems that occur in newly sequenced genomes, and to explore the functional roles of the set of genes within each subsystem. These computational predictions can considerably reduce the volume of experimental studies required to assess basic metabolic properties of multiple bacterial species. However, experimental validations are still required to resolve the apparent inconsistencies in the predictions by multiple resources. Here, we present combined computational-experimental analyses on eight completely sequenced Pseudomonas species. Comparative pathway analyses reveal that several pathways within the Pseudomonas species show high plasticity and versatility. Potential bypasses in 11 metabolic pathways were identified. We further confirmed the presence of the enzyme O-acetyl homoserine (thiol) lyase (EC: 2.5.1.49) in P. syringae pv. tomato that revealed inconsistent annotations in KEGG and in the recently published SYSTOMONAS database. These analyses connect and integrate systematic data generation, computational data interpretation, and experimental validation and represent a synergistic and powerful means for conducting biological research.