William W. Metcalf

Bio: William W. Metcalf is an academic researcher from University of Illinois at Urbana–Champaign. The author has contributed to research in topics: Methanosarcina acetivorans & Methanosarcina. The author has an hindex of 57, co-authored 156 publications receiving 9737 citations. Previous affiliations of William W. Metcalf include Purdue University & Howard Hughes Medical Institute.

The Genome of M. Acetivorans Reveals Extensive Metabolic and Physiological Diversity

Abstract: The Archaea remain the most poorly understood domain of life despite their importance to the biosphere. Methanogenesis, which plays a pivotal role in the global carbon cycle, is unique to the Archaea. Each year, an estimated 900 million metric tons of methane are biologically produced, representing the major global source for this greenhouse gas and contributing significantly to global warming (Schlesinger 1997). Methanogenesis is critical to the waste-treatment industry and biologically produced methane also represents an important alternative fuel source. At least two-thirds of the methane in nature is derived from acetate, although only two genera of methanogens are known to be capable of utilizing this substrate. We report here the first complete genome sequence of an acetate-utilizing (acetoclastic) methanogen, Methanosarcina acetivorans C2A. The Methanosarcineae are metabolically and physiologically the most versatile methanogens. Only Methanosarcina species possess all three known pathways for methanogenesis (Fig. ​(Fig.1)1) and are capable of utilizing no less than nine methanogenic substrates, including acetate. In contrast, all other orders of methanogens possess a single pathway for methanogenesis, and many utilize no more than two substrates. Among methanogens, the Methanosarcineae also display extensive environmental diversity. Individual species of Methanosarcina have been found in freshwater and marine sediments, decaying leaves and garden soils, oil wells, sewage and animal waste digesters and lagoons, thermophilic digesters, feces of herbivorous animals, and the rumens of ungulates (Zinder 1993). Figure 1 Three pathways for methanogenesis. Methanogenesis is a form of anaerobic respiration using a variety of one-carbon (C-1) compounds or acetic acid as a terminal electron acceptor. All three pathways converge on the reduction of methyl-CoM to methane (CH ... The Methanosarcineae are unique among the Archaea in forming complex multicellular structures during different phases of growth and in response to environmental change (Fig. ​(Fig.2).2). Within the Methanosarcineae, a number of distinct morphological forms have been characterized, including single cells with and without a cell envelope, as well as multicellular packets and lamina (Macario and Conway de Macario 2001). Packets and lamina display internal morphological heterogeneity, suggesting the possibility of cellular differentiation. Moreover, it has been suggested that cells within lamina may display differential production of extracellular material, a potential form of cellular specialization (Macario and Conway de Macario 2001). The formation of multicellular structures has been proposed to act as an adaptation to stress and likely plays a role in the ability of Methanosarcina species to colonize diverse environments. Figure 2 Different morphological forms of Methanosarcina acetivorans. Thin-section electron micrographs showing M. acetivorans growing as both single cells (center of micrograph) and within multicellular aggregates (top left, bottom right). Cells were harvested ... Significantly, powerful methods for genetic analysis exist for Methanosarcina species. These tools include plasmid shuttle vectors (Metcalf et al. 1997), very high efficiency transformation (Metcalf et al. 1997), random in vivo transposon mutagenesis (Zhang et al. 2000), directed mutagenesis of specific genes (Zhang et al. 2000), multiple selectable markers (Boccazzi et al. 2000), reporter gene fusions (M. Pritchett and W. Metcalf, unpubl.), integration vectors (Conway de Macario et al. 1996), and anaerobic incubators for large-scale growth of methanogens on solid media (Metcalf et al. 1998). Furthermore, and in contrast to other known methanogens, genetic analysis can be used to study the process of methanogenesis: Because Methanosarcina species are able to utilize each of the three known methanogenic pathways, mutants in a single pathway are viable (M. Pritchett and W. Metcalf, unpubl.). The availability of genetic methods allowing immediate exploitation of genomic sequence, coupled with the genetic, physiological, and environmental diversity of M. acetivorans make this species an outstanding model organism for the study of archaeal biology. For these reasons, we set out to study the genome of M. acetivorans.

A computational framework to explore large-scale biosynthetic diversity

Abstract: Genome mining has become a key technology to exploit natural product diversity. Although initially performed on a single-genome basis, the process is now being scaled up to mine entire genera, strain collections and microbiomes. However, no bioinformatic framework is currently available for effectively analyzing datasets of this size and complexity. In the present study, a streamlined computational workflow is provided, consisting of two new software tools: the ‘biosynthetic gene similarity clustering and prospecting engine’ (BiG-SCAPE), which facilitates fast and interactive sequence similarity network analysis of biosynthetic gene clusters and gene cluster families; and the ‘core analysis of syntenic orthologues to prioritize natural product gene clusters’ (CORASON), which elucidates phylogenetic relationships within and across these families. BiG-SCAPE is validated by correlating its output to metabolomic data across 363 actinobacterial strains and the discovery potential of CORASON is demonstrated by comprehensively mapping biosynthetic diversity across a range of detoxin/rimosamide-related gene cluster families, culminating in the characterization of seven detoxin analogues. Two bioinformatic tools, BiG-SCAPE and CORASON, enable sequence similarity network and phylogenetic analysis of gene clusters and their families across hundreds of strains and in large datasets, leading to the discovery of new natural products.

A roadmap for natural product discovery based on large-scale genomics and metabolomics

Abstract: Actinobacteria encode a wealth of natural product biosynthetic gene clusters, whose systematic study is complicated by numerous repetitive motifs. By combining several metrics, we developed a method for the global classification of these gene clusters into families (GCFs) and analyzed the biosynthetic capacity of Actinobacteria in 830 genome sequences, including 344 obtained for this project. The GCF network, comprising 11,422 gene clusters grouped into 4,122 GCFs, was validated in hundreds of strains by correlating confident mass spectrometric detection of known small molecules with the presence or absence of their established biosynthetic gene clusters. The method also linked previously unassigned GCFs to known natural products, an approach that will enable de novo, bioassay-free discovery of new natural products using large data sets. Extrapolation from the 830-genome data set reveals that Actinobacteria encode hundreds of thousands of future drug leads, and the strong correlation between phylogeny and GCFs frames a roadmap to efficiently access them.

Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria.

Abstract: A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn 5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1 x 10(-3) per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10(-3). Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonas stutzeri and in an Alcaligenes faecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Abstract: The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

“Bioinformatics” 특집을 내면서

Abstract: BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.

Fast Tree: Computing Large Minimum-Evolution Trees with Profiles instead of a Distance Matrix

Abstract: Gene families are growing rapidly, but standard methods for inferring phylogenies do not scale to alignments with over 10,000 sequences. We present FastTree, a method for constructing large phylogenies and for estimating their reliability. Instead of storing a distance matrix, FastTree stores sequence profiles of internal nodes in the tree. FastTree uses these profiles to implement neighbor-joining and uses heuristics to quickly identify candidate joins. FastTree then uses nearest-neighbor interchanges to reduce the length of the tree. For an alignment with N sequences, L sites, and a different characters, a distance matrix requires O(N^2) space and O(N^2 L) time, but FastTree requires just O( NLa + N sqrt(N) ) memory and O( N sqrt(N) log(N) L a ) time. To estimate the tree's reliability, FastTree uses local bootstrapping, which gives another 100-fold speedup over a distance matrix. For example, FastTree computed a tree and support values for 158,022 distinct 16S ribosomal RNAs in 17 hours and 2.4 gigabytes of memory. Just computing pairwise Jukes-Cantor distances and storing them, without inferring a tree or bootstrapping, would require 17 hours and 50 gigabytes of memory. In simulations, FastTree was slightly more accurate than neighbor joining, BIONJ, or FastME; on genuine alignments, FastTree's topologies had higher likelihoods. FastTree is available at http://microbesonline.org/fasttree.