Niels Bent Larsen

The Subsystems Approach to Genome Annotation and its Use in the Project to Annotate 1000 Genomes

The release of the 1000th complete microbial genome will occur in the next two to three years. In anticipation of this milestone, the Fellowship for Interpretation of Genomes (FIG) launched the Project to Annotate 1000 Genomes. The project is built around the principle that the key to improved accuracy in high-throughput annotation technology is to have experts annotate single subsystems over the complete collection of genomes, rather than having an annotation expert attempt to annotate all of the genes in a single genome. Using the subsystems approach, all of the genes implementing the subsystem are analyzed by an expert in that subsystem. An annotation environment was created where populated subsystems are curated and projected to new genomes. A portable notion of a populated subsystem was defined, and tools developed for exchanging and curating these objects. Tools were also developed to resolve conflicts between populated subsystems. The SEED is the first annotation environment that supports this model of annotation. Here, we describe the subsystem approach, and offer the first release of our growing library of populated subsystems. The initial release of data includes 180 177 distinct proteins with 2133 distinct functional roles. This data comes from 173 subsystems and 383 different organisms.

Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis

Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by diarrhoeal or emetic syndromes. It is closely related to the animal and human pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis, the former being used as a biological weapon and the latter as a pesticide. B. anthracis and B. thuringiensis are readily distinguished from B. cereus by the presence of plasmid-borne specific toxins (B. anthracis and B. thuringiensis) and capsule (B. anthracis). But phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B. cereus, B. anthracis and B. thuringiensis are varieties of the same species or different species. Here we report the sequencing and analysis of the type strain B. cereus ATCC 14579. The complete genome sequence of B. cereus ATCC 14579 together with the gapped genome of B. anthracis A2012 enables us to perform comparative analysis, and hence to identify the genes that are conserved between B. cereus and B. anthracis, and the genes that are unique for each species. We use the former to clarify the phylogeny of the cereus group, and the latter to determine plasmid-independent species-specific markers.

The genome sequence of the facultative intracellular pathogen Brucella melitensis

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other α-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.

Genome Sequence and Analysis of the Oral Bacterium Fusobacterium nucleatum Strain ATCC 25586

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H(2)S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth.

The ERGOTM genome analysis and discovery system

The ERGO (http://ergo.integratedgenomics.com/ERGO/) genome analysis and discovery suite is an integration of biological data from genomics, biochemistry, high-throughput expression profiling, genetics and peer-reviewed journals to achieve a comprehensive analysis of genes and genomes. Far beyond any conventional systems that facilitate functional assignments, ERGO combines pattern-based analysis with comparative genomics by visualizing genes within the context of regulation, expression profiling, phylogenetic clusters, fusion events, networked cellular pathways and chromosomal neighborhoods of other functionally related genes. The result of this multifaceted approach is to provide an extensively curated database of the largest available integration of genomes, with a vast collection of reconstructed cellular pathways spanning all domains of life. Although access to ERGO is provided only under subscription, it is already widely used by the academic community. The current version of the system integrates 500 genomes from all domains of life in various levels of completion, 403 of which are available for subscription.

Optical waveguide sensor for on-line monitoring of bacteria

A grating-coupled planar optical waveguide sensor is presented for sensing of bacteria by evanescent waves. The waveguide design results in increased depth of penetration into the sample volume, which makes it suitable for detecting micrometer-sized biological objects. We tested the sensors performance by monitoring the adhesion of Escherichia coli K12 cells to the sensor surface.

Lithography beyond light: microcontact printing with monolayer resists

We describe high-resolution lithography based on transfer of a pattern from an elastomeric stamp to a solid substrate by conformal contact : a nanoscale interaction between substrate and stamp on macroscopic scales that allows transport of material from stamp to substrate. The stamp is first formed by curing poly(dimethyl siloxane) (PDMS) on a master with the negative of the desired surface, resulting in an elastomeric solid with a pattern of reliefs, typically a few microns deep, on its surface. The stamp provides an ink that forms a self-assembled monolayer (SAM) on a solid surface by a covalent, chemical reaction. Because SAMs act as highly localized and efficient barriers to some wet etches, microcontact printing forms part of a convenient lithographic system not subject to diffraction or depth of focus limitations while still providing simultaneous transfer of patterned features. Our study helps to define the strengths and limitations of microcontact printing with SAMs, a process that is necessary to assess its worth to technology. We used lithography based on scanning tunneling microscopy (STM) to demonstrate that disruption of SAMs on gold allowed the formation of etched features as small as 20 nm using a CN - /O 2 etch. This result implied that etching occurred where damage of a few molecules in the ordered SAM allowed passage of cyanide, whereas adjacent molecules in the SAM remained unperturbed at this scale. Features as small as 30 nm 2 etched in gold over areas greater than 1 cm 2 resulted from microcontact printing with replicas of electron-beam-formed masters, with the transfer of these printed SAMs requiring only 1 s. STM studies of these transferred SAMs revealed an achievable order indistinguishable from that found for SAMs prepared from solution. Facile alignment of printing steps at submicron scales may result from new designs of stamps that exploit their limited deformability and lock-and-key-type approaches to mate stamp and substrate.

Single-molecule denaturation mapping of DNA in nanofluidic channels

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO®-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent “barcode” corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.

Monitoring of living cell attachment and spreading using reverse symmetry waveguide sensing

The effect of the attachment and spreading of living cells on the modes of a grating coupled reverse symmetry waveguide sensor is investigated in real time. The reverse symmetry design has an increased probing depth into the sample making it well suited for the monitoring of cell morphology. As a result, significant changes in the incoupling peak height and peak shape were observed during cell attachment and spreading. It is suggested that the area under the incoupling peaks reflects the initial cell attachment process, while the mean peak position is mostly governed by the spreading of the cells.

Confinement Spectroscopy: Probing Single DNA Molecules with Tapered Nanochannels

We demonstrate a confinement spectroscopy technique capable of probing small conformational changes of unanchored single DNA molecules in a manner analogous to force spectroscopy, in the regime corresponding to femtonewton forces. In contrast to force spectroscopy, various structural forms of DNA can easily be probed, as indicated by experiments on linear and circular DNA. The extension of circular DNA is found to scale according to the de Gennes exponent, unlike for linear DNA.