Tulika Prakash

Complete genome of the uncultured Termite Group 1 bacteria in a single host protist cell

Termites harbor a symbiotic gut microbial community that is responsible for their ability to thrive on recalcitrant plant matter. The community comprises diverse microorganisms, most of which are as yet uncultivable; the detailed symbiotic mechanism remains unclear. Here, we present the first complete genome sequence of a termite gut symbiont—an uncultured bacterium named Rs-D17 belonging to the candidate phylum Termite Group 1 (TG1). TG1 is a dominant group in termite guts, found as intracellular symbionts of various cellulolytic protists, without any physiological information. To acquire the complete genome sequence, we collected Rs-D17 cells from only a single host protist cell to minimize their genomic variation and performed isothermal whole-genome amplification. This strategy enabled us to reconstruct a circular chromosome (1,125,857 bp) encoding 761 putative protein-coding genes. The genome additionally contains 121 pseudogenes assigned to categories, such as cell wall biosynthesis, regulators, transporters, and defense mechanisms. Despite its apparent reductive evolution, the ability to synthesize 15 amino acids and various cofactors is retained, some of these genes having been duplicated. Considering that diverse termite-gut protists harbor TG1 bacteria, we suggest that this bacterial group plays a key role in the gut symbiotic system by stably supplying essential nitrogenous compounds deficient in lignocelluloses to their host protists and the termites. Our results provide a breakthrough to clarify the functions of and the interactions among the individual members of this multilayered symbiotic complex.

Genome of an Endosymbiont Coupling N2 Fixation to Cellulolysis Within Protist Cells in Termite Gut

Termites harbor diverse symbiotic gut microorganisms, the majority of which are as yet uncultivable and their interrelationships unclear. Here, we present the complete genome sequence of the uncultured Bacteroidales endosymbiont of the cellulolytic protist Pseudotrichonympha grassii, which accounts for 70% of the bacterial cells in the gut of the termite Coptotermes formosanus. Functional annotation of the chromosome (1,114,206 base pairs) unveiled its ability to fix dinitrogen and recycle putative host nitrogen wastes for biosynthesis of diverse amino acids and cofactors, and import glucose and xylose as energy and carbon sources. Thus, nitrogen fixation and cellulolysis are coupled within the protists cells. This highly evolved symbiotic system probably underlies the ability of the worldwide pest termites Coptotermes to use wood as their sole food.

Expression of conjoined genes: another mechanism for gene regulation in eukaryotes.

From the ENCODE project, it is realized that almost every base of the entire human genome is transcribed. One class of transcripts resulting from this arises from the conjoined gene, which is formed by combining the exons of two or more distinct (parent) genes lying on the same strand of a chromosome. Only a very limited number of such genes are known, and the definition and terminologies used for them are highly variable in the public databases. In this work, we have computationally identified and manually curated 751 conjoined genes (CGs) in the human genome that are supported by at least one mRNA or EST sequence available in the NCBI database. 353 representative CGs, of which 291 (82%) could be confirmed, were subjected to experimental validation using RT-PCR and sequencing methods. We speculate that these genes are arising out of novel functional requirements and are not merely artifacts of transcription, since more than 70% of them are conserved in other vertebrate genomes. The unique splicing patterns exhibited by CGs reveal their possible roles in protein evolution or gene regulation. Novel CGs, for which no transcript is available, could be identified in 80% of randomly selected potential CG forming regions, indicating that their formation is a routine process. Formation of CGs is not only limited to human, as we have also identified 270 CGs in mouse and 227 in drosophila using our approach. Additionally, we propose a novel mechanism for the formation of CGs. Finally, we developed a database, ConjoinG, which contains detailed information about all the CGs (800 in total) identified in the human genome. In summary, our findings reveal new insights about the functionality of CGs in terms of another possible mechanism for gene regulation and genomic evolution and the mechanism leading to their formation.

MetaBioME: a database to explore commercially useful enzymes in metagenomic datasets

Microbial enzymes have many known applications as biocatalysts in biotechnology, agriculture, medical and other industries. However, only a few enzymes are currently employed for such commercial applications. In this scenario, the current onslaught of metagenomic data provides a new unexplored treasure trove of genomic wealth that can not only enhance the enzyme repertoire by the discovery of novel commercially useful enzymes (CUEs) but can also reveal better functional variants for existing CUEs. We prepared a catalogue of CUEs using text mining of PubMed abstracts and other publicly available information, and manually curated the data to identify 510 CUEs. Further, in order to identify novel homologues of these CUEs, we identified potential ORFs in publicly available metagenomic datasets from 10 diverse sources. Using this strategy, we have developed a resource called MetaBioME (http://metasystems.riken.jp/metabiome/) that comprises (i) a database of CUEs and (ii) a comprehensive platform to facilitate homology-based computational identification of novel homologous CUEs from metagenomic and bacterial genomic datasets. Using MetaBioME, we have identified several novel homologues to known CUEs that can potentially serve as leads for further experimental verification.

Fast and Accurate Taxonomic Assignments of Metagenomic Sequences Using MetaBin

Taxonomic assignment of sequence reads is a challenging task in metagenomic data analysis, for which the present methods mainly use either composition- or homology-based approaches. Though the homology-based methods are more sensitive and accurate, they suffer primarily due to the time needed to generate the Blast alignments. We developed the MetaBin program and web server for better homology-based taxonomic assignments using an ORF-based approach. By implementing Blat as the faster alignment method in place of Blastx, the analysis time has been reduced by severalfold. It is benchmarked using both simulated and real metagenomic datasets, and can be used for both single and paired-end sequence reads of varying lengths (≥45 bp). To our knowledge, MetaBin is the only available program that can be used for the taxonomic binning of short reads (<100 bp) with high accuracy and high sensitivity using a homology-based approach. The MetaBin web server can be used to carry out the taxonomic analysis, by either submitting reads or Blastx output. It provides several options including construction of taxonomic trees, creation of a composition chart, functional analysis using COGs, and comparative analysis of multiple metagenomic datasets. MetaBin web server and a standalone version for high-throughput analysis are available freely at http://metabin.riken.jp/.

CoPS: Comprehensive Peptide Signature Database

UNLABELLED We present the development of a Comprehensive database of 12 076 invariant Peptide Signatures (CoPS) derived from 52 bacterial genomes with a minimum occurrence in at least seven organisms. These peptides were observed in functionally similar proteins and are distributed over nearly 1250 different functional proteins. The database provides function, structure and occurrence in biochemical pathways of the proteins containing these signature peptides. It houses additional information on the signature peptides, such as identical match in other motif/pattern (e.g. PROSITE, BLOCKS, PRINTS and Pfam) databases and the database of interacting proteins, human proteome and mutation effect on these signature peptides. There is a wide applicability of this database in the identification of critical functional residues in proteins. The database also facilitates the identification of folding nucleus/structural determinants in proteins and functional assignment to yet unknown proteins. We demonstrate functional assignment to 2605 hypothetical proteins in bacterial genomes and 112 unknown proteins in human using this database. AVAILABILITY The database can be freely accessed through the following URL: http://203.195.151.46/copsv2/index.html or http://203.90.127.70/copsv2/index.html

Recognition and analysis of protein-coding genes in severe acute respiratory syndrome associated coronavirus

Abstract Motivation: The recent outbreak of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) has necessitated an in-depth molecular understanding of the virus to identify new drug targets. The availability of complete genome sequence of several strains of SARS virus provides the possibility of identification of protein-coding genes and defining their functions. Computational approach to identify protein-coding genes and their putative functions will help in designing experimental protocols. Results: In this paper, a novel analysis of SARS genome using gene prediction method GeneDecipher developed in our laboratory has been presented. Each of the 18 newly sequenced SARS-CoV genomes has been analyzed using GeneDecipher. In addition to polyprotein 1ab1, polyprotein 1a and the four genes coding for major structural proteins spike (S), small envelope (E), membrane (M) and nucleocapsid (N), six to eight additional proteins have been predicted depending upon the strain analyzed. Their lengths range between 61 and 274 amino acids. Our method also suggests that polyprotein 1ab, polyprotein 1a, S, M and N are proteins of viral origin and others are of prokaryotic. Putative functions of all predicted protein-coding genes have been suggested using conserved peptides present in their open reading frames. Availability: Detailed results of GeneDecipher analysis of all the 18 strains of SARS-CoV genomes are available at http://www.igib.res.in/sarsanalysis.html

Correction: Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y

[This corrects the article DOI: 10.1371/journal.pone.0197448.].

Structural assessment of glycyl mutations in invariantly conserved motifs

Motifs that are evolutionarily conserved in proteins are crucial to their structure and function. In one of our earlier studies, we demonstrated that the conserved motifs occurring invariantly across several organisms could act as structural determinants of the proteins. We observed the abundance of glycyl residues in these invariantly conserved motifs. The role of glycyl residues in highly conserved motifs has not been studied extensively. Thus, it would be interesting to examine the structural perturbations induced by mutation in these conserved glycyl sites. In this work, we selected a representative set of invariant signature (IS) peptides for which both the PDB structure and mutation information was available. We thoroughly analyzed the conformational features of the glycyl sites and their local interactions with the surrounding residues. Using Ramachandran angles, we showed that the glycyl residues occurring in these IS peptides, which have undergone mutation, occurred more often in the L‐disallowed as compared with the L‐allowed region of the Ramachandran plot. Short range contacts around the mutation site were analyzed to study the steric effects. With the results obtained from our analysis, we hypothesize that any change of activity arising because of such mutations must be attributed to the long‐range interaction(s) of the new residue if the glycyl residue in the IS peptide occurred in the L‐allowed region of the Ramachandran plot. However, the mutation of those conserved glycyl residues that occurred in the L‐disallowed region of the Ramachandran plot might lead to an altered activity of the protein as a result of an altered conformation of the backbone in the immediate vicinity of the glycyl residue, in addition to long range effects arising from the long side chains of the new residue. Thus, the loss of activity because of mutation in the conserved glycyl site might either relate to long range interactions or to local perturbations around the site depending upon the conformational preference of the glycyl residue. Proteins 2007.