Libusha Kelly

MODBASE, a database of annotated comparative protein structure models, and associated resources

ModBase (http://salilab.org/modbase) is a database of annotated comparative protein structure models. The models are calculated by ModPipe, an automated modeling pipeline that relies primarily on Modeller for fold assignment, sequence-structure alignment, model building and model assessment (http://salilab.org/modeller/). ModBase currently contains almost 30 million reliable models for domains in 4.7 million unique protein sequences. ModBase allows users to compute or update comparative models on demand, through an interface to the ModWeb modeling server (http://salilab.org/modweb). ModBase models are also available through the Protein Model Portal (http://www.proteinmodelportal.org/). Recently developed associated resources include the AllosMod server for modeling ligand-induced protein dynamics (http://salilab.org/allosmod), the AllosMod-FoXS server for predicting a structural ensemble that fits an SAXS profile (http://salilab.org/allosmod-foxs), the FoXSDock server for protein–protein docking filtered by an SAXS profile (http://salilab.org/foxsdock), the SAXS Merge server for automatic merging of SAXS profiles (http://salilab.org/saxsmerge) and the Pose & Rank server for scoring protein–ligand complexes (http://salilab.org/poseandrank). In this update, we also highlight two applications of ModBase: a PSI:Biology initiative to maximize the structural coverage of the human alpha-helical transmembrane proteome and a determination of structural determinants of human immunodeficiency virus-1 protease specificity.

LS-SNP: large-scale annotation of coding non-synonymous SNPs based on multiple information sources

MOTIVATION The NCBI dbSNP database lists over 9 million single nucleotide polymorphisms (SNPs) in the human genome, but currently contains limited annotation information. SNPs that result in amino acid residue changes (nsSNPs) are of critical importance in variation between individuals, including disease and drug sensitivity. RESULTS We have developed LS-SNP, a genomic scale software pipeline to annotate nsSNPs. LS-SNP comprehensively maps nsSNPs onto protein sequences, functional pathways and comparative protein structure models, and predicts positions where nsSNPs destabilize proteins, interfere with the formation of domain-domain interfaces, have an effect on protein-ligand binding or severely impact human health. It currently annotates 28,043 validated SNPs that produce amino acid residue substitutions in human proteins from the SwissProt/TrEMBL database. Annotations can be viewed via a web interface either in the context of a genomic region or by selecting sets of SNPs, genes, proteins or pathways. These results are useful for identifying candidate functional SNPs within a gene, haplotype or pathway and in probing molecular mechanisms responsible for functional impacts of nsSNPs. AVAILABILITY http://www.salilab.org/LS-SNP CONTACT: rachelk@salilab.org SUPPLEMENTARY INFORMATION http://salilab.org/LS-SNP/supp-info.pdf.

Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments

T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.

Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism

Cyanophages infecting the marine cyanobacteria Prochlorococcus and Synechococcus encode and express genes for the photosynthetic light reactions. Sequenced cyanophage genomes lack Calvin cycle genes, however, suggesting that photosynthetic energy harvested via phage proteins is not used for carbon fixation. We report here that cyanophages carry and express a Calvin cycle inhibitor, CP12, whose host homologue directs carbon flux from the Calvin cycle to the pentose phosphate pathway (PPP). Phage CP12 was coexpressed with phage genes involved in the light reactions, deoxynucleotide biosynthesis, and the PPP, including a transaldolase gene that is the most prevalent PPP gene in cyanophages. Phage transaldolase was purified to homogeneity from several strains and shown to be functional in vitro, suggesting that it might facilitate increased flux through this key reaction in the host PPP, augmenting production of NADPH and ribose 5-phosphate. Kinetic measurements of phage and host transaldolases revealed that the phage enzymes have k(cat)/K(m) values only approximately one third of the corresponding host enzymes. The lower efficiency of phage transaldolase may be a tradeoff for other selective advantages such as reduced gene size: we show that more than half of host-like cyanophage genes are significantly shorter than their host homologues. Consistent with decreased Calvin cycle activity and increased PPP and light reaction activity under infection, the host NADPH/NADP ratio increased two-fold in infected cells. We propose that phage-augmented NADPH production fuels deoxynucleotide biosynthesis for phage replication, and that the selection pressures molding phage genomes involve fitness advantages conferred through mobilization of host energy stores.

Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria.

Our understanding of secondary metabolite production in bacteria has been shaped primarily by studies of attached varieties such as symbionts, pathogens, and soil bacteria. Here we show that a strain of the single-celled, planktonic marine cyanobacterium Prochlorococcus—which conducts a sizable fraction of photosynthesis in the oceans—produces many cyclic, lanthionine-containing peptides (lantipeptides). Remarkably, in Prochlorococcus MIT9313 a single promiscuous enzyme transforms up to 29 different linear ribosomally synthesized peptides into a library of polycyclic, conformationally constrained products with highly diverse ring topologies. Genes encoding this system are found in variable abundances across the oceans—with a hot spot in a Galapagos hypersaline lagoon—suggesting they play a habitat- and/or community-specific role. The extraordinarily efficient pathway for generating structural diversity enables these cyanobacteria to produce as many secondary metabolites as model antibiotic-producing bacteria, but with much smaller genomes.

The genome and structural proteome of an ocean siphovirus: a new window into the cyanobacterial 'mobilome'

Prochlorococcus, an abundant phototroph in the oceans, are infected by members of three families of viruses: myo-, podo- and siphoviruses. Genomes of myo- and podoviruses isolated on Prochlorococcus contain DNA replication machinery and virion structural genes homologous to those from coliphages T4 and T7 respectively. They also contain a suite of genes of cyanobacterial origin, most notably photosynthesis genes, which are expressed during infection and appear integral to the evolutionary trajectory of both host and phage. Here we present the first genome of a cyanobacterial siphovirus, P-SS2, which was isolated from Atlantic slope waters using a Prochlorococcus host (MIT9313). The P-SS2 genome is larger than, and considerably divergent from, previously sequenced siphoviruses. It appears most closely related to lambdoid siphoviruses, with which it shares 13 functional homologues. The ∼108 kb P-SS2 genome encodes 131 predicted proteins and notably lacks photosynthesis genes which have consistently been found in other marine cyanophage, but does contain 14 other cyanobacterial homologues. While only six structural proteins were identified from the genome sequence, 35 proteins were detected experimentally; these mapped onto capsid and tail structural modules in the genome. P-SS2 is potentially capable of integration into its host as inferred from bioinformatically identified genetic machinery int, bet, exo and a 53 bp attachment site. The host attachment site appears to be a genomic island that is tied to insertion sequence (IS) activity that could facilitate mobility of a gene involved in the nitrogen-stress response. The homologous region and a secondary IS-element hot-spot in Synechococcus RS9917 are further evidence of IS-mediated genome evolution coincident with a probable relic prophage integration event. This siphovirus genome provides a glimpse into the biology of a deep-photic zone phage as well as the ocean cyanobacterial prophage and IS element ‘mobilome’.

Comparison of human solute carriers

Solute carriers are eukaryotic membrane proteins that control the uptake and efflux of solutes, including essential cellular compounds, environmental toxins, and therapeutic drugs. Solute carriers can share similar structural features despite weak sequence similarities. Identification of sequence relationships among solute carriers is needed to enhance our ability to model individual carriers and to elucidate the molecular mechanisms of their substrate specificity and transport. Here, we describe a comprehensive comparison of solute carriers. We link the proteins using sensitive profile–profile alignments and two classification approaches, including similarity networks. The clusters are analyzed in view of substrate type, transport mode, organism conservation, and tissue specificity. Solute carrier families with similar substrates generally cluster together, despite exhibiting relatively weak sequence similarities. In contrast, some families cluster together with no apparent reason, revealing unexplored relationships. We demonstrate computationally and experimentally the functional overlap between representative members of these families. Finally, we identify four putative solute carriers in the human genome. The solute carriers include a biomedically important group of membrane proteins that is diverse in sequence and structure. The proposed classification of solute carriers, combined with experiment, reveals new relationships among the individual families and identifies new solute carriers. The classification scheme will inform future attempts directed at modeling the structures of the solute carriers, a prerequisite for describing the substrate specificities of the individual families.

Analysis of high-throughput sequencing and annotation strategies for phage genomes

Background Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. Methodology/Principal Findings To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. Conclusions/Significance These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis

Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world’s oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome—that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome.

Genomes of marine cyanopodoviruses reveal multiple origins of diversity

The marine cyanobacteria Prochlorococcus and Synechococcus are highly abundant in the global oceans, as are the cyanophage with which they co-evolve. While genomic analyses have been relatively extensive for cyanomyoviruses, only three cyanopodoviruses isolated on marine cyanobacteria have been sequenced. Here we present nine new cyanopodovirus genomes, and analyse them in the context of the broader group. The genomes range from 42.2 to 47.7 kb, with G+C contents consistent with those of their hosts. They share 12 core genes, and the pan-genome is not close to being fully sampled. The genomes contain three variable island regions, with the most hypervariable genes concentrated at one end of the genome. Concatenated core-gene phylogeny clusters all but one of the phage into three distinct groups (MPP-A and two discrete clades within MPP-B). The outlier, P-RSP2, has the smallest genome and lacks RNA polymerase, a hallmark of the Autographivirinae subfamily. The phage in group MPP-B contain photosynthesis and carbon metabolism associated genes, while group MPP-A and the outlier P-RSP2 do not, suggesting different constraints on their lytic cycles. Four of the phage encode integrases and three have a host integration signature. Metagenomic analyses reveal that cyanopodoviruses may be more abundant in the oceans than previously thought.