FunGeCo: a web-based tool for estimation of functional potential of bacterial genomes and microbiomes using gene context information.

MOTIVATION: Functional potential of genomes and metagenomes which are inferred using homology-based methods are often subjected to certain limitations, especially for proteins with homologs which function in multiple pathways. Augmenting the homology information with genomic location of the constituent genes can significantly improve the accuracy of estimated functions. This can help in distinguishing cognate homolog belonging to a candidate pathway from its other homologs functional in different pathways. RESULTS: In this article, we present a web-based analysis platform 'FunGeCo' to enable gene-context-based functional inference for microbial genomes and metagenomes. It is expected to be a valuable resource and complement the existing tools for understanding the functional potential of microbes which reside in an environment. AVAILABILITY AND IMPLEMENTATION: https://web.rniapps.net/fungeco [Freely available for academic use]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Polyvalent Proteins, a Pervasive Theme in the Intergenomic Biological Conflicts of Bacteriophages and Conjugative Elements.

Intense biological conflicts between prokaryotic genomes and their genomic parasites have resulted in an arms race in terms of the molecular "weaponry" deployed on both sides. Using a recursive computational approach, we uncovered a remarkable class of multidomain proteins with 2 to 15 domains in the same polypeptide deployed by viruses and plasmids in such conflicts. Domain architectures and genomic contexts indicate that they are part of a widespread conflict strategy involving proteins injected into the host cell along with parasite DNA during the earliest phase of infection. Their unique feature is the combination of domains with highly disparate biochemical activities in the same polypeptide; accordingly, we term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large fraction are enzymatic domains predicted to modify proteins, target nucleic acids, alter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal components. They further contain nucleic acid-binding domains, virion structural domains, and 40 novel uncharacterized domains. Analysis of their architectural network reveals both pervasive common themes and specialized strategies for conjugative elements and plasmids or (pro)phages. The themes include likely processing of multidomain polypeptides by zincin-like metallopeptidases and mechanisms to counter restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA-binding domains acquired by eukaryotes from such systems have been reused in XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kinetoplastids. Characterization of the novel domains discovered here, such as RNases and peptidases, are likely to aid in the development of new reagents and elucidation of the spread of antibiotic resistance.IMPORTANCE This is the first report of the widespread presence of large proteins, termed polyvalent proteins, predicted to be transmitted by genomic parasites such as conjugative elements, plasmids, and phages during the initial phase of infection along with their DNA. They are typified by the presence of multiple domains with disparate activities combined in the same protein. While some of these domains are predicted to assist the invasive element in replication, transcription, or protection of their DNA, several are likely to target various host defense systems or modify the host to favor the parasite's life cycle. Notably, DNA-binding domains from these systems have been transferred to eukaryotes, where they have been incorporated into DNA repair and mitochondrial genome replication systems.

The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.

Catalysis of NAD(+)-dependent ADP-ribosylation of proteins, nucleic acids, or small molecules has evolved in at least three structurally unrelated superfamilies of enzymes, namely ADP-ribosyltransferase (ART), the Sirtuins, and probably TM1506. Of these, the ART superfamily is the most diverse in terms of structure, active site residues, and targets that they modify. The primary diversification of the ART superfamily occurred in the context of diverse bacterial conflict systems, wherein ARTs play both offensive and defensive roles. These include toxin-antitoxin systems, virus-host interactions, intraspecific antagonism (polymorphic toxins), symbiont/parasite effectors/toxins, resistance to antibiotics, and repair of RNAs cleaved in conflicts. ARTs evolving in these systems have been repeatedly acquired by lateral transfer throughout eukaryotic evolution, starting from the PARP family, which was acquired prior to the last eukaryotic common ancestor. They were incorporated into eukaryotic regulatory/epigenetic control systems (e.g., PARP family and NEURL4), and also used as defensive (e.g., pierisin and CARP-1 families) or immunity-related proteins (e.g., Gig2-like ARTs). The ADP-ribosylation system also includes other domains, such as the Macro, ADP-ribosyl glycohydrolase, NADAR, and ADP-ribosyl cyclase, which appear to have initially diversified in bacterial conflict-related systems. Unlike ARTs, sirtuins appear to have a much smaller presence in conflict-related systems.

Probing the amino acids critical for protein oligomerisation and protein-nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches.

We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60-E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60-62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.

Modeling holo-ACP:DH and holo-ACP:KR complexes of modular polyketide synthases: a docking and molecular dynamics study.

BACKGROUND: Modular polyketide synthases are multifunctional megasynthases which biosynthesize a variety of secondary metabolites using various combinations of dehydratase (DH), ketoreductase (KR) and enoyl-reductase (ER) domains. During the catalysis of various reductive steps these domains act on a substrate moiety which is covalently attached to the phosphopantetheine (P-pant) group of the holo-Acyl Carrier Protein (holo-ACP) domain, thus necessitating the formation of holo-ACP:DH and holo-ACP:KR complexes. Even though three dimensional structures are available for DH, KR and ACP domains, no structures are available for DH or KR domains in complex with ACP or substrate moieties. Since Ser of holo-ACP is covalently attached to a large phosphopantetheine group, obtaining complexes involving holo-ACP by standard protein-protein docking has been a difficult task. RESULTS: We have modeled the holo-ACP:DH and holo-ACP:KR complexes for identifying specific residues on DH and KR domains which are involved in interaction with ACP, phosphopantetheine and substrate moiety. A novel combination of protein-protein and protein-ligand docking has been used to first model complexes involving apo-ACP and then dock the phosphopantetheine and substrate moieties using covalent connectivity between ACP, phosphopantetheine and substrate moiety as constraints. The holo-ACP:DH and holo-ACP:KR complexes obtained from docking have been further refined by restraint free explicit solvent MD simulations to incorporate effects of ligand and receptor flexibilities. The results from 50 ns MD simulations reveal that substrate enters into a deep tunnel in DH domain while in case of KR domain the substrate binds a shallow surface exposed cavity. Interestingly, in case of DH domain the predicted binding site overlapped with the binding site in the inhibitor bound crystal structure of FabZ, the DH domain from E.Coli FAS. In case of KR domain, the substrate binding site identified by our simulations was in proximity of the known stereo-specificity determining residues. CONCLUSIONS: We have modeled the holo-ACP:DH and holo-ACP:KR complexes and identified the specific residues on DH and KR domains which are involved in interaction with ACP, phosphopantetheine and substrate moiety. Analysis of the conservation profile of binding pocket residues in homologous sequences of DH and KR domains indicated that, these results can also be extrapolated to reductive domains of other modular PKS clusters.

SBSPKS: structure based sequence analysis of polyketide synthases.

Polyketide synthases (PKSs) catalyze biosynthesis of a diverse family of pharmaceutically important secondary metabolites. Bioinformatics analysis of sequence and structural features of PKS proteins plays a crucial role in discovery of new natural products by genome mining, as well as in design of novel secondary metabolites by biosynthetic engineering. The availability of the crystal structures of various PKS catalytic and docking domains, and mammalian fatty acid synthase module prompted us to develop SBSPKS software which consists of three major components. Model_3D_PKS can be used for modeling, visualization and analysis of 3D structure of individual PKS catalytic domains, dimeric structures for complete PKS modules and prediction of substrate specificity. Dock_Dom_Anal identifies the key interacting residue pairs in inter-subunit interfaces based on alignment of inter-polypeptide linker sequences to the docking domain structure. In case of modular PKS with multiple open reading frames (ORFs), it can predict the cognate order of substrate channeling based on combinatorial evaluation of all possible interface contacts. NRPS-PKS provides user friendly tools for identifying various catalytic domains in the sequence of a Type I PKS protein and comparing them with experimentally characterized PKS/NRPS clusters cataloged in the backend databases of SBSPKS. SBSPKS is available at http://www.nii.ac.in/sbspks.html.

Host-microbiome interactions: Gut-Liver axis and its connection with other organs.

An understanding of connections between gut microbiome and liver has provided important insights into the pathophysiology of liver diseases. Since gut microbial dysbiosis increases gut permeability, the metabolites biosynthesized by them can reach the liver through portal circulation and affect hepatic immunity and inflammation. The immune cells activated by these metabolites can also reach liver through lymphatic circulation. Liver influences immunity and metabolism in multiple organs in the body, including gut. It releases bile acids and other metabolites into biliary tract from where they enter the systemic circulation. In this review, the bidirectional communication between the gut and the liver and the molecular cross talk between the host and the microbiome has been discussed. This review also provides details into the intricate level of communication and the role of microbiome in Gut-Liver-Brain, Gut-Liver-Kidney, Gut-Liver-Lung, and Gut-Liver-Heart axes. These observations indicate a complex network of interactions between host organs influenced by gut microbiome.

Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase.

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.

'GutFeel': an in silico method for predicting gut health status based on the metabolic functional capabilities of the resident microbiome.

Dysbiosis or imbalance in the gut microbiome has been correlated with the etiology of a number of diseases/disorders. Thus, gut microbial communities can potentially be utilized for assessing the health of the human gut. Although the taxonomic composition of the microbiomes is dependent on factors such as diet, lifestyle, and geography, these microbes perform a specific set of common functions in the gut. In this study, metabolic pathway-based markers (agnostic to above-mentioned factors) specific to commensals and those specific to pathogens are utilized as indicators of gut health. Furthermore, this gut health assessment requires only a small set of features rather than complete sequencing of metagenomes. The proposed scheme can also be used to design personalized biotherapeutics, depending on functional aspects observed in an individual.

Corrigendum: FLIM-MAP: Gene Context Based Identification of Functional Modules in Bacterial Metabolic Pathways.

[This corrects the article DOI: 10.3389/fmicb.2018.02183.].

Diet, Microbiota and Gut-Lung Connection.

The gut microbial community (Gut microbiota) is known to impact metabolic functions as well as immune responses in our body. Diet plays an important role in determining the composition of the gut microbiota. Gut microbes help in assimilating dietary nutrients which are indigestible by humans. The metabolites produced by them not only modulate gastro-intestinal immunity, but also impact distal organs like lung and brain. Micro-aspiration of gut bacteria or movement of sensitized immune cells through lymph or bloodstream can also influence immune response of other organs. Dysbiosis in gut microbiota has been implicated in several lung diseases, including allergy, asthma and cystic fibrosis. The bi-directional cross-talk between gut and lung (termed as Gut-Lung axis) is best exemplified by intestinal disturbances observed in lung diseases. Some of the existing probiotics show beneficial effects on lung health. A deeper understanding of the gut microbiome which comprises of all the genetic material within the gut microbiota and its role in respiratory disorders is likely to help in designing appropriate probiotic cocktails for therapeutic applications.

FLIM-MAP: Gene Context Based Identification of Functional Modules in Bacterial Metabolic Pathways.

Prediction of functional potential of bacteria can only be ascertained by the accurate annotation of its metabolic pathways. Homology based methods decipher metabolic gene content but ignore the fact that homologs of same protein can function in different pathways. Therefore, mere presence of all constituent genes in an organism is not sufficient to indicate a pathway. Contextual occurrence of genes belonging to a pathway on the bacterial genome can hence be exploited for an accurate estimation of functional potential of a bacterium. In this communication, we present a novel annotation resource to accurately identify pathway presence by using gene context. Our tool FLIM-MAP (Functionally Important Modules in bacterial Metabolic Pathways) predicts biologically relevant functional units called 'GCMs' (Gene Context based Modules) from a given metabolic reaction network. We benchmark the accuracy of our tool on amino acids and carbohydrate metabolism pathways.

In silico methods for linking genes and secondary metabolites: The way forward.

In silico methods for linking genomic space to chemical space have played a crucial role in genomics driven discovery of new natural products as well as biosynthesis of altered natural products by engineering of biosynthetic pathways. Here we give an overview of available computational tools and then briefly describe a novel computational framework, namely retro-biosynthetic enumeration of biosynthetic reactions, which can add to the repertoire of computational tools available for connecting natural products to their biosynthetic gene clusters. Most of the currently available bioinformatics tools for analysis of secondary metabolite biosynthetic gene clusters utilize the "Genes to Metabolites" approach. In contrast to the "Genes to Metabolites" approach, the "Metabolites to Genes" or retro-biosynthetic approach would involve enumerating the various biochemical transformations or enzymatic reactions which would generate the given chemical moiety starting from a set of precursor molecules and identifying enzymatic domains which can potentially catalyze the enumerated biochemical transformations. In this article, we first give a brief overview of the presently available in silico tools and approaches for analysis of secondary metabolite biosynthetic pathways. We also discuss our preliminary work on development of algorithms for retro-biosynthetic enumeration of biochemical transformations to formulate a novel computational method for identifying genes associated with biosynthesis of a given polyketide or nonribosomal peptide.

Comparative In silico Analysis of Butyrate Production Pathways in Gut Commensals and Pathogens.

Biosynthesis of butyrate by commensal bacteria plays a crucial role in maintenance of human gut health while dysbiosis in gut microbiome has been linked to several enteric disorders. Contrastingly, butyrate shows cytotoxic effects in patients with oral diseases like periodontal infections and oral cancer. In addition to these host associations, few syntrophic bacteria couple butyrate degradation with sulfate reduction and methane production. Thus, it becomes imperative to understand the distribution of butyrate metabolism pathways and delineate differences in substrate utilization between pathogens and commensals. The bacteria utilize four pathways for butyrate production with different initial substrates (Pyruvate, 4-aminobutyrate, Glutarate and Lysine) which follow a polyphyletic distribution. A comprehensive mining of complete/draft bacterial genomes indicated conserved juxtaposed genomic arrangement in all these pathways. This gene context information was utilized for an accurate annotation of butyrate production pathways in bacterial genomes. Interestingly, our analysis showed that inspite of a beneficial impact of butyrate in gut, not only commensals, but a few gut pathogens also possess butyrogenic pathways. The results further illustrated that all the gut commensal bacteria (Faecalibacterium, Roseburia, Butyrivibrio, and commensal species of Clostridia etc) ferment pyruvate for butyrate production. On the contrary, the butyrogenic gut pathogen Fusobacterium utilizes different amino acid metabolism pathways like those for Glutamate (4-aminobutyrate and Glutarate) and Lysine for butyrogenesis which leads to a concomitant release of harmful by-products like ammonia in the process. The findings in this study indicate that commensals and pathogens in gut have divergently evolved to produce butyrate using distinct pathways. No such evolutionary selection was observed in oral pathogens (Porphyromonas and Filifactor) which showed presence of pyruvate as well as amino acid fermenting pathways which might be because the final product butyrate is itself known to be cytotoxic in oral diseases. This differential utilization of butyrogenic pathways in gut pathogens and commensals has an enormous ecological impact taking into consideration the immense influence of butyrate on different disorders in humans. The results of this study can potentially guide bioengineering experiments to design therapeutics/probiotics by manipulation of butyrate biosynthesis gene clusters in bacteria.

Prediction of inter domain interactions in modular polyketide synthases by docking and correlated mutation analysis.

Polyketide synthases (PKSs) are huge multi-enzymatic protein complexes involved in the biosynthesis of one of the largest families of bioactive natural products, namely polyketides. The specificity of interactions between various catalytic domains of these megasynthases is one of the pivotal factors which control the precise order in which the extender units are joined during the biosynthetic process. Hence, understanding the molecular details of protein-protein interactions in the PKS megasynthases would be crucial for rational design of novel polyketides by domain swapping experiments involving engineered combinations of PKS catalytic domains. We have developed a computational method for exploring the binding interface between two proteins, and used it to identify the interacting residue pairs, which govern the specificity of recognition between acyl carrier protein (ACP) domain and two core catalytic domains, namely the ketosynthase (KS) and acyl transferase (AT). Both of these domain interactions i.e. the KS-ACP and the AT-ACP, are likely to play a major role in channelling of substrates and control of specificity during polyketide biosynthesis. The method, called interface scan, uses a combination of geometric docking and evolutionary information for the identification of the most appropriate mode of association between two proteins. The parameters of interface scan have been standardized based on analysis of contacts in the crystal structure of ACP in complex with ACP synthase (AcpS). Many of the contacts predicted for PKS domains are in agreement with available experiments.

Novel autoproteolytic and DNA-damage sensing components in the bacterial SOS response and oxidized methylcytosine-induced eukaryotic DNA demethylation systems.

The bacterial SOS response is an elaborate program for DNA repair, cell cycle regulation and adaptive mutagenesis under stress conditions. Using sensitive sequence and structure analysis, combined with contextual information derived from comparative genomics and domain architectures, we identify two novel domain superfamilies in the SOS response system. We present evidence that one of these, the SOS response associated peptidase (SRAP; Pfam: DUF159) is a novel thiol autopeptidase. Given the involvement of other autopeptidases, such as LexA and UmuD, in the SOS response, this finding suggests that multiple structurally unrelated peptidases have been recruited to this process. The second of these, the ImuB-C superfamily, is linked to the Y-family DNA polymerase-related domain in ImuB, and also occurs as a standalone protein. We present evidence using gene neighborhood analysis that both these domains function with different mutagenic polymerases in bacteria, such as Pol IV (DinB), Pol V (UmuCD) and ImuA-ImuB-DnaE2 and also other repair systems, which either deploy Ku and an ATP-dependent ligase or a SplB-like radical SAM photolyase. We suggest that the SRAP superfamily domain functions as a DNA-associated autoproteolytic switch that recruits diverse repair enzymes upon DNA damage, whereas the ImuB-C domain performs a similar function albeit in a non-catalytic fashion. We propose that C3Orf37, the eukaryotic member of the SRAP superfamily, which has been recently shown to specifically bind DNA with 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, is a sensor for these oxidized bases generated by the TET enzymes from methylcytosine. Hence, its autoproteolytic activity might help it act as a switch that recruits DNA repair enzymes to remove these oxidized methylcytosine species as part of the DNA demethylation pathway downstream of the TET enzymes.

Inter-domain movements in polyketide synthases: a molecular dynamics study.

Insights into the structure and dynamics of modular polyketide synthases (PKS) are essential for understanding the mechanistic details of the biosynthesis of a large number of pharmaceutically important secondary metabolites. The crystal structures of the KS-AT di-domain from erythromycin synthase have revealed the relative orientation of various catalytic domains in a minimal PKS module. However, the relatively large distance between catalytic centers of KS and AT domains in the static structure has posed certain intriguing questions regarding mechanistic details of substrate transfer during polyketide biosynthesis. In order to investigate the role of inter-domain movements in substrate channeling, we have carried out a series of explicit solvent MD simulations for time periods ranging from 10 to 15 ns on the KS-AT di-domain and its sub-fragments. Analyses of these MD trajectories have revealed that both the catalytic domains and the structured inter-domain linker region remain close to their starting structures. Inter-domain movements at KS-linker and linker-AT interfaces occur around hinge regions which connect the structured linker region to the catalytic domains. The KS-linker interface was found to be more flexible compared to the linker-AT interface. However, inter-domain movements observed during the timescale of our simulations do not significantly reduce the distance between catalytic centers of KS and AT domains for facilitating substrate channeling. Based on these studies and prediction of intrinsic disorder we propose that the intrinsically unstructured linker stretch preceding the ACP domain might be facilitating movement of ACP domains to various catalytic centers.