Genomic profiling and characteristics of a C1 degrading heterotrophic fresh-water bacterium Paracoccus sp. strain DMF.

Paracoccus species are metabolically versatile gram-negative, aerobic facultative methylotrophic bacteria showing enormous promise for environmental and bioremediation studies. Here we report, the complete genome analysis of Paracoccus sp. strain DMF (P. DMF) that was isolated from a domestic wastewater treatment plant in Kanpur, India (26.4287 °N, 80.3891 °E) based on its ability to degrade a recalcitrant organic solvent N, N-dimethylformamide (DMF). The results reveal a genome size of 4,202,269 base pairs (bp) with a G + C content of 67.9%. The assembled genome comprises 4141 coding sequences (CDS), 46 RNA sequences, and 2 CRISPRs. Interestingly, catabolic operons related to the conventional marine-based methylated amines (MAs) degradation pathway were functionally annotated within the genome of an obligated aerobic heterotroph that is P. DMF. The genomic data-based characterization presented here for the novel heterotroph P. DMF aims to improve the understanding of the phenotypic gene products, enzymes, and pathways involved with greater emphasis on facultative methylotrophic motility-based latent pathogenicity.

Exploring the medicinally important secondary metabolites landscape through the lens of transcriptome data in fenugreek (Trigonella foenum graecum L.).

Fenugreek (Trigonella foenum-graecum L.) is a self-pollinated leguminous crop belonging to the Fabaceae family. It is a multipurpose crop used as herb, spice, vegetable and forage. It is a traditional medicinal plant in India attributed with several nutritional and medicinal properties including antidiabetic and anticancer. We have performed a combined transcriptome assembly from RNA sequencing data derived from leaf, stem and root tissues. Around 209,831 transcripts were deciphered from the assembly of 92% completeness and an N50 of 1382 bases. Whilst secondary metabolites of medicinal value, such as trigonelline, diosgenin, 4-hydroxyisoleucine and quercetin, are distributed in several tissues, we report transcripts that bear sequence signatures of enzymes involved in the biosynthesis of such metabolites and are highly expressed in leaves, stem and roots. One of the antidiabetic alkaloid, trigonelline and its biosynthesising enzyme, is highly abundant in leaves. These findings are of value to nutritional and the pharmaceutical industry.

Data enabled prediction analysis assigns folate/biopterin transporter (BT1) family to 36 hypothetical membrane proteins in Leishmania donovani.

Leishmaniasis is a neglected tropical disease caused by the pathogenic protozoan Leishmania donovani and it is transmitted by an infected sand fly. Approximately 0.4 million cases of Visceral Leishmaniasis are reported across the globe every year, of which 67% is from the Indian subcontinent. The currently available drugs have not been effective owing to their high toxicity levels, inadequate specificity, drug resistance, extended treatment periods and/or prohibitive prices. For this reason, hypothetical proteins in this pathogen, which constitute about 67% of its proteome, must be distinctly characterized and studied for their potential role as drug targets for Leishmaniasis. Domain information from PFAM and functional information from GO has been used to assign putative functions to 36 hypothetical membrane proteins in this protozoan. Furthermore, as a case study, we have performed a thorough sequence level characterization of a hypothetical protein E9BPD7 from the BT1 family of membrane proteins that transports folate/biopterin. Phylogenetic analyses of E9BPD7 have revealed interesting evolutionary correlations to BT1 family and MFS superfamily, which have significant roles in a number of diseases and drug resistance pathways.

Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway.

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

RAPD assisted selection of black gram (Vigna mungo L. Hepper) towards the development of multiple disease resistant germplasm.

Black gram (Vigna mungo L. Hepper), is an extensively studied food crop which is affected by many abiotic and biotic factors, especially diseases. The yield potential of Black gram is shallow due to lack of genetic variability and biotic stress susceptibility. Core biotic stress factors include mung bean yellow mosaic virus (MYMV), urdbean leaf crinkle virus (UCLV), wilt (Fusarium oxysporum) and powdery mildew (Erysiphe polygoni DC). Although many studies determine resistant varieties to a particular disease, however, it is often complimented by low yield and susceptibility to other diseases. Hence, this study focuses on investigating the genetic relationships among three varieties and nine accessions of black gram having disease resistance to previously described diseases and susceptibility using random amplified polymorphic deoxyribonucleic acid (RAPD) markers. A total of 33 RAPD primers were used for diversity analysis and yielded 206 fragments. Number of amplified fragments ranged from two (OPN-1) to 13 (OPF-1). The highest similarity coefficient was observed between IC-145202 and IC-164118 (0.921), while lowest similarity was between PU-31 and IC-145202 (0.572). The genetic diversity obtained in this study along with disease analysis suggests PU31as a useful variety for the development of markers linked to MYMV, UCLV, wilt and powdery mildew resistance by marker-assisted back cross breeding and facilitates the production of crosses with multiple disease resistance.

Characterization of a hypothetical protein YVRE from Bacillus subtilis indicates its key role as glucono-lactonase in pentose phosphate pathway and glucose metabolism.

Hypothetical proteins are functionally uncharacterized proteins with assigned function using sequence annotation tools. Almost half of the coding regions of several genomes are hypothetical proteins. Therefore, it is of our interest to characterize a hypothetical protein YVRE from the model system Bacillus subtilis using known data. YVRE is assigned the function as a glucono-lactonase using prediction and phylogenetic analysis. A molecular dynamics simulated homology model of YVRE (with calcium) using human senescence marker protein 30 /SMP30 (PDB ID: 3G4E) as template is reported for functional inference. It is observed that the protein possesses bivalent metal binding domain. Molecular docking studies with the substrate glucono-δ-lactone show YVRE binding with the substrate. This data was further validated using cloning and sub-cloning in pUC57 and pET22b+ respectively, followed by expression and purification using nickel affinity chromatography. The activity of YVRE using the substrate glucono-δ-lactone was calculated. The results show the function of YVRE as a gluconolactonase, with higher preference to zinc than calcium or magnesium. Thus, YVRE is shown to play key role in three metabolic pathways namely, pentose phosphate pathway, ascorbate and aldarate metabolism, and caprolactam degradation.

Structure of a heterogeneous, glycosylated, lipid-bound, in vivo-grown protein crystal at atomic resolution from the viviparous cockroach Diploptera punctata.

Macromolecular crystals for X-ray diffraction studies are typically grown in vitro from pure and homogeneous samples; however, there are examples of protein crystals that have been identified in vivo. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown in cellulo. Here, an atomic resolution (1.2 Å) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These in vivo-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, Diploptera punctata. The milk proteins crystallized in space group P1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single in vivo-grown crystal of a natural protein in its native environment at atomic resolution.

Characterization of hypothetical protein VNG0128C from Halobacterium NRC-1 reveals GALE like activity and its involvement in Leloir pathway of galactose metabolism.

VNG0128C, a hypothetical protein from Halobacterium NRC-1, was chosen for detailed insilico and experimental investigations. Computational exercises revealed that VNG0128C functions as NAD(+) binding protein. The phylogenetic analysis with the homolog sequences of VNG0128C suggested that it could act as UDP-galactose 4-epimerase. Hence, the VNG0128C sequence was modeled using a suitable template and docking studies were performed with NAD and UDP-galactose as ligands. The binding interactions strongly indicate that VNG0128C could plausibly act as UDP-galactose 4-epimerase. In order to validate these insilico results, VNG0128C was cloned in pUC57, subcloned in pET22b(+), expressed in BL21 cells and purified using nickel affinity chromatography. An assay using blue dextran was performed to confirm the presence of NAD binding domain. To corroborate the epimerase like enzymatic role of the hypothetical protein, i.e. the ability of the enzyme to convert UDP-galactose to UDP-glucose, the conversion of NAD to NADH was measured. The experimental assay significantly correlated with the insilico predictions, indicating that VNG0128C has a NAD(+) binding domain with epimerase activity. Consequently, its key role in nucleotide-sugar metabolism was thus established. Additionally, the work highlights the need for a methodical characterization of hypothetical proteins (less studied class of biopolymers) to exploit them for relevant applications in the field of biology.

Insilico analysis of hypothetical proteins unveils putative metabolic pathways and essential genes in Leishmania donovani.

Leishmaniasis is a parasitic disease caused by the protozoan Leishmania, which is active in two broad forms namely, Visceral Leishmaniasis (VL or Kala Azar) and Cutaneous Leishmaniasis (CL). The disease is most prevalent in the tropical regions and poses a threat to over 70 countries across the globe. About 200 million people are estimated to be at risk of developing VL in the Indian subcontinent, and this refers to around 67% of the global VL disease burden. The Indian state of Bihar alone accounts for 50% of the total VL cases. While no vaccination exists, several pentavalent antimonials and drugs like Paromomycin, Amphotericin, Miltefosine etc. are used in the treatment of Leishmaniasis. However, due to their low efficacies and the resistance developed by the bug to these medications, there is an urgent need to look into newer species specific targets. The proteome information available suggests that among the 7960 proteins in Leishmania donavani, a staggering 65% remains classified as a hypothetical uncharacterized set. In this background, we have attempted to assign probable functions to these hypothetical sequences present in this parasite, to explore their plausible roles as druggable receptors. Thus, putative functions have been defined to 105 hypothetical proteins, which exhibited a GO term correlation and PFAM domain coverage of more than 50% over the query sequence length. Of these, 27 sequences were found to be associated with a reference pathway in KEGG as well. Further, using homology approaches, four pathways viz., Ubiquinone biosynthesis, Fatty acid elongation in Mitochondria, Fatty Acid Elongation in ER and Seleno-cysteine Metabolism have been reconstructed. In addition, 7 new putative essential genes have been mined with the help of Eukaryotic Database of Essential Genes (DEG). All these information related to pathways and essential genes indeed show promise for exploiting the select molecules as potential therapeutic targets.

Genome wide survey and molecular modeling of hypothetical proteins containing 2Fe-2S and FMN binding domains suggests Rieske Dioxygenase Activity highlighting their potential roles in bioremediation.

'Conserved hypothetical' proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a 'complete' understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 17 proteins with N-terminus FNR domain and C-terminus 2Fe-2S domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicate their plausible roles in degradation of aromatic moieties.

Analysis of multi-domain hypothetical proteins containing iron-sulphur clusters and fad ligands reveal rieske dioxygenase activity suggesting their plausible roles in bioremediation.

'Conserved hypothetical' proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a 'complete' understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 40 proteins with N-terminus 2Fe-2S domain and C-terminus FNR domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicating their plausible roles in degradation of aromatic moieties.