Homology modeling and assigned functional annotation of an uncharacterized antitoxin protein from Streptomyces xinghaiensis

Streptomyces xinghaiensis is a Gram-positive, aerobic and non-motile bacterium. The bacterial genome is known. Therefore, it is ofinterest to study the uncharacterized proteins in the genome. An uncharacterized protein (gi|518540893|86 residues) in thegenome was selected for a comprehensive computational sequence-structure-function analysis using available data and tools. Subcellularlocalization of the targeted protein with conserved residues and assigned secondary structures is documented. Sequencehomology search against the protein data bank (PDB) and non-redundant GenBank proteins using BLASTp showed differenthomologous proteins with known antitoxin function. A homology model of the target protein was developed using a knowntemplate (PDB ID: 3CTO:A) with 62% sequence similarity in HHpred after assessment using programs PROCHECK and QMEAN6.The predicted active site using CASTp is analyzed for assigned anti-toxin function. This information finds specific utility inannotating the said uncharacterized protein in the bacterial genome.     

Structure and functional annotation of hypothetical proteins having putative Rubisco activase function from Vitis vinifera

Rubisco is a very large, complex and one of the most abundant proteins in the world and comprises up to 50% of all soluble protein in plants. The activity of Rubisco, the enzyme that catalyzes CO₂ assimilation in photosynthesis, is regulated by Rubisco activase (Rca). In the present study, we searched for hypothetical protein of Vitis vinifera which has putative Rubisco activase function. The Arabidopsis and tobacco Rubisco activase protein sequences were used as seed sequences to search against Vitis vinifera in UniprotKB database. The selected hypothetical proteins of Vitis vinifera were subjected to sequence, structural and functional annotation. Subcellular localization predictions suggested it to be cytoplasmic protein. Homology modelling was used to define the three-dimensional (3D) structure of selected hypothetical proteins of Vitis vinifera. Template search revealed that all the hypothetical proteins share more than 80% sequence identity with structure of green-type Rubisco activase from tobacco, indicating proteins are evolutionary conserved. The homology modelling was generated using SWISS-MODEL. Several quality assessment and validation parameters computed indicated that homology models are reliable. Further, functional annotation through PFAM, CATH, SUPERFAMILY, CDART suggested that selected hypothetical proteins of Vitis vinifera contain ATPase family associated with various cellular activities (AAA) and belong to the AAA+ super family of ring-shaped P-loop containing nucleoside triphosphate hydrolases. This study will lead to research in the optimization of the functionality of Rubisco which has large implication in the improvement of plant productivity and resource use efficiency.     

Homology modeling of Mycoplasma pneumoniae enolase and its molecular interaction with human plasminogen

Alpha (α)-enolase (e), a glycolytic enzyme, has an alternative role as a surface receptor of several bacteria mediating plasminogen (pg) binding. It is also recognized as a virulence factor of some pathogenic bacteria facilitating plasminogen activation and host cell invasion. A mycoplasmal α-enolase is also a plasminogen binding protein. Molecular interactions of enolase from Mycoplasma pneumoniae with host plasminogen would be useful for exploring the pathogen-host interaction. In an attempt to identify plasminogen binding sites of M. pneumoniae enolase, homology modeling and docking studies were conducted to obtain modeled structures of the M. pneumoniae enolase-plasminogen complex. The refined model was validated further by standard methods. Molecular docking revealed hydrogen bonding of eLys70-pgTyr50, eAsn165-pgThr66, eAla168-pgGlu21, eAsp17-pgLys70, and eAsn213-pgPro68/pgAsn69. Substantial decreases in accessible surface area (ASA) were observed and in concurrence with hydrogen bond pattern. These findings provide a detailed prediction of key residues that interact at the protein-protein interface. Our theoretical prediction is consistent with known biochemical data. The predicted interaction complex can be of great assistance in understanding structural insights, which is necessary to pathogen and host-component interaction. The ability of M. pneumoniae enolase to bind plasminogen may be indicative of an important role in invasion of this pathogen to host.     

Crystal structure of a putative type I restriction-modification S subunit from Mycoplasma genitalium

The crystal structure of the eubacteria Mycoplasma genitalium ORF MG438 polypeptide, determined by multiple anomalous dispersion and refined at 2.3 A resolution, reveals the organization of S subunits from the Type I restriction and modification system. The structure consists of two globular domains, with about 150 residues each, separated by a pair of 40 residue long antiparallel alpha-helices. The globular domains correspond to the variable target recognition domains (TRDs), as previously defined for S subunits on sequence analysis, while the two helices correspond to the central (CR1) and C-terminal (CR2) conserved regions, respectively. The structure of the MG438 subunit presents an overall cyclic topology with an intramolecular 2-fold axis that superimposes the N and the C-half parts, each half containing a globular domain and a conserved helix. TRDs are found to be structurally related with the small domain of the Type II N6-adenine DNA MTase TaqI. These relationships together with the structural peculiarities of MG438, in particular the presence of the intramolecular quasi-symmetry, allow the proposal of a model for S subunits recognition of their DNA targets in agreement with previous experimental results. In the crystal, two subunits of MG438 related by a crystallographic 2-fold axis present a large contact area mainly involving the symmetric interactions of a cluster of exposed hydrophobic residues. Comparison with the recently reported structure of an S subunit from the archaea Methanococcus jannaschii highlights the structural features preserved despite a sequence identity below 20%, but also reveals important differences in the globular domains and in their disposition with respect to the conserved regions.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

Novel one-step mechanism for tRNA 3'-end maturation by the exoribonuclease RNase R of Mycoplasma genitalium

Mycoplasma genitalium is expected to metabolize RNA using unique pathways because its minimal genome encodes very few ribonucleases. In this work, we report that the only exoribonuclease identified in M. genitalium, RNase R, is able to remove tRNA 3'-trailers and generate mature 3'-ends. Several sequence and structural features of a tRNA precursor determine its precise processing at the 3'-end by RNase R in a purified system. The aminoacyl-acceptor stem plays a major role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA by RNase R, whereas extension of the stem results in the formation of a product terminating downstream at the new mature 3'-end. In addition, the 3'-terminal CCA sequence and the discriminator residue influence the ability of RNase R to stop at the mature 3'-end. RNase R-mediated generation of the mature 3'-end prefers a sequence of RCCN at the 3' terminus of tRNA. Variations of this sequence may cause RNase R to trim further and remove terminal CA residues from the mature 3'-end. Therefore, M. genitalium RNase R can precisely remove the 3'-trailer of a tRNA precursor by recognizing features in the terminal domains of tRNA, a process requiring multiple RNases in most bacteria.     

Annotation of hypothetical proteins orthologous in Pongo abelii and Sus scrofa

A hypothetical protein is predicted to be expressed from an open reading frame without known experimental evidence of translation. They constitute a substantial fraction of proteomes. Domain extraction from these hypothetical sequences helps to search for protein coding genes for protein structural and functional annotation. We describe the analysis of prediction data in a sequence dataset of hypothetical protein orthologs of Pongo abelii (orangutan) and Sus scrofa (pig). It should be noted that these orangutan-pig orthologs are also non-homologous to human proteins. These predicted data find application in the genome wide annotation of proteins in poorly understood genomes.

Abbreviations

PDB - Protein Data Bank, DEG - Database of Essential Genes, CDD - Conserved Domain Database, IUCN - International Union for Conservation of Nature.     

Homology modeling, docking studies and functional analysis of various azoreductase accessory interacting proteins of Nostoc sp.PCC7120

Azo dyes have become a threat to public health because of its toxicity and carcinogenicity. Azoreductase enzyme plays a pivotal role in the degradation of azodyes released by industrial effluents and other resources. The degradation pathway has to be studied in detail for increasing the activity of azoreductase and for better degradation of azo dyes. But the data available on cyanobacterial azoreductase enzyme and its degradation pathway are still very less. Therefore the present work explored the azoreductase pathway of the cyanobacterium Nostoc sp. PCC7120 for better understanding of the degradation pathway and the other accessory interacting proteins involved. The accessory interacting proteins of azoreductase from cyanobacterium Nostoc sp. PCC7120 were obtained from STRING database. The proteins do not have a comprehensive three dimensional structure and are hypothetical. The secondary structure and functional analysis indicated that the proteins are all soluble proteins, without disulphide bonds and have alpha helices only. The structural prediction and docking study showed that alr2106, alr1063 and alr2326 have best docking result which tally with the STRING database confidence score and thus these proteins could possibly enhance the azoreductase activity and better dye degradation. These results will pave way for further increase in azoreductase activity and for better understanding of the dye degradation pathway.     

Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzae

HI1506 is a 128-residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85-residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full-length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram-negative bacteria, and H. influenzae in particular, we report here the three-dimensional solution NMR structure for the corrected full-length HI1506 protein. The structure consists of two well-defined domains, an alpha/beta 50-residue N-domain and a 3-alpha 32-residue C-domain, separated by an unstructured 30-residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites.     

Computational structural and functional analysis of hypothetical proteins of Staphylococcus aureus

Genome sequencing projects has led to an explosion of large amount of gene products in which many are of hypothetical proteins with unknown function. Analyzing and annotating the functions of hypothetical proteins is important in Staphylococcus aureus which is a pathogenic bacterium that cause multiple types of diseases by infecting various sites in humans and animals. In this study, ten hypothetical proteins of Staphylococcus aureus were retrieved from NCBI and analyzed for their structural and functional characteristics by using various bioinformatics tools and databases. The analysis revealed that some of them possessed functionally important domains and families and protein-protein interacting partners which were ABC transporter ATP-binding protein, Multiple Antibiotic Resistance (MAR) family, export proteins, Helix-Turn-helix domains, arsenate reductase, elongation factor, ribosomal proteins, Cysteine protease precursor, Type-I restriction endonuclease enzyme and plasmid recombination enzyme which might have the same functions in hypothetical proteins. The structural prediction of those proteins and binding sites prediction have been done which would be useful in docking studies for aiding in the drug discovery.     

NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins

The NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima represents an alpha/beta-topology formed by the regular secondary structures alpha1-beta1-beta2-alpha2-beta3-beta4-alpha3- beta5-3(10)-alpha4, with a small anti-parallel beta-sheet of beta-strands 1 and 2, and a mixed parallel/anti-parallel beta-sheet of beta-strands 3-5. Similar folds have previously been observed in other proteins, with amino acid sequence identity as low as 3% and a variety of different functions. There are also 216 sequence homologs of TM0487, which all have the signature sequence of domains of unknown function 59 (DUF59), for which no three-dimensional structures have as yet been reported. The TM0487 structure thus presents a platform for homology modeling of this large group of DUF59 proteins. Conserved among most of the DUF59s are 13 hydrophobic residues, which are clustered in the core of TM0487. A putative active site of TM0487 consisting of residues D20, E22, L23, T51, T52, and C55 is conserved in 98 of the 216 DUF59 sequences. Asp20 is buried within the proposed active site without any compensating positive charge, which suggests that its pK(a) value may be perturbed. Furthermore, the DUF59 family includes ORFs that are part of a conserved chromosomal group of proteins predicted to be involved in Fe-S cluster metabolism.     

Virulence related sequences; insights provided by comparative genomics of Streptococcus uberis of differing virulence

Background

Streptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains.

Results

Pan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection.

Conclusion

The gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first “whole-genome” comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1512-6) contains supplementary material, which is available to authorized users.     

Comparative genomics of two newly isolated Dehalococcoides strains and an enrichment using a genus microarray

Comparative genomics of Dehalococcoides strains and an enrichment were performed using a microarray targeting genes from all available sequenced genomes of the Dehalococcoides genus. The microarray was designed with 4305 probe sets to target 98.6% of the open-reading frames from strains 195, CBDB1, BAV1 and VS. The microarrays were validated and applied to query the genomes of two recently isolated Dehalococcoides strains, ANAS1 and ANAS2, and their enrichment source (ANAS) to understand the genome–physiology relationships. Strains ANAS1 and ANAS2 can both couple the reduction of trichloroethene, cis-dichloroethene (DCE) and 1,1-DCE, but not tetrachloroethene and trans-DCE with growth, whereas only strain ANAS2 couples vinyl chloride reduction to growth. Comparative genomic analysis showed that the genomes of both strains are similar to each other and to strain 195, except for genes that are within the previously defined integrated elements or high-plasticity regions. Combined results of the two isolates closely matched the results obtained using genomic DNA of the ANAS enrichment. The genome similarities, together with the distinct chlorinated ethene usage of strains ANAS1, ANAS2 and 195 demonstrate that closely phylogenetically related strains can be physiologically different. This incongruence between physiology and core genome phylogeny seems to be related to the presence of distinct reductive dehalogenase-encoding genes with assigned chlorinated ethene functions (pceA, tceA in strain 195; tceA in strain ANAS1; vcrA in strain ANAS2). Overall, the microarrays are a valuable high-throughput tool for comparative genomics of unsequenced Dehalococcoides-containing samples to provide insights into their gene content and dechlorination functions.     

Structure modeling and antidiabetic activity of a seed protein of Momordica charantia in non-obese diabetic (NOD) mice

Momordica charantia is a well known medicinal plant used in the traditional medicinal system for the treatment of various diseases including diabetes mellitus. Recently, a novel protein termed as ADMc1 from the seed extract of M. charantia has been identified and isolated showing significant antihyperglycemic activity in type 1 diabetic rats in which diabetes was induced. However, the structure of this protein has not yet been analyzed. Homology modeling approach was used to generate a high quality protein 3D structure for the amino acid sequence of the ADMc1 protein in this study. The comparative assessment of secondary structures revealed ADMc1 as an all-alpha helix protein with random coils. Tertiary structure predicted on the template structure of Napin of B. Napus (PDB ID: 1SM7) with which the ADMc1 showed significant sequence similarity, was validated using protein structure validation tools like PROCHECK, WHAT_CHECK, VERIFY3D and ProSA. Arrangement of disulfide bridges formed by cysteine residues were predicted by the Dianna 1.1 server. The presence of multiple disulfide bond confers the stable nature of the ADMc1 protein. Further, the biological activity of the ADMc1 was assessed in non-obese diabetic (NOD) mice which are spontaneous model of type 1 diabetes. Significant reduction in the blood glucose levels of NOD mice was observed up to 8 h post administration of the rADMc1 protein. Overall, the structural characterizations with antihyperglycemic activity of this seed protein of Momordica charantia demonstrate its potential as an antidiabetic agent.     

Single-cell genomics reveals the lifestyle of Poribacteria,a candidate phylum symbiotically associated with marine sponges

In this study, we present a single-cell genomics approach for the functional characterization of the candidate phylum Poribacteria, members of which are nearly exclusively found in marine sponges. The microbial consortia of the Mediterranean sponge Aplysina aerophoba were singularized by fluorescence-activated cell sorting, and individual microbial cells were subjected to phi29 polymerase-mediated ‘whole-genome amplification''. Pyrosequencing of a single amplified genome (SAG) derived from a member of the Poribacteria resulted in nearly 1.6 Mb of genomic information distributed among 554 contigs analyzed in this study. Approximately two-third of the poribacterial genome was sequenced. Our findings shed light on the functional properties and lifestyle of a possibly ancient bacterial symbiont of marine sponges. The Poribacteria are mixotrophic bacteria with autotrophic CO₂-fixation capacities through the Wood–Ljungdahl pathway. The cell wall is of Gram-negative origin. The Poribacteria produce at least two polyketide synthases (PKSs), one of which is the sponge-specific Sup-type PKS. Several putative symbiosis factors such as adhesins (bacterial Ig-like domains, lamininin G domain proteins), adhesin-related proteins (ankyrin, fibronectin type III) and tetratrico peptide repeat domain-encoding proteins were identified, which might be involved in mediating sponge–microbe interactions. The discovery of genes coding for 24-isopropyl steroids implies that certain fossil biomarkers used to date the origins of metazoan life on earth may possibly be of poribacterial origin. Single-cell genomic approaches, such as those shown herein, contribute to a better understanding of beneficial microbial consortia, of which most members are, because of the lack of cultivation, inaccessible by conventional techniques.     

五株克雷伯氏菌噬菌体的生物学特性及比较基因组学研究

【目的】耐药性克雷伯氏菌属(Klebsiella)的细菌作为人类感染的重要病原,是临床治疗重要的挑战。本研究对多株克雷伯氏菌裂解性噬菌体的生物学特性和基因组特征进行比较分析,为其应用提供更多科学数据。【方法】使用双层平板法从人类和动物新鲜粪便、污水中分离纯化裂解性克雷伯氏菌噬菌体;通过磷钨酸染色和透射电镜观察其形态;采用双层平板噬菌斑法确定其宿主范围,测定温度和pH稳定性、一步生长曲线和体外抑菌效果等生物学特性;基于全基因组测序对分离株进行比较基因组学分析;通过体内抑菌试验评估噬菌体对多重耐药变栖克雷伯氏菌(Klebsiella variicola)BS375-3感染的大蜡螟(Galleria mellonella)幼虫的保护作用。【结果】5株噬菌体分别属于Schitoviridae(pKP-BM327-1.2)、Autographiviridae(pKP-M186-2.1、pKP-M186-2.2和pKV-BS375-3.1)、Drexlerviridae(pKP-BS317-1.1)家族;噬菌体pKV-BS375-3.1可裂解受试菌中的8株,pKP-BM327-1.2可裂解受试菌中的3株,pKP-M186-2.1、pKP-M186-2.2和pKP-BS317-1.1则分别裂解受试菌中的1株;5株噬菌体感染10–20 min后即进入指数增长期,在–20–37℃、pH 6–10环境下均能够保持稳定活性;感染变栖克雷伯氏菌BS375-3后经噬菌体pKV-BS375-3.1处理[感染复数(multiplicity of infection,MOI)=100]的大蜡螟幼虫96 h内存活率达到80%(8/10);5株噬菌体基因组长度在42–77 kb之间,未携带抗生素抗性基因和毒力基因,基于内溶素(endolysin)的溯源分析显示该蛋白在克雷伯氏菌噬菌体中呈现多样性,属内呈保守性。【结论】5株克雷伯氏菌噬菌体均具有较好的体外抑菌活性,生物学特性稳定,endolysin在噬菌体属内呈现保守性。宿主谱宽、潜伏期短的噬菌体pKV-BS375-3.1在治疗Klebsiella pneumoniae和K.variicola临床感染方面具有潜在应用前景。     

Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved β-sheet on top of a long and a short α-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k _cat of 1.1 × 10² sec⁻¹. The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.