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Abstract The situation of global warming imparts negative impacts on crop growth and development. Cotton is the most important fiber crop around the globe. However, frequent drought episodes pose serious threats to cotton production worldwide. Due to the complex genetic structure of drought tolerance, the development of a tolerant cultivar is cumbersome via conventional breeding. Multiple omics techniques have appeared as successful tool for cotton improvement in drought tolerance. Advanced omics-based biotechniques have paved the way for generation of omics data like transcriptomics, genomics, metabolomics and proteomics, which greatly expand the knowledge of cotton response to drought stress. Omics methodologies and have provided ways for the identification of quantitative trait loci (QTLs), gene regulatory networks, and other regulatory pathways against drought stress in cotton. These resources could speed up the discovery and incorporation of drought tolerant traits in the elite genotypes. The genome wide association study (GWAS), gene-editing system CRISPER/Cas9, gene silencing through RNAi are efficient tools to explore the molecular mechanism of drought tolerance and facilitate the identification of mechanisms and candidate genes for the improvement of drought tolerance in cotton.

The situation of global warming imparts negative impacts on crop growth and development. Cotton is the most important fiber crop around the globe. However, frequent drought episodes pose serious threats to cotton production worldwide. Due to the complex genetic structure of drought tolerance, the development of a tolerant cultivar is cumbersome via conventional breeding. Multiple omics techniques have appeared as successful tool for cotton improvement in drought tolerance. Advanced omics-based biotechniques have paved the way for generation of omics data like transcriptomics, genomics, metabolomics and proteomics, which greatly expand the knowledge of cotton response to drought stress. Omics methodologies and have provided ways for the identification of quantitative trait loci (QTLs), gene regulatory networks, and other regulatory pathways against drought stress in cotton. These resources could speed up the discovery and incorporation of drought tolerant traits in the elite genotypes. The genome wide association study (GWAS), gene-editing system CRISPER/Cas9, gene silencing through RNAi are efficient tools to explore the molecular mechanism of drought tolerance and facilitate the identification of mechanisms and candidate genes for the improvement of drought tolerance in cotton. [ABSTRACT FROM AUTHOR]

Brucella ovis is an etiologic agent of ovine epididymitis and brucellosis that causes global devastation in sheep, rams, goats, small ruminants and deer. There are no cost‐effective methods for the worldwide eradication of ovine brucellosis. B. ovis and other protein targets from various Brucella species are currently in the pipeline for high‐throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), with the aim of identifying new therapeutic targets. Furthermore, the wealth of structures generated are effective tools for teaching scientific communication, structural science and biochemistry. One of these structures, B. ovis leucine‐, isoleucine‐, valine‐, threonine‐ and alanine‐binding protein (BoLBP), is a putative periplasmic amino acid‐binding protein. BoLBP shares less than 29% sequence identity with any other structure in the Protein Data Bank. The production, crystallization and high‐resolution structures of BoLBP are reported. BoLBP is a prototypical bacterial periplasmic amino acid‐binding protein with the characteristic Venus flytrap topology of two globular domains encapsulating a large central cavity containing the peptide‐binding region. The central cavity contains small molecules usurped from the crystallization milieu. The reported structures reveal the conformational flexibility of the central cavity in the absence of bound peptides. The structural similarity to other LBPs can be exploited to accelerate drug repurposing. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae (Kp) is an infectious disease pathogen that poses a significant global health threat due to its potential to cause severe infections and its tendency to exhibit multidrug resistance. Understanding the enzymatic mechanisms of the oxygen‐insensitive nitroreductases (Kp‐NRs) from Kp is crucial for the development of effective nitrofuran drugs, such as nitrofurantoin, that can be activated as antibiotics. In this paper, three crystal structures of two Kp‐NRs (PDB entries 7tmf/7tmg and 8dor) are presented, and an analysis of their crystal structures and their flavin mononucleotide (FMN)‐binding mode is provided. The structures with PDB codes 7tmf (Kp‐NR1a), 7tmg (Kp‐NR1b) and 8dor (Kp‐NR2) were determined at resolutions of 1.97, 1.90 and 1.35 Å, respectively. The Kp‐NR1a and Kp‐NR1b structures adopt an αβ fold, in which four‐stranded antiparallel β‐sheets are surrounded by five helices. With domain swapping, the β‐sheet was expanded with a β‐strand from the other molecule of the dimer. The difference between the structures lies in the loop spanning Leu173–Ala185: in Kp‐NR1a the loop is disordered, whereas the loop adopts multiple conformations in Kp‐NR1b. The FMN interactions within Kp‐NR1/NR2 involve hydrogen‐bond and π‐stacking interactions. Kp‐NR2 contains four‐stranded antiparallel β‐sheets surrounded by eight helices with two short helices and one β‐sheet. Structural and sequence alignments show that Kp‐NR1a/b and Kp‐NR2 are homologs of the Escherichia coli oxygen‐insensitive NRs YdjA and NfnB and of Enterobacter cloacae NR, respectively. By homology inference from E. coli, Kp‐NR1a/b and Kp‐NR2 may detoxify polynitroaromatic compounds and Kp‐NR2 may activate nitrofuran drugs to cause bactericidal activity through a ping‐pong bi‐bi mechanism, respectively. [ABSTRACT FROM AUTHOR]

The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID‐19, with multi‐drug‐resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty‐acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty‐acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high‐molecular‐weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low‐molecular‐weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low‐molecular‐weight FabG. [ABSTRACT FROM AUTHOR]

The effector proteins produced by plant pathogens are one of the essential components of host–pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the diversity in their primary sequence generated by the high selection pressure of the host immune system. However to maintain the primary function in the infection process, these effectors may tend to maintain their native protein fold to perform the corresponding biological function. In the present study, unannotated candidate secretory effector proteins of sixteen major plant fungal pathogens were analyzed to find the conserved known protein folds using homology, ab initio, and Alpha Fold/Rosetta Fold protein dimensional (3D) structure approaches. Several unannotated candidate effector proteins were found to match various known conserved protein families potentially involved in host defense manipulation in different plant pathogens. Surprisingly a large number of plant Kiwellin proteins fold like secretory proteins (> 100) were found in studied rust fungal pathogens. Many of them were predicted as potential effector proteins. Furthermore, template independent modelling using Alpha Fold/Rosetta Fold analysis and structural comparison of these candidates also predicted them to match with plant Kiwellin proteins. We also found plant Kiwellin matching proteins outside rusts including several non-pathogenic fungi suggesting the broad function of these proteins. One of the highest confidently modeled Kiwellin matching candidates effectors, Pstr_13960 (97.8%), from the Indian P. striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pstr_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the Kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the Kiwellin core fold in rust fungi. Molecular docking showed that Pstr_13960 can interact with plant Chorismate mutases (CMs) using three loops conserved in plant and rust Kiwellins. Further analysis of Pstr_13960 showed to contain Intrinsically disordered regions (IDRs) in place of the N-terminal β1/β2 region found in plant Kiwellins suggesting the evolution of rust Kiwellins-like effectors (KLEs). Overall, this study reports the presence of a Kiwellin protein–like fold containing a novel effector protein family in rust fungi depicting a classical example of the evolution of effectors at the structure level as Kiwellin effectors show very low significant similarity to plant Kiwellin at the sequence level. [ABSTRACT FROM AUTHOR]

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti‐infective agents due to the reduced chances of off‐target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP‐bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13–19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate Tm values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors. [ABSTRACT FROM AUTHOR]

Circularity confers protection to viral genomes where linearity falls short, thereby fulfilling the form follows function aphorism. However, a shift away from morphology-based classification toward the molecular and ecological classification of viruses is currently underway within the field of virology. Recent years have seen drastic changes in the International Committee on Taxonomy of Viruses' operational definitions of viruses, particularly for the tailed phages that inhabit the human gut. After the abolition of the order Caudovirales, these tailed phages are best defined as members of the class Caudoviricetes. To determine the epistemological value of genome topology in the context of the human gut virome, we designed a set of seven experiments to assay the impact of genome topology and representative viral selection on biological interpretation. Using Oxford Nanopore long reads for viral genome assembly coupled with Illumina short-read polishing, we showed that circular and linear virus genomes differ remarkably in terms of genome quality, GC skew, transfer RNA gene frequency, structural variant frequency, cross-reference functional annotation (COG, KEGG, Pfam, and TIGRfam), state-of-the-art marker-based classification, and phage–host interaction. Furthermore, the disparity profile changes during dereplication. In particular, our phage–host interaction results demonstrated that proportional abundances cannot be meaningfully compared without due regard for genome topology and dereplication threshold, which necessitates the need for standardized reporting. As a best practice guideline, we recommend that comparative studies of the human gut virome always report the ratio of circular to linear viral genomes along with the dereplication threshold so that structural and functional metrics can be placed into context when assessing biologically relevant metagenomic properties such as proportional abundance. [ABSTRACT FROM AUTHOR]

Inorganic pyrophosphate (PPi) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PPi must be regulated as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PPi into two orthophosphates (Pi), preventing the toxic accumulation of the PPi byproduct in cells and making Pi available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0 Å resolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide‐binding (OB) β‐barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg2+ being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest. [ABSTRACT FROM AUTHOR]

The compound ethyl‐adenosyl monophosphate ester (ethyl‐AMP) has been shown to effectively inhibit acetyl‐CoA synthetase (ACS) enzymes and to facilitate the crystallization of fungal ACS enzymes in various contexts. In this study, the addition of ethyl‐AMP to a bacterial ACS from Legionella pneumophila resulted in the determination of a co‐crystal structure of this previously elusive structural genomics target. The dual functionality of ethyl‐AMP in both inhibiting ACS enzymes and promoting crystallization establishes its significance as a valuable resource for advancing structural investigations of this class of proteins. [ABSTRACT FROM AUTHOR]

Circularity confers protection to viral genomes where linearity falls short, thereby fulfilling the form follows function aphorism. However, a shift away from morphology-based classification toward the molecular and ecological classification of viruses is currently underway within the field of virology. Recent years have seen drastic changes in the International Committee on Taxonomy of Viruses’ operational definitions of viruses, particularly for the tailed phages that inhabit the human gut. After the abolition of the order Caudovirales, these tailed phages are best defined as members of the class Caudoviricetes. To determine the epistemological value of genome topology in the context of the human gut virome, we designed a set of seven experiments to assay the impact of genome topology and representative viral selection on biological interpretation. Using Oxford Nanopore long reads for viral genome assembly coupled with Illumina short-read polishing, we showed that circular and linear virus genomes differ remarkably in terms of genome quality, GC skew, transfer RNA gene frequency, structural variant frequency, cross-reference functional annotation (COG, KEGG, Pfam, and TIGRfam), state-of-the-art marker-based classification, and phage–host interaction. Furthermore, the disparity profile changes during dereplication. In particular, our phage–host interaction results demonstrated that proportional abundances cannot be meaningfully compared without due regard for genome topology and dereplication threshold, which necessitates the need for standardized reporting. As a best practice guideline, we recommend that comparative studies of the human gut virome always report the ratio of circular to linear viral genomes along with the dereplication threshold so that structural and functional metrics can be placed into context when assessing biologically relevant metagenomic properties such as proportional abundance.

Free-standing single-layer β-sheets are extremely rare in naturally occurring proteins, even though β-sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single-layer, anti-parallel β-sheet proteins, comprised of three or four twisted β-hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β-sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent-exposed tyrosine residues, was identified on the concave surface of the β-sheet. These new modular single-layer β-sheet proteins may serve as a new model system for studying folding and design of β-rich proteins.

Crystal structure determination requires solving the phase problem. This can be accomplished using ab initio direct methods for small molecules and macromolecules at resolutions higher than 1.2 Å, whereas macromolecular structure determination at lower resolution requires either molecular replacement using a homologous structure or experimental phases using a derivative such as covalent labeling (for example selenomethionine or mercury derivatization) or heavy-atom soaking (for example iodide ions). Here, a case is presented in which crystals were obtained from a 30.8 kDa protein sample and yielded a 1.6 Å resolution data set with a unit cell that could accommodate approximately 8 kDa of protein. Thus, it was unclear what had been crystallized. Molecular replacement with pieces of homologous proteins and attempts at iodide ion soaking failed to yield a solution. The crystals could not be reproduced. Sequence-independent molecular replacement using the structures available in the Protein Data Bank also failed to yield a solution. Ultimately, ab initio structure solution proved successful using the program ARCIMBOLDO, which identified two α-helical elements and yielded interpretable maps. The structure was the C-terminal dimerization domain of the intended target from Mycobacterium smegmatis. This structure is presented as a user-friendly test case in which an unknown protein fragment could be determined using ARCIMBOLDO.

Structural genomics began as a global effort in the 1990s to determine the tertiary structures of all protein families as a response to large-scale genome sequencing projects. The immediate outcome was an influx of tens of thousands of protein structures, many of which had unknown functions. At the time, the value of structural genomics was controversial. However, the structures themselves were only the most obvious output. In addition, these newly solved structures motivated the emergence of huge data science and infrastructure efforts, which, together with advances in Deep Learning, have brought about a revolution in computational molecular biology. Here, we review some of the computational research carried out at the Protein Data Bank Japan (PDBj) during the Protein 3000 project under the leadership of Haruki Nakamura, much of which continues to flourish today. [ABSTRACT FROM AUTHOR]

DNA from four sweet cherry seedlings derived from gamma-irradiated female parents was sequenced via nanopore technology (Oxford Nanopore MinION). Total data yield was 8.07 Gb, ranging from 0.92 to 3.36 Gb per sample, with the average length of mapped reads ranging from 22 Kbp–24 Kbp. Sequence data was then analysed to identify and characterize variants using a published sweet cherry reference genome. Small and medium-sized indels (55–135 bp), as well as structural variants, including several large indels and complex variants were detected. Of these, 20 variants were localized within protein-coding gene sequences, including those encoding a putative F-box protein, an ADP-ribose glyxohydrolase protein, a predicted 26S protease regulatory subunit, an E3 ubiquitin protein ligase, a UDP-galactose/UDP-blucose transporter, an alpha/beta hydrolase domain-containing protein, a rhodanese-like domain-containing protein, a cytochrome p450 protein, phosphoinositide phosphatase, cysteine synthase-like, phosphoenolpyruvate carboxylase 4, and several uncharacterized proteins. These variations could have functional and phenotypic consequences that are useful in basic research and breeding.

Structural genomics involves the advent of three-dimensional structures of the genome encoded proteins through various techniques available. Numerous structural genomics research groups have been developed across the globe and they contribute enormously to the identification of three-dimensional structures of various proteins. In this review, we have discussed the applications of the structural genomics approach towards the discovery of potential lead-like molecules against the genomic drug targets of three vector-borne diseases, namely, Dengue, Chikungunya and Zika. Currently, all these three diseases are associated with the most important global public health problems and significant economic burden in tropical countries. Structural genomics has accelerated the identification of novel drug targets and inhibitors for the treatment of these diseases. We start with the current development status of the drug targets and antiviral drugs against these three diseases and conclude by describing challenges that need to be addressed to overcome the shortcomings in the process of drug discovery. [ABSTRACT FROM AUTHOR]

Since the completion of the rice genome sequencing project in 2005, we have entered the era of rice genomics, which is still in its ascendancy. Rice genomics studies can be classified into three stages: structural genomics, functional genomics, and quantitative genomics. Structural genomics refers primarily to genome sequencing for the construction of a complete map of rice genome sequence. This is fundamental for rice genetics and molecular biology research. Functional genomics aims to decode the functions of rice genes. Quantitative genomics is large-scale sequence- and statistics-based research to define the quantitative traits and genetic features of rice populations. Rice genomics has been a transformative influence on rice biological research and contributes significantly to rice breeding, making rice a good model plant for studying crop sciences. This review chronologically and comprehensively summarizes progress in rice genomics over the past 20 years, including rice genome, pan-genome and genetic variation research, genetic resources for functional genomics, development of approaches for gene identification, and genes characterized to control agronomically important traits. It also presents contemporary views on important scientific questions and discusses ongoing challenges and future directions in rice genomics research. [ABSTRACT FROM AUTHOR]

A few empirical examples document fixed alternative male mating strategies in animals. Here we focus on the polymorphism of male mating strategies in the ruff (Calidris pugnax, Aves Charadriiformes). In ruffs, three fixed alternative male mating strategies coexist and are signaled by extreme plumage polymorphism. We first present relevant data on the biology of the species. Then we review the available knowledge of the behavioral ecology of ruffs during the breeding season, and we detail the characteristics of each of the three known fixed male mating strategies. We next turn to the results of exceptional quality accumulated on both the structural and functional genomics of the ruff over the past few years. We show how much these genomic data can shed new, mechanistic light on the evolution and maintenance of the three fixed alternative male mating strategies. We then look if there is sufficient indication to support frequency-dependent selection as a key mechanism in maintaining these three strategies. Specifically, we search for evidence of equal fitness among individuals using each of the three strategies. Finally, we propose three lines of research avenues that will help to understand the eco-evolutionary dynamics of phenotypic differences within natural populations of this iconic model species. [ABSTRACT FROM AUTHOR]

The multidrug resistance developed in many organisms due to the prolonged use of antibiotics has been an increasing global health crisis. Klebsiella pneumoniae is a causal organism for various infections, including respiratory, urinary tract and biliary diseases. Initially, immunocompromised individuals are primarily affected by K. pneumoniae. Due to the emergence of hypervirulent strains recently, both healthy and immunocompetent individuals are equally susceptible to K. pneumoniae infections. The infections caused by multidrug-resistant and hypervirulent K. pneumoniae strains are complicated to treat, illustrating an urgent need to develop novel and more practical approaches to combat the pathogen. We focused on the previously performed high-throughput analyses by other groups to discover several novel enzymes that may be considered attractive drug targets of K. pneumoniae. These targets qualify most of the selection criteria for drug targeting, including an absence of its homolog's gene in the host. The capsule, lipopolysaccharide, fimbriae, siderophores and essential virulence factors facilitate the pathogen entry, infection and survival inside the host. This review discusses K. pneumoniae pathophysiology, including its virulence determinants and further the potential drug targets that might facilitate the discovery of novel drugs and effective treatment regimens shortly. [ABSTRACT FROM AUTHOR]

Burkholderia phymatum is an important symbiotic nitrogen‐fixing betaproteobacterium. B. phymatum is beneficial, unlike other Burkholderia species, which cause disease or are potential bioagents. Structural genomics studies at the SSGCID include characterization of the structures of short‐chain dehydrogenases/reductases (SDRs) from multiple Burkholderia species. The crystal structure of a short‐chain dehydrogenase from B. phymatum (BpSDR) was determined in space group C2221 at a resolution of 1.80 Å. BpSDR shares less than 38% sequence identity with any known structure. The monomer is a prototypical SDR with a well conserved cofactor‐binding domain despite its low sequence identity. The substrate‐binding cavity is unique and offers insights into possible functions and likely inhibitors of the enzymatic functions of BpSDR. [ABSTRACT FROM AUTHOR]

Giardiasis is the most prevalent diarrheal disease globally and affects humans and animals. It is a significant problem in developing countries, the number one cause of travelers' diarrhea and affects children and immunocompromised individuals, especially HIV‐infected individuals. Giardiasis is treated with antibiotics (tinidazole and metronidazole) that are also used for other infections such as trichomoniasis. The ongoing search for new therapeutics for giardiasis includes characterizing the structure and function of proteins from the causative protozoan Giardia lamblia. These proteins include hypothetical proteins that share 30% sequence identity or less with proteins of known structure. Here, the atomic resolution structure of a 15.6 kDa protein was determined by molecular replacement. The structure has the two‐layer αβ‐sandwich topology observed in the prototypical endoribonucleases L‐PSPs (liver perchloric acid‐soluble proteins) with conserved allosteric active sites containing small molecules from the crystallization solution. This article is an educational collaboration between Hampton University and the Seattle Structural Genomics Center for Infectious Disease. [ABSTRACT FROM AUTHOR]

Paraburkholderia xenovorans degrades organic wastes, including polychlorinated biphenyls. The atomic structure of a putative dehydrogenase/reductase (SDR) from P. xenovorans (PxSDR) was determined in space group P21 at a resolution of 1.45 Å. PxSDR shares less than 37% sequence identity with any known structure and assembles as a prototypical SDR tetramer. As expected, there is some conformational flexibility and difference in the substrate‐binding cavity, which explains the substrate specificity. Uniquely, the cofactor‐binding cavity of PxSDR is not well conserved and differs from those of other SDRs. PxSDR has an additional seven amino acids that form an additional unique loop within the cofactor‐binding cavity. Further studies are required to determine how these differences affect the enzymatic functions of the SDR. [ABSTRACT FROM AUTHOR]

Amina Nazir,1,2,* Yachao Zhao,2,* Manli Li,2 Rakia Manzoor,3,4 Rana Adnan Tahir,1 Xianglilan Zhang,2 Hong Qing,1 Yigang Tong2 1Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Sciences, Beijing Institute of Technology, Beijing, People’s Republic of China; 2State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People’s Republic of China; 3State Key Laboratory of Molecular Development Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China; 4School of Life Science, University of Chinese Academy of Sciences, Beijing 100101, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hong QingKey Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Sciences, Beijing Institute of Technology, Beijing, People’s Republic of ChinaTel +8613264286266Email hqing@bit.edu.cnYigang TongState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, People’s Republic of ChinaEmail tong.yigang@gmail.comBackground: Multidrug-resistant plasmids carrying replication genes have been widely present in various strains of Klebsiella pneumoniae. RepA and repB1 were found in plasmids belong to the IncFIB, but their detailed structural and genomic characterization was not reported yet. This is the first study that delivers structural and functional insights of repA- and repB1-carrying IncFIB plasmids.Methods: Klebsiella pneumoniae strains A1705, 911021, and 1642 were isolated from the human urine samples and bronchoalveolar fluids collected from different hospitals of China. Antibacterial susceptibility and plasmid transfer ability were tested to characterize the resistant phenotypes mediated by the pA1705-qnrS, p911021-tetA, and p1642-tetA. The complete nucleotide sequences of these plasmids were determined through high-throughput sequencing technology and comparative genomic analyses of plasmids belong to the same incompatibility group were executed to extract the genomic variations and features.Results: The pA1705-qnrS, p911021-tetA, and p1642-tetA are defined as non-conjugative plasmids, having two replication genes, repA and repB1 associated with IncFIB family, and unknown incompatible group, respectively. Comparative genomic analysis revealed that relatively small backbones of IncFIB plasmids integrated massive accessory module at one “hotspot” that was located between orf312 and repB1. These IncFIB plasmids exhibited the distinct profiles of accessory modules including one or two multidrug-resistant regions, many complete and remnant mobile elements comprising integrons, transposons and insertion sequences. The clusters of resistant genes were recognized in this study against different classes of antibiotics including β-lactam, phenicol, aminoglycoside, tetracycline, quinolone, trimethoprim, sulfonamide, tunicamycin, and macrolide. It has been observed that all resistant genes were located in multidrug resistance regions.Conclusion: It is concluded that multidrug-resistant repA and repB1-carrying IncFIB plasmids are a key source to mediate the resistance through mobile elements among Klebsiella pneumoniae. Current findings provide a deep understanding of horizontal gene transfer among plasmids of the IncFIB family via mobile elements that will be utilized in further in vitro studies.Keywords: plasmids, repA, repB1, multidrug resistance, structural genomics, bioinformatics

BackgroundOne of the recent challenges of computational biology is development of new algorithms, tools and software to facilitate predictive modeling of big data generated by high-throughput technologies in biomedical research.ResultsTo meet these demands we developed PROPER - a package for visual evaluation of ranking classifiers for biological big data mining studies in the MATLAB environment.ConclusionPROPER is an efficient tool for optimization and comparison of ranking classifiers, providing over 20 different two- and three-dimensional performance curves.

Structural-genetic characterization of protease producing genes and enzymes from microbial sources are seldom appreciated despite having its substantial utilization in protein engineering or genetic manipulation for biotechnological applications. Aeromonas veronii CMF, a mesophilic bacterium isolated from the gut of Chrysomya megacephala, was found to exhibited significant level of protease activity. For the revelation of genetic potential in relation to protease production, whole genome of this organism was sequenced and analysed while structure–function of different protease enzyme was predicated using various in silico analysis. The 4.5 mb CMF genome was found to encompass various types of protease and mostly they are neutral in nature. Enzyme production was highest in an optimum pH and temperature of 6.0 (32.09 ± 1.015 U/ml) and 35ºC (41.65 ± 1.152 U/ml), respectively. Other culture parameters for optimum production of protease were determined to be inoculum size (1%), incubation period (72 h), shaking condition (125 rpm), carbon and nitrogen source [2% lactose (92.21 ± 3.16 U/ml) and 0.5% urea (163.62 ± 4.31 U/ml), respectively] and effect of surfactants [0.02 mg/ml Tween 80 (174.72 ± 4.48 U/ml)]. Furthermore, A. veronii CMF exhibited significant enzyme production like serine protease (15.22 ± 0.563 U/ml), aspartate protease (33.16 ± 0.762 U/ml) and collagenase (17.26 ± 0.626 U/ml). Genomic information and results of physio-biochemical assays indicate its cost-effective potential use in different enzyme-industry. [ABSTRACT FROM AUTHOR]

Protein domain repeats are known to arise due to tandem duplications of internal genes. However, the understanding of the underlying mechanisms of this process is incomplete. The goal of this work was to investigate the mechanism of occurrence of repeat expansion based on studying the sequences of 1324 rpsA genes of bacterial S1 ribosomal proteins containing different numbers of S1 structural domains. The rpsA gene encodes ribosomal S1 protein, which is essential for cell viability as it interacts with both mRNA and proteins. Gene ontology (GO) analysis of S1 domains in ribosomal S1 proteins revealed that bacterial protein sequences in S1 mainly have 3 types of molecular functions: RNA binding activity, nucleic acid activity, and ribosome structural component. Our results show that the maximum value of rpsA gene identity for full-length proteins was found for S1 proteins containing six structural domains (58%). Analysis of consensus sequences showed that parts of the rpsA gene encoding separate S1 domains have no a strictly repetitive structure between groups containing different numbers of S1 domains. At the same time, gene regions encoding some conserved residues that form the RNA-binding site remain conserved. The detected phylogenetic similarity suggests that the proposed fold of the rpsA translation initiation region of Escherichia coli has functional value and is important for translational control of rpsA gene expression in other bacterial phyla, but not only in gamma Proteobacteria. [ABSTRACT FROM AUTHOR]

BackgroundCA_C2195 from Clostridium acetobutylicum is a protein of unknown function. Sequence analysis predicted that part of the protein contained a metallopeptidase-related domain. There are over 200 homologs of similar size in large sequence databases such as UniProt, with pairwise sequence identities in the range of ~40-60%. CA_C2195 was chosen for crystal structure determination for structure-based function annotation of novel protein sequence space.ResultsThe structure confirmed that CA_C2195 contained an N-terminal metallopeptidase-like domain. The structure revealed two extra domains: an α+β domain inserted in the metallopeptidase-like domain and a C-terminal circularly permuted winged-helix-turn-helix domain.ConclusionsBased on our sequence and structural analyses using the crystal structure of CA_C2195 we provide a view into the possible functions of the protein. From contextual information from gene-neighborhood analysis, we propose that rather than being a peptidase, CA_C2195 and its homologs might play a role in biosynthesis of a modified cell-surface carbohydrate in conjunction with several sugar-modification enzymes. These results provide the groundwork for the experimental verification of the function.

The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5'-monophosphate-3-deoxy-D-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of D-ribulose 5-phosphate to D-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections.

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.

Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas-specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure-based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family.

Obtaining diffraction quality crystals remains one of the major bottlenecks in structural biology. The ability to predict the chances of crystallization from the amino-acid sequence of the protein can, at least partly, address this problem by allowing a crystallographer to select homologs that are more likely to succeed and/or to modify the sequence of the target to avoid features that are detrimental to successful crystallization. In 2007, the now widely used XtalPred algorithm [Slabinski et al. (2007), Protein Sci. 16, 2472-2482] was developed. XtalPred classifies proteins into five `crystallization classes' based on a simple statistical analysis of the physicochemical features of a protein. Here, towards the same goal, advanced machine-learning methods are applied and, in addition, the predictive potential of additional protein features such as predicted surface ruggedness, hydrophobicity, side-chain entropy of surface residues and amino-acid composition of the predicted protein surface are tested. The new XtalPred-RF (random forest) achieves significant improvement of the prediction of crystallization success over the original XtalPred. To illustrate this, XtalPred-RF was tested by revisiting target selection from 271 Pfam families targeted by the Joint Center for Structural Genomics (JCSG) in PSI-2, and it was estimated that the number of targets entered into the protein-production and crystallization pipeline could have been reduced by 30% without lowering the number of families for which the first structures were solved. The prediction improvement depends on the subset of targets used as a testing set and reaches 100% (i.e. twofold) for the top class of predicted targets.

A few empirical examples document fixed alternative male mating strategies in animals. Here we focus on the polymorphism of male mating strategies in the ruff (Calidris pugnax, Aves Charadriiformes). In ruffs, three fixed alternative male mating strategies coexist and are signaled by extreme plumage polymorphism. We first present relevant data on the biology of the species. Then we review the available knowledge of the behavioral ecology of ruffs during the breeding season, and we detail the characteristics of each of the three known fixed male mating strategies. We next turn to the results of exceptional quality accumulated on both the structural and functional genomics of the ruff over the past few years. We show how much these genomic data can shed new, mechanistic light on the evolution and maintenance of the three fixed alternative male mating strategies. We then look if there is sufficient indication to support frequency-dependent selection as a key mechanism in maintaining these three strategies. Specifically, we search for evidence of equal fitness among individuals using each of the three strategies. Finally, we propose three lines of research avenues that will help to understand the eco-evolutionary dynamics of phenotypic differences within natural populations of this iconic model species.

Rickettsia felis, a Gram‐negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl‐CoA reductase from R. felis is reported at a resolution of 2.0 Å. While R. felis acetoacetyl‐CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short‐chain dehydrogenase/reductase (SDR) family members, such as the fatty‐acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies. [ABSTRACT FROM AUTHOR]

Despite the tremendous success of X‐ray cryo‐crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal‐mounting workflow from a rate‐limiting manual activity to a controllable, high‐throughput semi‐automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100–240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X‐ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment‐screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography. [ABSTRACT FROM AUTHOR]