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Flavin transferase: the maturation factor of flavin-containing oxidoreductases.
Bogachev, AV, Baykov, AA, Bertsova, YV
Biochemical Society transactions. 2018;(5):1161-1169
Abstract
Flavins, cofactors of many enzymes, are often covalently linked to these enzymes; for instance, flavin adenine mononucleotide (FMN) can form a covalent bond through either its phosphate or isoalloxazine group. The prevailing view had long been that all types of covalent attachment of flavins occur as autocatalytic reactions; however, in 2013, the first flavin transferase was identified, which catalyzes phosphoester bond formation between FMN and Na+-translocating NADHquinone oxidoreductase in certain bacteria. Later studies have indicated that this post-translational modification is widespread in prokaryotes and is even found in some eukaryotes. Flavin transferase can occur as a separate ∼40 kDa protein or as a domain within the target protein and recognizes a degenerate DgxtsAT/S motif in various target proteins. The purpose of this review was to summarize the progress already achieved by studies of the structure, mechanism, and specificity of flavin transferase and to encourage future research on this topic. Interestingly, the flavin transferase gene (apbE) is found in many bacteria that have no known target protein, suggesting the presence of yet unknown flavinylation targets.
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Safety of Pseudomonas chlororaphis as a gene source for genetically modified crops.
Anderson, JA, Staley, J, Challender, M, Heuton, J
Transgenic research. 2018;(1):103-113
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Abstract
Genetically modified crops undergo extensive evaluation to characterize their food, feed and environmental safety prior to commercial introduction, using a well-established, science-based assessment framework. One component of the safety assessment includes an evaluation of each introduced trait, including its source organism, for potential adverse pathogenic, toxic and allergenic effects. Several Pseudomonas species have a history of safe use in agriculture and certain species represent a source of genes with insecticidal properties. The ipd072Aa gene from P. chlororaphis encodes the IPD072Aa protein, which confers protection against certain coleopteran pests when expressed in maize plants. P. chlororaphis is ubiquitous in the environment, lacks known toxic or allergenic properties, and has a history of safe use in agriculture and in food and feed crops. This information supports, in part, the safety assessment of potential traits, such as IPD072Aa, that are derived from this source organism.
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A Comprehensive Computational Analysis of Mycobacterium Genomes Pinpoints the Genes Co-occurring with YczE, a Membrane Protein Coding Gene Under the Putative Control of a MocR, and Predicts its Function.
Milano, T, Angelaccio, S, Tramonti, A, di Salvo, ML, Nogues, I, Contestabile, R, Pascarella, S
Interdisciplinary sciences, computational life sciences. 2018;(1):111-125
Abstract
Bacterial proteins belonging to the YczE family are predicted to be membrane proteins of yet unknown function. In many bacterial species, the yczE gene coding for the YczE protein is divergently transcribed with respect to an adjacent transcriptional regulator of the MocR family. According to in silico predictions, proteins named YczR are supposed to regulate the expression of yczE genes. These regulators linked to the yczE genes are predicted to constitute a subfamily within the MocR family. To put forward hypotheses amenable to experimental testing about the possible function of the YczE proteins, a phylogenetic profile strategy was applied. This strategy consists in searching for those genes that, within a set of genomes, co-occur exclusively with a certain gene of interest. Co-occurrence can be suggestive of a functional link. A set of 30 mycobacterial complete proteomes were collected. Of these, only 16 contained YczE proteins. Interestingly, in all cases each yczE gene was divergently transcribed with respect to a yczR gene. Two orthology clustering procedures were applied to find proteins co-occurring exclusively with the YczE proteins. The reported results suggest that YczE may be involved in the membrane translocation and metabolism of sulfur-containing compounds mostly in rapidly growing, low pathogenicity mycobacterial species. These observations may hint at potential targets for therapies to treat the emerging opportunistic infections provoked by the widespread environmental mycobacterial species and may contribute to the delineation of the genomic and physiological differences between the pathogenic and non-pathogenic mycobacterial species.
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Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27.
Norambuena, J, Wang, Y, Hanson, T, Boyd, JM, Barkay, T
Applied and environmental microbiology. 2018;(2)
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Abstract
Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 μM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA.IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.
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Diffusible signal factor signaling regulates multiple functions in the opportunistic pathogen Stenotrophomonas maltophilia.
An, SQ, Tang, JL
BMC research notes. 2018;(1):569
Abstract
OBJECTIVE Stenotrophomonas maltophilia is a Gram-negative bacterium commonly isolated from nosocomial infections. Analysis of the genome of the clinical S. maltophilia isolate K279a indicates that it encodes a diffusible signal factor (DSF)-dependent cell-cell signaling mechanism that is highly similar to the system previously described in phytopathogens from the genera Xanthomonas and Xylella. Our objective was to study the function of DSF signaling in the clinical strain S. maltophilia K279a using genetic and functional genomic analyses. RESULTS We compared the wild-type strain with a mutant deficient in the rpfF (regulation of pathogenicity factors) gene that is essential for the synthesis of DSF. The effects of disruption of DSF signaling were pleiotropic with an impact on virulence, biofilm formation and pathogenesis. The phenotypic effects of rpfF mutation in S. maltophilia could be reversed by addition of exogenous DSF. Taken together, we demonstrate that DSF signaling regulates factors contributing to virulence, biofilm formation and motility of this important opportunistic pathogen.
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6.
Regulated Proteolysis in Bacteria.
Mahmoud, SA, Chien, P
Annual review of biochemistry. 2018;:677-696
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Abstract
Regulated proteolysis is a vital process that affects all living things. Bacteria use energy-dependent AAA+ proteases to power degradation of misfolded and native regulatory proteins. Given that proteolysis is an irreversible event, specificity and selectivity in degrading substrates are key. Specificity is often augmented through the use of adaptors that modify the inherent specificity of the proteolytic machinery. Regulated protein degradation is intricately linked to quality control, cell-cycle progression, and physiological transitions. In this review, we highlight recent work that has shed light on our understanding of regulated proteolysis in bacteria. We discuss the role AAA+ proteases play during balanced growth as well as how these proteases are deployed during changes in growth. We present examples of how protease selectivity can be controlled in increasingly complex ways. Finally, we describe how coupling a core recognition determinant to one or more modifying agents is a general theme for regulated protein degradation.
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Redox Sensing by Fe2+ in Bacterial Fur Family Metalloregulators.
Pinochet-Barros, A, Helmann, JD
Antioxidants & redox signaling. 2018;(18):1858-1871
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Abstract
SIGNIFICANCE Iron is required for growth and is often redox active under cytosolic conditions. As a result of its facile redox chemistry, iron homeostasis is intricately involved with oxidative stress. Bacterial adaptation to iron limitation and oxidative stress often involves ferric uptake regulator (Fur) proteins: a diverse set of divalent cation-dependent, DNA-binding proteins that vary widely in both metal selectivity and sensitivity to metal-catalyzed oxidation. Recent Advances: Bacteria contain two Fur family metalloregulators that use ferrous iron (Fe2+) as their cofactor, Fur and PerR. Fur functions to regulate iron homeostasis in response to changes in intracellular levels of Fe2+. PerR also binds Fe2+, which enables metal-catalyzed protein oxidation as a mechanism for sensing hydrogen peroxide (H2O2). CRITICAL ISSUES To effectively regulate iron homeostasis, Fur has an Fe2+ affinity tuned to monitor the labile iron pool of the cell and may be under selective pressure to minimize iron oxidation, which would otherwise lead to an inappropriate increase in iron uptake under oxidative stress conditions. Conversely, Fe2+ is bound more tightly to PerR but exhibits high H2O2 reactivity, which enables a rapid induction of peroxide stress genes. FUTURE DIRECTIONS The features that determine the disparate reactivity of these proteins with oxidants are still poorly understood. A controlled, comparative analysis of the affinities of Fur/PerR proteins for their metal cofactors and their rate of reactivity with H2O2, combined with structure/function analyses, will be needed to define the molecular mechanisms that have facilitated this divergence of function between these two paralogous regulators.
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Prediction of human-Bacillus anthracis protein-protein interactions using multi-layer neural network.
Ahmed, I, Witbooi, P, Christoffels, A
Bioinformatics (Oxford, England). 2018;(24):4159-4164
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Abstract
MOTIVATION Triplet amino acids have successfully been included in feature selection to predict human-HPV protein-protein interactions (PPI). The utility of supervised learning methods is curtailed due to experimental data not being available in sufficient quantities. Improvements in machine learning techniques and features selection will enhance the study of PPI between host and pathogen. RESULTS We present a comparison of a neural network model versus SVM for prediction of host-pathogen PPI based on a combination of features including: amino acid quadruplets, pairwise sequence similarity, and human interactome properties. The neural network and SVM were implemented using Python Sklearn library. The neural network model using quadruplet features and other network features outperformance the SVM model. The models are tested against published predictors and then applied to the human-B.anthracis case. Gene ontology term enrichment analysis identifies immunology response and regulation as functions of interacting proteins. For prediction of Human-viral PPI, our model (neural network) is a significant improvement in overall performance compared to a predictor using the triplets feature and achieves a good accuracy in predicting human-B.anthracis PPI. AVAILABILITY AND IMPLEMENTATION All code can be downloaded from ftp://ftp.sanbi.ac.za/machine_learning/. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.
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Biochemical and Biophysical Characterization of the Enolase from Helicobacter pylori.
López-López, MJ, Rodríguez-Luna, IC, Lara-Ramírez, EE, López-Hidalgo, M, Benítez-Cardoza, CG, Guo, X
BioMed research international. 2018;:9538193
Abstract
Enolase, which catalyses the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate, is an important enzyme in the classic glycolysis pathway in cells. Enolase is highly conserved in organisms from bacteria to humans, indicating its importance in cells. Thus, enolase is a good target for developing new drugs. In the last decade, new functions of this enzyme have been found. Helicobacter pylori is a common human pathogen that causes gastric diseases and even gastric cancer. In this study, the sequence of H. pylori enolase (HpEno) was analysed; the conservation (at least partial) of binding sites for cofactor, plasminogen, and host extracellular RNA, as well as catalytic site, indicates that HpEno should be capable of performing the functions. Recombinant HpEno was overexpressed and purified from E. coli. Compared to the enolases from other species, HpEno had similar characteristics for its secondary structure. The temperature-induced profiles indicate that HpEno is quite stable to temperature, compared to other homologs. Regarding the kinetics of the unfolding reaction, we found that the activation enthalpy associated with the thermal unfolding reaction is equivalent to the reported activation enthalpy for yeast enolase, indicating a similar scaffold and kinetic stability. Although a wide range of experimental conditions were assayed, it was not possible to detect any enzymatic activity of HpEno. To prove the lack of activity, still a much wider range of experiments should be carried out.
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Production of thermotolerant, detergent stable alkaline protease using the gut waste of Sardinella longiceps as a substrate: Optimization and characterization.
Ramkumar, A, Sivakumar, N, Gujarathi, AM, Victor, R
Scientific reports. 2018;(1):12442
Abstract
The gut wastes of Sardinella longiceps were used as substrate for protease production. The gut waste has 61.6% proteins, 21.8% lipids, 8.5% carbohydrates on dry weight basis and trace elements. The significant factors of protease fermentation were screened by Plackett-Burman design. A protease activity of 68.56 U/ml was predicted at 46.31 °C, incubation time 71.11 h, inoculum 4.86% (v/v) and substrate concentration 2.66% (w/v), using response surface methodology. However, the validation experiment showed 73.52 U/ml activity. The artificial neural network was found as a better tool to predict the experimental results. The partially purified protease showed higher activity at pH 9 and 10 and retained 90% activity after 120 h at pH 9. It showed maximum activity at 50 °C and retained 88% residual activity until 90 min at 50 °C. Zn++ enhanced the protease activity by 40%. The protease retained an activity of 93, 103, 90 and 98% against urea, β-mercaptoethanol, SDS and tween 80 respectively. The alkaline protease was compatible with all the commercial detergents tested with the residual activity above 90%. The alkaline protease exhibited 22% higher activity on the tryptone soya substrate. The gut waste of S. longiceps is a worthy low cost substrate for the production of industrially important alkaline protease.