scholarly journals Functional Metagenomics Reveals an Overlooked Diversity and Novel Features of Soil-Derived Bacterial Phosphatases and Phytases

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. e01966-18 ◽  
Author(s):  
Genis Andrés Castillo Villamizar ◽  
Heiko Nacke ◽  
Marc Boehning ◽  
Kristin Herz ◽  
Rolf Daniel
Keyword(s):  
Phosphatase Activity ◽  
High Performance ◽  
Cell Metabolism ◽  
Screening Method ◽  
Phosphorus Cycle ◽  
Functional Metagenomics ◽  
Phosphorus Pollution ◽  
Metagenome Analysis ◽  
Genes Encoding ◽  
Novel Target

ABSTRACTPhosphatases, including phytases, play a major role in cell metabolism, phosphorus cycle, biotechnology, and pathogenic processes. Nevertheless, their discovery by functional metagenomics is challenging. Here, soil metagenomic libraries were successfully screened for genes encoding phosphatase activity. In this context, we report the largest number and diversity of phosphatase genes derived from functional metagenome analysis. Two of the detected gene products carry domains which have never been associated with phosphatase activity before. One of these domains, the SNARE-associated domain DedA, harbors a so-far-overlooked motif present in numerous bacterial SNARE-associated proteins. Our analysis revealed a previously unreported phytase activity of the alkaline phosphatase and sulfatase superfamily (cl23718) and of purple acid phosphatases from nonvegetal origin. This suggests that the classical concept comprising four classes of phytases should be modified and indicates high performance of our screening method for retrieving novel types of phosphatases/phytases hidden in metagenomes of complex environments.IMPORTANCEPhosphorus (P) is a key element involved in numerous cellular processes and essential to meet global food demand. Phosphatases play a major role in cell metabolism and contribute to control the release of P from phosphorylated organic compounds, including phytate. Apart from the relationship with pathogenesis and the enormous economic relevance, phosphatases/phytases are also important for reduction of phosphorus pollution. Almost all known functional phosphatases/phytases are derived from cultured individual microorganisms. We demonstrate here for the first time the potential of functional metagenomics to exploit the phosphatase/phytase pools hidden in environmental soil samples. The recovered diversity of phosphatases/phytases comprises new types and proteins exhibiting largely unknown characteristics, demonstrating the potential of the screening method for retrieving novel target enzymes. The insights gained into the unknown diversity of genes involved in the P cycle highlight the power of function-based metagenomic screening strategies to study Earth’s phosphatase pools.

scholarly journals Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claudia Durall ◽  
Kateryna Kukil ◽  
Jeffrey A. Hawkes ◽  
Alessia Albergati ◽  
Peter Lindblad ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Cell Metabolism ◽  
Tca Cycle ◽  
Malate Synthase ◽  
Glyoxylate Shunt ◽  
Control Strain ◽  
Genes Encoding ◽  
Pcc 6803

Abstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

scholarly journals Chemical and Toxicological Analysis of Antiretroviral Drugs

2019 ◽  
Vol 8 (4) ◽  
pp. 53-60
Author(s):  
T. N. Komarov ◽  
M. V. Belova ◽  
D. D. Stolyarova ◽  
I. E. Shohin ◽  
D. S. Bogdanova ◽  
...  
Keyword(s):  
Side Effects ◽  
Sample Preparation ◽  
Highly Active Antiretroviral Therapy ◽  
High Performance ◽  
Biological Fluids ◽  
Screening Method ◽  
Acute Poisoning ◽  
Antiretroviral Drugs ◽  
Precipitation Method ◽  
Relevant Today

Introduction. Human Immunodeficiency Virus (HIV) is one of the main socially significant infection all over the world. HIV-positive patients take medical care, including antiretroviral drugs (ARVs) pharmacotherapy. Like all drugs, ARVs have lots of side effects that should be taken when prescribing drugs as part of highly active antiretroviral therapy. There are many cases when side effects of ARVs caused patients to enter the toxicology department. Therefore, the development of new methods for the analysis of ARV in biological fluids for the timely diagnosis of treatment of poisoning of this group of drugs is relevant today.Aim. The aim of this study is development of screening analysis of atazanavir, abacavir, nevirapine, ritonavir, lopinavir, zidovudine, darunavir and efavirenz in the urine to identify these drugs as possible toxicants for poisoning by high-performance liquid chromatography with tandem massselective detection (HPLC-MS/MS).Materials and methods. Identification of ARV was performed by HPLC-MS/MS. Methanol precipitation method was used as a sample preparation.Results and discussion. The optimal conditions for sample preparation, chromatographic separation, and mass-spectrometric detection were selected to determine the studied ARVs. This method was tested on urine samples from patients in the Department of Acute Poisoning and Somatopsychiatric Disorders (OOSPD) with acute ARV poisoning.Conclusion. This screening method for analyse atazanavir, abacavir, nevirapine, ritonavir, lopinavir, zidovudine, darunavir and efavirenz in human urine has been developed by HPLC-MS/MS. The developed method can be used to identify these drugs as possible toxicants in case of poisoning. The prospect for the development of the topic is the inclusion of new molecules in the method and quantitative determination of the studied ARVs. 

scholarly journals Functional metagenomic analysis of dust-associated microbiomes above the Red Sea

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nojood A. Aalismail ◽  
David K. Ngugi ◽  
Rubén Díaz-Rúa ◽  
Intikhab Alam ◽  
Michael Cusack ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Dna Sequences ◽  
Biofilm Formation ◽  
Atmospheric Transport ◽  
Taxonomic Diversity ◽  
Functional Metagenomics ◽  
Genetic Traits ◽  
Gene Catalogue ◽  
Range Transport ◽  
Genes Encoding

Abstract Atmospheric transport is a major vector for the long-range transport of microbial communities, maintaining connectivity among them and delivering functionally important microbes, such as pathogens. Though the taxonomic diversity of aeolian microorganisms is well characterized, the genomic functional traits underpinning their survival during atmospheric transport are poorly characterized. Here we use functional metagenomics of dust samples collected on the Global Dust Belt to initiate a Gene Catalogue of Aeolian Microbiome (GCAM) and explore microbial genetic traits enabling a successful aeolian lifestyle in Aeolian microbial communities. The GCAM reported here, derived from ten aeolian microbial metagenomes, includes a total of 2,370,956 non-redundant coding DNA sequences, corresponding to a yield of ~31 × 106 predicted genes per Tera base-pair of DNA sequenced for the aeolian samples sequenced. Two-thirds of the cataloged genes were assigned to bacteria, followed by eukaryotes (5.4%), archaea (1.1%), and viruses (0.69%). Genes encoding proteins involved in repairing UV-induced DNA damage and aerosolization of cells were ubiquitous across samples, and appear as fundamental requirements for the aeolian lifestyle, while genes coding for other important functions supporting the aeolian lifestyle (chemotaxis, aerotaxis, germination, thermal resistance, sporulation, and biofilm formation) varied among the communities sampled.

scholarly journals Dramatic Increase in Content of Diverse Flavonoids Accompanied with Down-Regulation of F-Box Genes in a Chrysanthemum (Chrysanthemum × morifolium (Ramat.) Hemsl.) Mutant Cultivar Producing Dark-Purple Ray Florets

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 865
Author(s):  
Yeong Deuk Jo ◽  
Jaihyunk Ryu ◽  
Ye-Sol Kim ◽  
Kyung-Yun Kang ◽  
Min Jeong Hong ◽  
...  
Keyword(s):  
Translational Regulation ◽  
High Performance ◽  
Flower Color ◽  
Chrysanthemum Morifolium ◽  
Ionization Mass ◽  
Photodiode Array Detection ◽  
Genes Encoding ◽  
Ray Florets ◽  
Chrysanthemum Morifolium Ramat ◽  
Flavonoid Accumulation

Anthocyanins (a subclass of flavonoids) and flavonoids are crucial determinants of flower color and substances of pharmacological efficacy, respectively, in chrysanthemum. However, metabolic and transcriptomic profiling regarding flavonoid accumulation has not been performed simultaneously, thus the understanding of mechanisms gained has been limited. We performed HPLC-DAD-ESI-MS (high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry) and transcriptome analyses using “ARTI-Dark Chocolate” (AD), which is a chrysanthemum mutant cultivar producing dark-purple ray florets, and the parental cultivar “Noble Wine” for metabolic characterization and elucidation of the genetic mechanism determining flavonoid content. Among 26 phenolic compounds identified, three cyanidins and eight other flavonoids were detected only in AD. The total amounts of diverse flavonoids were 8.0 to 10.3 times higher in AD. Transcriptome analysis showed that genes in the flavonoid biosynthetic pathway were not up-regulated in AD at the early flower stage, implying that the transcriptional regulation of the pathway did not cause flavonoid accumulation. However, genes encoding post-translational regulation-related proteins, especially F-box genes in the mutated gene, were enriched among down-regulated genes in AD. From the combination of metabolic and transcriptomic data, we suggest that the suppression of post-translational regulation is a possible mechanism for flavonoid accumulation in AD. These results will contribute to research on the regulation and manipulation of flavonoid biosynthesis in chrysanthemum.

scholarly journals Exploiting the Natural Diversity of RhlA Acyltransferases for the Synthesis of the Rhamnolipid Precursor 3-(3-Hydroxyalkanoyloxy)Alkanoic Acid

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Andrea Germer ◽  
Till Tiso ◽  
Conrad Müller ◽  
Beate Behrens ◽  
Christian Vosse ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Performance ◽  
Serratia Plymuthica ◽  
Dickeya Dadantii ◽  
Transfer Event ◽  
Alkanoic Acid ◽  
Content Type ◽  
Genes Encoding ◽  
Natural Diversity ◽  
Burkholderia Ambifaria

ABSTRACT While rhamnolipids of the Pseudomonas aeruginosa type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new rhlA genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in Betaproteobacteria and Gammaproteobacteria. A likely horizontal gene transfer event into Actinobacteria is the only identified exception. The phylogeny of the RhlA homologs from Pseudomonas and Burkholderia species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the Enterobacterales do not follow the organisms’ phylogeny but form their own branch. Furthermore, in many Enterobacterales and Halomonas from the Oceanospirillales, an isolated rhlA homolog can be found in the genome. The RhlAs from Pseudomonas aeruginosa PA01, Pseudomonas fluorescens LMG 05825, Pantoea ananatis LMG 20103, Burkholderia plantarii PG1, Burkholderia ambifaria LMG 19182, Halomonas sp. strain R57-5, Dickeya dadantii Ech586, and Serratia plymuthica PRI-2C were expressed in Escherichia coli and tested for HAA production. Indeed, except for the Serratia RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C10, C12, and C14 fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity. IMPORTANCE The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.

‘Candidatus Oscillochloris kuznetsovii’ a novel mesophilic filamentous anoxygenic phototrophic Chloroflexales bacterium from Arctic coastal environments

2020 ◽  
Vol 367 (19) ◽  
Author(s):  
Vasil A Gaisin ◽  
Denis S Grouzdev ◽  
Maria S Krutkina ◽  
Aleksandr A Ashikhmin ◽  
Maria A Sinetova ◽  
...  
Keyword(s):  
Microbial Communities ◽  
High Performance ◽  
Hot Spring ◽  
Calvin Cycle ◽  
Phylogenomic Analysis ◽  
Coastal Environments ◽  
Anoxygenic Phototrophs ◽  
Thermal Environments ◽  
Genes Encoding ◽  
Kandalaksha Gulf

ABSTRACT Chloroflexales bacteria are mostly known as filamentous anoxygenic phototrophs that thrive as members of the microbial communities of hot spring cyanobacterial mats. Recently, we described many new Chloroflexales species from non-thermal environments and showed that mesophilic Chloroflexales are more diverse than previously expected. Most of these species were isolated from aquatic environments of mid-latitudes. Here, we present the comprehensive characterization of a new filamentous multicellular anoxygenic phototrophic Chloroflexales bacterium from an Arctic coastal environment (Kandalaksha Gulf, the White Sea). Phylogenomic analysis and 16S rRNA phylogeny indicated that this bacterium belongs to the Oscillochloridaceae family as a new species. We propose that this species be named ‘Candidatus Oscillochloris kuznetsovii’. The genomes of this species possessed genes encoding sulfide:quinone reductase, the nitrogenase complex and the Calvin cycle, which indicate potential for photoautotrophic metabolism. We observed only mesophilic anaerobic anoxygenic phototrophic growth of this novel bacterium. Electron microphotography showed the presence of chlorosomes, polyhydroxyalkanoate-like granules and polyphosphate-like granules in the cells. High-performance liquid chromatography also revealed the presence of bacteriochlorophylls a, c and d as well as carotenoids. In addition, we found that this bacterium is present in benthic microbial communities of various coastal environments of the Kandalaksha Gulf.

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