scholarly journals Three Genomes from the Phylum Acidobacteria Provide Insight into the Lifestyles of These Microorganisms in Soils

2009 ◽  
Vol 75 (7) ◽  
pp. 2046-2056 ◽  
Author(s):  
Naomi L. Ward ◽  
Jean F. Challacombe ◽  
Peter H. Janssen ◽  
Bernard Henrissat ◽  
Pedro M. Coutinho ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Carbon Sources ◽  
Major Facilitator Superfamily ◽  
Nitrite Reduction ◽  
Cellulose Synthesis ◽  
Novel Proteins ◽  
Siderophore Receptors ◽  
Major Facilitator ◽  
Nitrate And Nitrite ◽  
Nutrient Conditions

ABSTRACT The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria, the Cyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

scholarly journals Pyridoxal-5′-phosphate–dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi

2019 ◽  
Vol 117 (2) ◽  
pp. 1174-1180 ◽  
Author(s):  
Guang Zhi Dai ◽  
Wen Bo Han ◽  
Ya Ning Mei ◽  
Kuang Xu ◽  
Rui Hua Jiao ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Complex Structure ◽  
Genome Mining ◽  
Biosynthetic Gene Cluster ◽  
Major Facilitator Superfamily ◽  
Molecular Architecture ◽  
Widespread Occurrence ◽  
Indolizidine Alkaloids ◽  
Major Facilitator ◽  
Insight Into

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C–C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A−I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature’s arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.

scholarly journals Two Nitrate/Nitrite Transporters Are Encoded within the Mobilizable Plasmid for Nitrate Respiration of Thermus thermophilus HB8

2000 ◽  
Vol 182 (8) ◽  
pp. 2179-2183 ◽  
Author(s):  
Sandra Ramírez ◽  
Renata Moreno ◽  
Olga Zafra ◽  
Pablo Castán ◽  
Cristina Vallés ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Average Distance ◽  
Thermus Thermophilus ◽  
Major Facilitator Superfamily ◽  
Nitrate Respiration ◽  
Mobilizable Plasmid ◽  
Excretion Rates ◽  
Major Facilitator ◽  
Nitrate And Nitrite

ABSTRACT Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes,narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed.

scholarly journals Glycerol-driven Denitratation: Process Kinetics, Microbial Ecology, and Operational Controls

2021 ◽  
Author(s):  
Matthew P. Baideme ◽  
Chenghua Long ◽  
Luke T. Plante ◽  
Jeffrey A. Starke ◽  
Michael A. Butkus ◽  
...  
Keyword(s):  
Organic Carbon ◽  
Microbial Ecology ◽  
Nitrate Reduction ◽  
Carbon Sources ◽  
Selective Reduction ◽  
Nitrite Reduction ◽  
Nitrite Accumulation ◽  
Ammonium Oxidation ◽  
Order Of Magnitude ◽  
Nitrate And Nitrite

ABSTRACTDenitratation, the selective reduction of nitrate to nitrite, is a novel process when coupled with anaerobic ammonium oxidation (anammox) could achieve resource-efficient biological nitrogen removal of ammonium- and nitrate-laden waste streams. Using a fundamentally-based, first principles approach, this study optimized a stoichiometrically-limited, glycerol-driven denitratation process and characterized mechanisms supporting nitrite accumulation with results that aligned with expectations. Glycerol supported selective nitrate reduction to nitrite and near-complete nitrate conversion, indicating its viability in a denitratation system. Glycerol-supported specific rates of nitrate reduction (135.3 mg-N/g-VSS/h) were at least one order of magnitude greater than specific rates of nitrite reduction (14.9 mg-N/g-VSS/h), potentially resulting in transient nitrite accumulation and indicating glycerol’s superiority over other organic carbon sources in denitratation systems. pH and ORP inflection points in nitrogen transformation assays corresponded to maximum nitrite accumulation, indicating operational setpoints to prevent further nitrite reduction. Denitratation conditions supported enrichment of Thauera sp. as the dominant genus. Stoichiometric limitation of influent organic carbon, coupled with differential nitrate and nitrite reduction kinetics, optimized operational controls, and a distinctively enriched microbial ecology, was identified as causal in glycerol-driven denitratation.

scholarly journals Aspergillus niger mstA encodes a high-affinity sugar/H+ symporter which is regulated in response to extracellular pH

2004 ◽  
Vol 379 (2) ◽  
pp. 375-383 ◽  
Author(s):  
Patricia A. vanKUYK ◽  
Jasper A. DIDERICH ◽  
Andrew P. MacCABE ◽  
Oscar HERERRO ◽  
George J. G. RUIJTER ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Ph Regulation ◽  
Functional Characterization ◽  
Carbon Sources ◽  
Sugar Transport ◽  
Major Facilitator Superfamily ◽  
Northern Analysis ◽  
High Affinity ◽  
Hexose Uptake ◽  
Major Facilitator

A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger. To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake. Uptake experiments using 14C-labelled monosaccharides demonstrated that although able to transport d-fructose (Km, 4.5±1.0 mM), d-xylose (Km, 0.3±0.1 mM) and d-mannose (Km, 60±20 µM), MSTA has a preference for d-glucose (Km, 25±10 µM). pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport. Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for d-glucose, d-mannose and d-xylose. Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC. A. niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1–60 mM d-glucose, d-mannose, d-fructose or d-xylose. This indicates that A. niger possesses other transporters capable of compensating for the absence of MSTA.

scholarly journals In Silico and Transcriptional Analysis of Carbohydrate Uptake Systems of Streptomyces coelicolor A3(2)

2004 ◽  
Vol 186 (5) ◽  
pp. 1362-1373 ◽  
Author(s):  
Ralph Bertram ◽  
Maximilian Schlicht ◽  
Kerstin Mahr ◽  
Harald Nothaft ◽  
Milton H. Saier ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Essential Feature ◽  
Carbon Sources ◽  
Gene Clusters ◽  
Major Facilitator Superfamily ◽  
Transcriptional Analysis ◽  
Phosphotransferase System ◽  
Function Similarity ◽  
Major Facilitator

ABSTRACT Streptomyces coelicolor is the prototype for the investigation of antibiotic-producing and differentiating actinomycetes. As soil bacteria, streptomycetes can metabolize a wide variety of carbon sources and are hence vested with various specific permeases. Their activity and regulation substantially determine the nutritional state of the cell and, therefore, influence morphogenesis and antibiotic production. We have surveyed the genome of S. coelicolor A3(2) to provide a thorough description of the carbohydrate uptake systems. Among 81 ATP-binding cassette (ABC) permeases that are present in the genome, we found 45 to encode a putative solute binding protein, an essential feature for carbohydrate permease function. Similarity analysis allowed the prediction of putative ABC systems for transport of cellobiose and cellotriose, α-glucosides, lactose, maltose, maltodextrins, ribose, sugar alcohols, xylose, and β-xylosides. A novel putative bifunctional protein composed of a substrate binding and a membrane-spanning moiety is likely to account for ribose or ribonucleoside uptake. Glucose may be incorporated by a proton-driven symporter of the major facilitator superfamily while a putative sodium-dependent permease of the solute-sodium symporter family may mediate uptake of galactose and a facilitator protein of the major intrinsic protein family may internalize glycerol. Of the predicted gene clusters, reverse transcriptase PCRs showed active gene expression in 8 of 11 systems. Together with the previously surveyed permeases of the phosphotransferase system that accounts for the uptake of fructose and N-acetylglucosamine, the genome of S. coelicolor encodes at least 53 potential carbohydrate uptake systems.

scholarly journals Biosynthesis and Role in Virulence of the Histone Deacetylase Inhibitor Depudecin from Alternaria brassicicola

2009 ◽  
Vol 22 (10) ◽  
pp. 1258-1267 ◽  
Author(s):  
Wanessa D. Wight ◽  
Kwang-Hyung Kim ◽  
Christopher B. Lawrence ◽  
Jonathan D. Walton
Keyword(s):  
Gene Cluster ◽  
Histone Deacetylase ◽  
Virulence Factor ◽  
Polyketide Synthase ◽  
Pathogenic Fungus ◽  
Major Facilitator Superfamily ◽  
Alternaria Brassicicola ◽  
Pyrenophora Tritici Repentis ◽  
Hc Toxin ◽  
Major Facilitator

Depudecin, an eleven-carbon linear polyketide made by the pathogenic fungus Alternaria brassicicola, is an inhibitor of histone deacetylase (HDAC). A chemically unrelated HDAC inhibitor, HC toxin, was earlier shown to be a major virulence factor in the interaction between Cochliobolus carbonum and its host, maize. In order to test whether depudecin is also a virulence factor for A. brassicicola, we identified the genes for depudecin biosynthesis and created depudecin-minus mutants. The depudecin gene cluster contains six genes (DEP1 to DEP6), which are predicted to encode a polyketide synthase (AbPKS9 or DEP5), a transcription factor (DEP6), two monooxygenases (DEP2 and DEP4), a transporter of the major facilitator superfamily (DEP3), and one protein of unknown function (DEP1). The involvement in depudecin production of DEP2, DEP4, DEP5, and DEP6 was demonstrated by targeted gene disruption. DEP6 is required for expression of DEP1 through DEP5 but not the immediate flanking genes, thus defining a coregulated depudecin biosynthetic cluster. The genes flanking the depudecin gene cluster but not the cluster itself are conserved in the same order in the related fungi Stagonospora nodorum and Pyrenophora tritici-repentis. Depudecin-minus mutants have a small (10%) but statistically significant reduction in virulence on cabbage (Brassica oleracea) but not on Arabidopsis. The role of depudecin in virulence is, therefore, less dramatic than that of HC toxin.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

scholarly journals Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Sushant Kumar ◽  
Arunabh Athreya ◽  
Ashutosh Gulati ◽  
Rahul Mony Nair ◽  
Ithayaraja Mahendran ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pathogenic Bacteria ◽  
Major Facilitator Superfamily ◽  
Single Domain ◽  
Fluoroquinolone Resistance ◽  
Structural Basis ◽  
Antibody Complex ◽  
Complementarity Determining Regions ◽  
Major Facilitator ◽  
Alternating Access

AbstractTransporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

scholarly journals Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

2021 ◽  
Vol 7 (2) ◽  
pp. 138
Author(s):  
Min Liang ◽  
Wei Li ◽  
Landa Qi ◽  
Guocan Chen ◽  
Lei Cai ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Genetic Manipulation ◽  
Major Facilitator Superfamily ◽  
Protoplast Regeneration ◽  
Transformation System ◽  
Regeneration Rate ◽  
Bioactive Natural Products ◽  
Genetics Research ◽  
Genetic Transformation System ◽  
Major Facilitator

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

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