Characterization of organic nitrogen transport in the ectomycorrhizal fungus Hebeloma cylindrosporum

Ektomykorrhizen haben in borealen Nadelwäldern und temperierten Laubwäldern der Nordhemisphäre eine entscheidene Aufgabe in der Versorgung ihrer pflanzlichen Partner mit Stickstoff, der wichtigste wachstumslimitierende Nährstoff. In diesen Waldböden ist Stickstoff hauptsächlich in organischen Verbindungen vorhanden, welche nicht für die Pflanze, aber für den Ektomykorrhizapilz verwertbar sind. Die mit der Pflanze in Symbiose lebenden Pilze können mit Hilfe von extrazellulären Proteinasen Proteine in diesen Böden spalten. Dies ermöglicht dem Pilz organischen Stickstoff in Form von freien Aminosäuren und Peptiden aufzunehmen, zu assimilieren und schliesslich (zum Teil) an die Pflanze abzugeben. Da der Ektomykorrhizapilz einen wichtigen Beitrag zur Stickstoffversorgung für die mit ihm assoziierten Pflanze leistet, ist es von Bedeutung, die Aufnahmemechanismen von organischen Stickstoff vom Boden in den Pilz (und schliesslich Transport in die Pflanze) und deren Regulation zu verstehen. Um Transporter zu identifizieren, die an der Aufnahme von organischen Stickstoffverbindungen aus dem Boden in die Pilzhyphen bzw. am Transport vom Pilz in die Pflanze, beteiligt sind wurde eine cDNA Bibliothek vom Myzel des Ektomykorrhizapilzes Hebeloma cylindrosporum hergestellt. In dieser Arbeit wurde die Qualität dieser Bibliothek getestet, indem ca. 500 ESTs sequenziert wurden und damit eine Sequenzdatenbank für den Modellorganismus H. cylindrosporum hergestellt (www.uni-tuebingen.de/plantphys /hebeloma/index.html). Von dieser cDNA Bibliothek wurde ein Gen, das für einen Aminosäuretransporter (HcGAP1) kodiert isoliert und charakterisiert. Zusätzlich wurden zwei Peptidtransporter (HcPTR2A, -B) charakterisiert. Die Analyse dieser Transporter durch Aufnahmeexperimenten zeigt, das sie am Import von organischen Stickstoffverbindungen beteiligt sind. Weiterhin konnte durch Expressionsstudien gezeigt werden, dass diese Transporter durch verschiedene Stickstoffquellen reguliert werden. Da extrazelluläre Proteinasen eine wichtige Rolle für die Verfügbarkeit von organischen Stickstoff für den Pilz bzw die Pflanze spielen, wurde die Proteaseaktivität von Hebeloma getestet und ein Gen das möglicherweise für eine Proteinase kodiert isoliert. Schliesslich wurde eine Methode zur Transformation von Hebeloma via Agrobakterium tumefaciens etabliert.Ectomycorrhizal trees dominate boreal and temperate forest ecosystems in which nitrogen is generally accepted to be the most important growth-limiting nutrient. In these forest soils nitrogen is mainly available as organic compounds which are not accessible to plants but to the ectomycorrhizal fungi. The fungal partners are able to break down proteins present in these soils by using extracellular proteinase. Thus, they can take up and assimilate organic nitrogen in the form of free amino acids and peptides which can then be transferred to the plant. Since ectomycorrhizal fungi strongly participate in nitrogen nutrition of the plant in these soils, it is necessary to understand uptake of organic nitrogen from the soil by the fungus (its subsequent transport to the plant) and its regulation. To identify the transporters involved in the uptake of organic nitrogen compounds by the fungal hyphae and their transfer to the plant, it was necessary to develop genomic tools. An oriented expression library was constructed from the mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum. In this work, the quality of this library was tested by DNA sequencing of ~500 ESTs and a sequence database was generated for the model fungus H. cylindrosporum (www.uni-tuebingen.de/plantphys/hebeloma/index.html). Furthermore the suitability of the library to identify Hebeloma genes via their function was demonstrated. Using Hebeloma cDNA libraries a gene encoding for an amino acid transporter (HcGAP1) was isolated and characterized. Two peptide transporters (HcPTR2A, -B) were also characterized. The characterization of these transporters by uptake experiments shows that they play a role in the import of organic nitrogen compounds into Hebeloma. Expression studies demonstrated that these transporters are regulated by different nitrogen sources. As extracellular proteinases play an important role in organic nitrogen availability, the proteinase activity of Hebeloma was characterized and a gene encoding for a putative extracellular proteinase was isolated. Finally, a method for Agrobacterium tumefaciens mediated transformation of Hebeloma was successfully established

Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase

The radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair. By rational engineering, we demonstrate that it is possible to rewire its HAT pathway, a first step toward the development of novel catalysts based on the radical SAM enzyme scaffold

The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress

The epeXEPAB (formerly yydFGHIJ) locus of Bacillus subtilis encodes a minimalistic biosynthetic pathway for a linear antimicrobial epipeptide, EpeX, which is ribosomally produced and post-translationally processed by the action of the radical-SAM epimerase, EpeE, and a membrane-anchored signal 2 peptide peptidase, EpeP. The ABC transporter EpeAB provides intrinsic immunity against self-produced EpeX, without conferring resistance against extrinsically added EpeX. EpeX specifically targets, and severely perturbs the integrity of the cytoplasmic membrane, which leads to the induction of the Lia-dependent envelope stress response. Here, we provide new insights into the distribution, expression, and regulation of the minimalistic epeXEPAB locus of B. subtilis, as well as the biosynthesis and biological efficiency of the produced epipeptide EpeX*. A comprehensive comparative genomics study demonstrates that the epe-locus is restricted to but widely distributed within the phylum Firmicutes. The gene products of epeXEP are necessary and sufficient for the production of the mature antimicrobial peptide EpeX*. In B. subtilis, the epeXEPAB locus is transcribed from three different promoters, one upstream of epeX (PepeX) and two within epeP (PepeA1 and PepeA2). While the latter two are mostly constitutive, PepeX shows a growth phase-dependent induction at the onset of stationary phase. We demonstrate that this regulation is the result of the antagonistic action of two global regulators: The transition state regulator AbrB keeps the epe locus shut off during exponential growth by direct binding. This tight repression is relieved by the master regulator of sporulation, Spo0A, which counteracts the AbrB-dependent repression of epeXEPAB expression during the transition to stationary phase. The net result of these three ­promoters is an expression pattern that ensures EpeAB-dependent autoimmunity prior to EpeX* production. In the absence of EpeAB, the general envelope stress response proteins LiaIH can compensate for the loss of specific autoimmunity by providing sufficient protection against the membrane-perturbating action of EpeX*. Hence, the transcriptional regulation of epe expression and the resulting intrinsic induction of the two corresponding resistance functions, encoded by epeAB and liaIH, are well balanced to provide a need-based immunity against mature EpeX*.Peer Reviewe

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four C-alpha-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-alpha-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by C-alpha H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an alpha,beta-dehydro-amino acid intermediate during C-alpha-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation

Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

<p>Abstract</p> <p>Background</p> <p>Enzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses.</p> <p>Results</p> <p>An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from <it>Mycobacterium tuberculosis</it>. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ). Partial Phylogenetic Profiling (PPP) based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P), but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR) and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P)-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity <it>in vitro </it>requires an artificial electron acceptor such as N,N-dimethyl-4-nitrosoaniline (NDMA) for the enzyme to cycle.</p> <p>Conclusions</p> <p>Taken together, these findings suggest that the mycofactocin precursor is modified by the Rv0693 family rSAM protein and other enzymes in its cluster. It becomes an electron carrier molecule that serves <it>in vivo </it>as NDMA and other artificial electron acceptors do <it>in vitro</it>. Subclasses from three different nicotinoprotein families show "only-if" relationships to mycofactocin because they require its presence. This framework suggests a segregated redox pool in which mycofactocin mediates communication among enzymes with non-exchangeable cofactors.</p

A combined metabolomic and phylogenetic study reveals putatively prebiotic effects of high molecular weight arabino-oligosaccharides when assessed by in vitro fermentation in bacterial communities derived from humans

AbstractPrebiotic oligosaccharides are defined by their selective stimulation of growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial for health. However, apart from the short chain fatty acids, little is known about bacterial metabolites created by fermentation of prebiotics, and the significance of the size of the oligosaccharides remains largely unstudied.By in vitro fermentations in human fecal microbial communities (derived from six different individuals), we studied the effects of high-mass (HA, >1 kDa), low-mass (LA, <1 kDa) and mixed (BA) sugar beet arabino-oligosaccharides (AOS) as carbohydrate sources. Fructo-oligosaccharides (FOS) were included as reference. The changes in bacterial communities and the metabolites produced in response to incubation with the different carbohydrates were analyzed by quantitative PCR (qPCR) and Liquid Chromatography–Mass Spectrometry (LC–MS), respectively.All tested carbohydrate sources resulted in a significant increase of Bifidobacterium spp. between 1.79 fold (HA) and 1.64 fold (FOS) in the microbial populations after fermentation, and LC–MS analysis suggested that the bifidobacteria contributed to decomposition of the arabino-oligosaccharide structures, most pronounced in the HA fraction, resulting in release of the essential amino acid phenylalanine. Abundance of Lactobacillus spp. correlated with the presence of a compound, most likely a flavonoid, indicating that lactobacilli contribute to release of such health-promoting substances from plant structures.Additionally, the combination of qPCR and LC–MS revealed a number of other putative interactions between intestinal microbes and the oligosaccharides, which contributes to the understanding of the mechanisms behind prebiotic impact on human health

Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions

Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors: ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B 12 , AdoCbl) or by single-electron reduction of S -adenosylmethionine (AdoMet) complexed to an iron–sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine's role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen, which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations; nevertheless, mechanistic insights gained from studying these enzymes have proved extremely valuable in understanding how enzymes generate and control highly reactive free radical intermediates. In contrast, there has been a recent explosion in the number of radical-AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions; here there is much still to learn about their mechanisms. Although all the radical-AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69165/1/604_ftp.pd