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Translation is an important step in gene expression. Initiation of translation is rate-limiting, and it is phylogenetically more diverse than elongation or termination. Bacteria contain only three initiation factors. In stark contrast, eukaryotes contain more than 10 (subunits of) initiation factors (eIFs). The genomes of archaea contain many genes that are annotated to encode archaeal homologs of eukaryotic initiation factors (aIFs). However, experimental characterization of aIFs is scarce and mostly restricted to very few species. To broaden the view, the protein-protein interaction network of aIFs in the halophilic archaeon Haloferax volcanii has been characterized. To this end, tagged versions of 14 aIFs were overproduced, affinity isolated, and the co-isolated binding partners were identified by peptide mass fingerprinting and MS/MS analyses. The aIF-aIF interaction network was resolved, and it was found to contain two interaction hubs, (1) the universally conserved factor aIF5B, and (2) a protein that has been annotated as the enzyme ribose-1,5-bisphosphate isomerase, which we propose to rename to aIF2Bα. Affinity isolation of aIFs also led to the co-isolation of many ribosomal proteins, but also transcription factors and subunits of the RNA polymerase (Rpo). To analyze a possible coupling of transcription and translation, seven tagged Rpo subunits were overproduced, affinity isolated, and co-isolated proteins were identified. The Rpo interaction network contained many transcription factors, but also many ribosomal proteins as well as the initiation factors aIF5B and aIF2Bα. These results showed that transcription and translation are coupled in haloarchaea, like in Escherichia coli . It seems that aIF5B and aIF2Bα are not only interaction hubs in the translation initiation network, but also key players in the transcription-translation coupling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schramm, Borst, Linne and Soppa.)

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs., (© 2021. The Author(s).)

Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis . Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements., Competing Interests: All authors have seen and approved the manuscript. All authors have contributed significantly to the work, and the manuscript has not been published and is not being considered for publication elsewhere. All authors declare that they have no competing financial interests., (© 2021 The Author(s).)

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was Alistipes which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including A. finegoldii showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.

Mannosylerythritol lipids (MELs) are surface active molecules produced by many basidiomycetous fungi. MELs consist of a mannosylerythritol disaccharide, which is acylated with short and medium chain fatty acids at the mannosyl moiety. A gene cluster composed of five genes is required for MEL biosynthesis. Here we show that the plant pathogenic fungus Ustilago hordei secretes these glycolipids under nitrogen starvation conditions. In contrast to MELs produced by the closely related fungus Ustilago maydis those secreted by U. hordei are mostly mono-acetylated and contain a different mixture of acyl groups. Cross-species complementation between these fungi revealed that these differences result from different catalytic activities of the acetyltransferase Mat1 and the acyltransferases Mac1 and Mac2. U. maydis mat1 mutants expressing the homologous mat1 gene from U. hordei produced mostly mono-acetylated variants and lack di-acetylated MELs normally produced by U. maydis. Furthermore, we determined that the acyltransferase Mac1 acylates the mannosylerythritol moiety at position C2 while Mac2 acylates C3. The identification of decorating enzymes with different substrate specificities will allow the tailor-made production of novel subsets of MELs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium Aquifex aeolicus : Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued the growth of Escherichia coli and Saccharomyces cerevisiae strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P. Homologs of Aquifex RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that A. aeolicus and related Aquificaceae likely acquired HARP by horizontal gene transfer from an archaeon., Competing Interests: The authors declare no conflict of interest.

Bacterial trans-translation is the main quality control mechanism employed to relieve stalled ribosomes. Trans-translation is mediated by the small protein B (SmpB) and transfer-mRNA (tmRNA) ribonucleoprotein complex, which interacts with translational complexes stalled at the 3' end of non-stop mRNAs to release the stalled ribosomes thereby targeting the nascent polypeptides and truncated mRNAs for degradation. The trans-translation system exists with a few exceptions in all bacteria. In the present study, we assessed the contribution of SmpB to the growth and virulence of Listeria monocytogenes, a human intracellular food-borne pathogen that colonizes host tissues to cause severe invasive infections. A smpB knockout significantly decreased the intracellular growth rate of L. monocytogenes during infection of murine macrophages. In addition, the mutant strain was attenuated for virulence when examined with the Galleria mellonella larvae killing assay and the organ colonisation model of mice following infection. Proteomic analysis of whole cell extracts of ΔsmpB deletion mutant revealed elevated protein levels of several proteins involved in ribosome assembly and interaction with tRNA substrates. These included the elongation factor Tu [EF-Tu] which promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis as well as the CysK which is known to interact with bacterial toxins that cleave tRNA substrates. The data presented here shed light on the role of SmpB and trans-translation during intracellular growth of L. monocytogenes., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo- and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ebola virus (EBOV) transcription is essentially regulated via dynamic dephosphorylation of its viral transcription activator VP30 by the host phosphatase PP2A. The nucleoprotein NP has emerged as a third key player in the regulation of this process by recruiting both the regulatory subunit B56 of PP2A and its substrate VP30 to initiate VP30 dephosphorylation and hence viral transcription. Both binding sites are located in close proximity to each other in NP's C-terminal-disordered region. This study investigates NP's role in VP30 dephosphorylation and transcription activation, focussing on the spatial requirements of NP's binding sites. Increasing the distance between PP2A-B56 and VP30 at the NP interface revealed that close spatial and orientational contact is necessary for efficient VP30 dephosphorylation and viral transcription. Longer distances were lethal for recombinant EBOV except when a compensatory mutation, NP-T603I, occurred. This mutation, located between the NP binding sites for PP2A-B56 and VP30, fully restored functionality. Mass spectrometry showed that T603 is phosphorylated in recEBOV-NPwt virions. Mutational analysis indicated that T603I facilitates VP30 dephosphorylation in otherwise lethal recEBOV and that dynamic phosphorylation of NP-T603 is important for efficient primary viral transcription in the WT context. These findings emphasize the critical and evolutionarily pressured interplay between VP30 and PP2A-B56 within the NP C-terminal-disordered region and highlight the important role of NP on the regulation of viral transcription during the EBOV life cycle.

The carbonic anhydrase II mutant His64Cys was prepared and applied to tethered alkyne/azide cycloaddition reactions. The azide component could be tethered to the enzyme surface through a disulfide bridge, while the alkyne component was reversibly coordinated through a sulfonamide anchor to the zinc ion in the original catalytic center of the enzyme. The incipient orientation of the reactants in the binding site and of the formed triazole product were characterized by crystallography. The reaction progression could be monitored by HPLC-MS analysis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Capistruin is a 19-residue ribosomally synthesized lasso peptide encoded by the capABCD gene cluster in Burkholderia thailandensis. It is composed of an N-terminal 9-residue macrolactam ring, through which the 10-residue C-terminal tail is threaded. Using a heterologous capistruin production system in Escherichia coli, we have generated 48 mutants of the precursor protein CapA to gain insights into capistruin biosynthesis. Only 4 residues (Gly1, Arg11, Val12, and Ile13) of the lasso sequence were found to be critical for maturation. Tandem mass spectrometric fragmentation studies of capistruin F16A/F18A proved Arg15 to be responsible for the trapping of the C-terminal tail. Substituting Arg15 and Phe16 by alanine revealed a temperature-sensitive capistruin derivative, which unfolds into a branched cyclic peptide upon heating. In conclusion, our global mutagenic approach revealed a low overall specificity of the biosynthetic machinery and important structure-stability correlations., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The attachment of modulators to a trimeric porin ion channel was investigated (see picture of the trimer with a crown ether modulator (orange)). The interplay of modulator and protein is essential for the conformational heterogeneity of the hybrid channel. Single-site attachment in large pores is not sufficient to change the electrophysiological characteristics of the pores-such change requires additional noncovalent interactions or second-site attachments.

Here, we report a new experimental approach utilizing an amide ligation reaction for the characterization of acyl carrier protein (ACP)-bound reaction intermediates, which are otherwise difficult to analyze by traditional biochemical methods. To explore fatty acid tailoring enzymes of the calcium-dependent antibiotic (CDA) biosynthetic pathway, this strategy enabled the transformation of modified fatty acids, covalently bound as thioesters to an ACP, into amide ligation products that can be directly analyzed and compared to synthetic standards by HPLC-MS. The driving force of the amide formation is the thermodynamic activation inherent to thioester-bound compounds. Using this novel method, we were able to characterize the ACP-mediated biosynthesis of the unique 2,3-epoxyhexanoyl moiety of CDA, revealing a new type of FAD-dependent oxidase HxcO with intrinsic enoyl-ACP epoxidase activity, as well as a second enoyl-ACP epoxidase, HcmO. In general, our approach should be widely applicable for the in vitro characterization of other biosynthetic systems acting on carrier proteins, such as integrated enzymes from NRPS and PKS assembly lines or tailoring enzymes of fatty and amino acid precursor synthesis.

Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.

The intermolecular communication within NRPS complexes relies on the coordinated interplay of donor and acceptor communication-mediating (COM) domains. In this study, the potential of COM domains was exploited in vivo by establishing a system for the true biocombinatorial synthesis of lipopeptides via directed reprogramming of a natural NRP biosynthetic assembly line (i.e., surfactin). By means of COM domain swapping, these experiments verified the decisive role of COM domains for the control of protein-protein interactions between NRPSs, demonstrated the functionality of COM domain pairs even in the context of a heterologous host and NRPS system, and allowed for the intended skipping of a biosynthetic enzyme within a multienzymatic biosynthetic complex. Ultimately, abrogation of the selectivity barrier provided by COM domains afforded the successful simultaneous, biocombinatorial synthesis of distinct lipopeptide products.

Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that emt1 is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of mat1 resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in U. maydis.

DNA and RNA hairpins, which represent well-folded oligonucleotide structures, were irradiated and the amount of damaged hairpins was directly quantified by using ion-exchange HPLC. The types of photoproducts formed in the hairpins were determined by ESI-HPLC-MS/MS experiments. Irradiation of hairpins with systematically varied sequences and conformations (A versus B) revealed remarkable differences regarding the amount of photolesions formed. UV-damage formation is, therefore, a strongly sequence and conformation dependent process.

ESI-MS(/MS) has been used as a method which allows the fast, unambiguous and sensitive simultaneous detection and relative stability approximation of supramolecular assemblies in mixtures. In spite of the obvious fundamental differences between solution and gas phase, ESI-MS in the case of self-assembled molecular capsules has been shown to produce very similar results to single binding experiments monitored by NMR titrations as well as conformational searches performed by Monte-Carlo simulations. MS/MS experiments reveal the same relative order of gas phase stabilities as previously found in solution. Moreover, proton transfer reactions which lead to new molecular capsules, are not detectable in the time-averaged NMR spectrum. However, the newly produced species are found in the complex mixtures by ESI-MS and can be conveniently characterized by subsequent MS/MS experiments: in a collision-induced dissociation the single half-spheres are easily discovered and structurally assigned. Thus, ESI-MS has worked as a valuable tool for the rapid screening of complex supramolecular mixtures and in combination with MS/MS experiments elucidated both the path of unexpected side reactions as well as the thermodynamic gas-phase stabilities of all components in the mixture.