Bacteriophages (phages) are well known to be one of the major driving forces in bacterial evolution. This also applies to virulent microorganisms, such as the major human pathogen Vibrio cholerae , whose pathogenic potential and epidemic proliferation largely depends on the interaction with environmental phages. Specifically, integration of the CTXϕ phage genome into the first chromosome of V. cholerae also introduced the ctxAB genes, encoding the primary toxin responsible for the severe acute diarrheal disease, cholera. Whereas the mechanisms underlying CTXϕ-associated horizontal gene transfer and transcriptional control of the ctxAB genes have been intensively studied over the past years, posttranscriptional regulation affecting the CTXϕ life cycle has not been documented. Here, we report the identification and characterization of the CisR small RNA (sRNA) that is produced from the 3’UTR (untranslated region) of the prtV gene and inhibits the expression of the CTXϕ-encoded cep mRNA. CisR-mediated repression of cep involves Hfq-assisted base-pairing of the two transcripts and results in reduced CTXϕ production under stress conditions. We further demonstrate that transcription of prtV-cisR requires both the master quorum-sensing regulator HapR and CRP (cAMP receptor protein), a global regulator of carbon metabolism. Taken together, our work provides evidence that V. cholerae employs sRNA-mediated posttranscriptional gene regulation to coordinate CTXϕ activation with both cell density and nutrient availability.

Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG , which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆ mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

Bacteriophages (phages) are well known to be one of the major driving forces in bacterial evolution. This also applies to virulent microorganisms, such as the major human pathogen Vibrio cholerae, whose pathogenic potential and epidemic proliferation largely depends on the interaction with environmental phages. Specifically, integration of the CTXphi; phage genome into the first chromosome of V. cholerae also introduced the ctxAB genes, encoding the primary toxin responsible for the severe acute diarrheal disease, cholera. Whereas the mechanisms underlying CTXphi-associated horizontal gene transfer and transcriptional control of the ctxAB genes have been intensively studied over the past years, post-transcriptional regulation affecting the CTXphi life-cycle has not been documented. Here, we report the discovery and characterization of the CisR small RNA (sRNA) that is produced from the 3UTR (untranslated region) of the prtV gene and inhibits the expression of the CTXphi-encoded cep mRNA. CisR-mediated repression of cep involves Hfq-assisted base-pairing of the two transcripts and results in reduced CTXphi production under stress conditions. We further demonstrate that transcription of prtV-cisR requires both the master quorum-sensing regulator HapR and CRP, a global regulator of carbon metabolism. Taken together, our work provides evidence that V. cholerae employs sRNA-mediated post-transcriptional gene regulation to coordinate CTXphi activation with both cell density and nutrient availability. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, CRC1127‐3 – Project‐ID 239748522, EXC 2051 – Project‐ID 390713860 European Research Council, CoG‐101088027

In bacteria, regulatory networks controlling the adaptation of gene expression in response to stress are frequently complemented by base-pairing small regulatory RNAs (sRNAs) that act at the post-transcriptional level. While many regulatory circuits governing stress resilience have been studied in the model bacterium Caulobacter crescentus, only a small fraction of its diverse sRNA repertoire has been characterized. In this study, we globally identify interacting RNA-RNA pairs associated with the major RNA-binding protein Hfq in C. crescentus. In addition to numerous connections between sRNAs and mRNAs, we also recover RNA-RNA pairs consisting of two non-coding transcripts. Our results indicate that the sRNA CrfA acts as a sponge to inactivate a family of four conserved sRNAs, SisA-D. When induced by carbon starvation, CrfA redirects gene expression towards the utilization of distinct energy sources, and loss of the RNA sponge is linked to a severe growth defect in environments with fluctuating nutrient availability.

Many, if not all, bacteria use quorum sensing (QS) to control collective behaviors, and more recently, QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, to switch between lytic and lysogenic modes of infection. Here, we study the interaction of Vibrio cholerae with the lysogenic phage VP882, which is activated by the QS molecule DPO. We discover that induction of VP882 results in the binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompetes and downregulates host-encoded small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs, and we demonstrate that one of these sRNAs, named VpdS, promotes phage replication by regulating host and phage mRNA levels. We further show that host-encoded sRNAs can antagonize phage replication by downregulating phage mRNA expression and thus might be part of the host’s phage defense arsenal.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological pro-cesses in many species across the bacterial kingdom. In the opportunistic human path-ogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA- binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co- immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species- specific RNAs bound by Hfq and to monitor Hfq- mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq- dependent small regulatory RNA (sRNA), DinR, which is processed from the 3 ' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA- based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA- controlled division inhibition during the SOS response

Gene regulation at the post-transcriptional level is prevalent in all domains of life. In bacteria, ProQ-like proteins have emerged as important RNA chaperones facilitating RNA stability and RNA duplex formation. In the major human pathogen Vibrio cholerae, post-transcriptional gene regulation is key for virulence, biofilm formation, and antibiotic resistance, yet the role of ProQ has not been studied. Here, we show that ProQ interacts with hundreds of transcripts in V. cholerae, including the highly abundant FlaX small RNA (sRNA). Global analyses of RNA duplex formation using RIL-Seq (RNA interaction by ligation and sequencing) revealed a vast network of ProQ-assisted interactions and identified a role for FlaX in motility regulation. Specifically, FlaX base-pairs with multiple sites on the flaB flagellin mRNA, preventing 30S ribosome binding and translation initiation. V. cholerae cells lacking flaX display impaired motility gene expression, altered flagella composition and reduced swimming in liquid environments. Our results provide a global view on ProQ-associated RNA duplex formation and pinpoint the mechanistic and phenotypic consequences associated with ProQ-associated sRNAs in V. cholerae.

ABSTRACT RNA-RNA interactions are key for post-transcriptional gene regulation in all domains of life. While ever more experimental protocols are being developed to study RNA-RNA interactions on a genome-wide scale, computational methods to analyze the underlying data are lagging behind. Here, we present ChimericFragments, an analysis and visualization framework for RNA-seq experiments producing chimeric RNA molecules. ChimericFragments implements a novel statistical method based on the complementarity of the base-pairing RNAs around their ligation site and is compatible with several widely used experimental procedures. We demonstrate that ChimericFragments enables the systematic identification of RNA regulators and RNA-RNA pairs and outperforms existing approaches.

Membrane transporters and receptors often rely on conserved hydrogen bonds to assemble transient paths for ion transfer or long-distance conformational couplings. For transporters and receptors that use proton binding and proton transfer for function, inter-helical hydrogen bonds of titratable protein sidechains that could change protonation are of central interest to formulate hypotheses about reaction mechanisms. Knowledge of hydrogen bonds common at sites of potential interest for proton binding could thus inform and guide studies on functional mechanisms of protonation-coupled membrane proteins. Here we apply graph-theory approaches to identify hydrogen-bond motifs of carboxylate and histidine sidechains in a large data set of static membrane protein structures. We find that carboxylate-hydroxyl hydrogen bonds are present in numerous structures of the dataset, and can be part of more extended H-bond clusters that could be relevant to conformational coupling. Carboxylate-carboxyamide and imidazole-imidazole hydrogen bonds are represented in comparably fewer protein structures of the dataset. Atomistic simulations on two membrane transporters in lipid membranes suggest that many of the hydrogen bond motifs present in static protein structures tend to be robust, and can be part of larger hydrogen-bond clusters that recruit additional hydrogen bonds.

Small regulatory RNAs (sRNAs) acting in concert with the RNA chaperone Hfq are prevalent in many bacteria and typically act by base-pairing with multiple target transcripts. In the human pathogen Vibrio cholerae , sRNAs play roles in various processes including antibiotic tolerance, competence, and quorum sensing (QS). Here, we use RIL-seq (RNA-interaction-by-ligation-and-sequencing) to identify Hfq-interacting sRNAs and their targets in V. cholerae . We find hundreds of sRNA-mRNA interactions, as well as RNA duplexes formed between two sRNA regulators. Further analysis of these duplexes identifies an RNA sponge, termed QrrX, that base-pairs with and inactivates the Qrr1-4 sRNAs, which are known to modulate the QS pathway. Transcription of qrrX is activated by QrrT, a previously uncharacterized LysR-type transcriptional regulator. Our results indicate that QrrX and QrrT are required for rapid conversion from individual to community behaviours in V. cholerae .

Dynamic hydrogen bonds and hydrogen bond networks give proteins structural plasticity required for function. This includes long-distance conformational coupling between remote regions of the protein, and transfer of protons across long distances by membrane transporters. In the case of large proteins and macromolecular complexes, identifying hydrogen-bond networks that assemble transiently in fluid environments brings about the challenge of large numbers of interactions that need to be evaluated.

Dynamic hydrogen-bond networks are key determinants of protein conformational dynamics. In the case of macromolecular protein complexes, which can have a large number of hydrogen bonds giving rise to extensive hydrogen-bond networks, efficient algorithms are required to analyze interactions that could be important for the dynamics and biological function of the complex. We present here a highly efficient, standalone interface designed for analyses of dynamical hydrogen-bond networks of biomolecules and macromolecular complexes. To facilitate a comprehensive description of protein dynamics, the interface includes analyses of hydrophobic interactions. We illustrate the usefulness and workflow of the interface by dissecting the dynamics of the ectodomain of SARS-CoV-2 protein S in its closed conformation. We find that protein S contains numerous local clusters of dynamic hydrogen bonds and identify hydrogen bonds that are sampled persistently. The receptor binding domain of the spike protein hosts only a handful of persistent hydrogen-bond clusters, suggesting structural plasticity. Our data analysis interface is released here for open use.

Although the outward-directed proton transport across biological membranes is well studied and its importance for bioenergetics is clearly understood, inward-directed light-driven proton pumping by microbial rhodopsins has remained a mystery both physiologically and mechanistically. A new family of Antarctic rhodopsins, which is a subgroup within a novel class of schizorhodopsins reported recently, includes a member, denoted as AntR, which proved amenable to extensive characterization with experiments and computation. Phylogenetic analyses identify AntR as distinct from the well-studied microbial rhodopsins that function as outward-directed ion pumps, and bioinformatics sequence analyses reveal amino acid substitutions at conserved sites essential for outward proton pumping. Modeling and numerical simulations of AntR, combined with advanced analyses using the graph theory and centrality measures from social sciences, identify the dynamic three-dimensional network of hydrogen-bonded water molecules and amino acid residues that function as communication hubs in AntR. This network undergoes major rearrangement upon retinal isomerization, showing important changes in the connectivity of the active center, retinal Schiff base, to the opposing sides of the membrane, as required for proton transport. Numerical simulations and experimental studies of the photochemical cycle of AntR by spectroscopy and sitedirected mutagenesis allowed us to identify pathways that could conduct protons in the direction opposite to that commonly known for outward-directed pumps.

Membrane-embedded proton transporters often expose to the bulk clusters of closely spaced carboxylate groups that might function as a proton antenna, binding a proton that could then be released to the bulk or transferred to an internal protein group. To characterize mechanisms of proton binding and proton transfer at protein and membrane interfaces we study membranes and proteins in membranes. Extensive computations of a proton-antenna model using classical mechanical and quantum mechanical descriptions indicate that the surface proton-binding site is part of an extensive hydrogen-bond cluster that includes caged waters. The water-mediated bridges between carboxylate groups are, however, highly dynamic, with lifetimes comparable to that of water hydrogen bonding. The energetic barrier for proton transfer within the proton antenna cluster is somewhat high, suggesting that a proton bound at this site could indeed be stored. At the interface of a membrane with negatively charged lipids, we find a rich network of dynamic lipid-water hydrogen bonds and transient clusters of water-bridged phosphate groups.We developed algorithms inspired from graph theory and social sciences and applied these algorithms to study protein conformational dynamics. Using such algorithms, we found that hydrogen-bonded waters might assist long-distance conformational coupling of the SecA protein motor. Financial support was provided in part by the DFG Collaborative Research Center SFB 1078 Project C4 (to A.-N.B.) and by the Freie Universität Berlin within the Excellence Initiative of the German Research Foundation. Computing time was provided by the HLRN, the North-German Supercomputing Alliance.

Corona virus spike protein S is a large homo-trimeric protein anchored in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, and contribute symmetrically to a relatively large H-bond cluster at the receptor binding domain, and to a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.

Membrane proteins that function as transporters or receptors must communicate with both sides of the lipid bilayer in which they sit. This long distance communication enables transporters to move protons or other ions and small molecules across the bilayer and receptors to transmit an external signal to the cell. Hydrogen bonds, hydrogen-bond networks, and lipid-protein interactions are essential for the motions and functioning of the membrane protein and, consequently, of outmost interest to structural biology and numerical simulations. We present here Bridge, an algorithm tailored for efficient analyses of hydrogen-bond networks in membrane transporter and receptor proteins. For channelrhodopsin, a membrane protein whose functioning involves proton-transfer reactions, Bridge identifies extensive networks of protein-water hydrogen bonds and an unanticipated network that can bridge transiently two proton donors across a distance of ∼20 Å. Graphs of the protein hydrogen bonds reveal rapid propagation of structural changes within hydrogen-bond networks of mutant transporters and identify protein groups potentially important for the proton transfer activity. The algorithm is made available as a plugin for PyMol.

Motor and transporter proteins couple protein conformational dynamics to chemical reactions such as binding and cleavage of a nucleotide, or proton transfer. Hydrogen bond networks are particularly important here, because they can mediate coupling between remote regions of the protein. These networks can be rather dynamic, and involve water molecules that visit transiently the interior of the protein. Algorithms inspired from graph theory allow us to analyze efficiently the dynamics of hydrogen bond networks in complex protein environments and at lipid membrane interfaces. Computations on a set of proteins whose functioning requires allosteric coupling between a chemical reaction, protein and water dynamics, suggest mechanisms by which dynamic hydrogen bond networks shape protein conformational dynamics. Research was supported in part by the German Research Foundation (DFG) Collaborative Research Center SFB 1078 Protonation Dynamics in Protein Function (Project C4), by the Freie Universität Berlin within the Excellence Initiative of the German Research Foundation, and by computing time from the North-German Supercomputing Alliance, HLRN.

Photosystem II uses the energy of absorbed light to split water molecules, generating molecular oxygen, electrons, and protons. The four protons generated during each reaction cycle are released to the lumen via mechanisms that are poorly understood. Given the complexity of photosystem II, which consists of multiple protein subunits and cofactor molecules and hosts numerous waters, a fundamental issue is finding transient networks of hydrogen bonds that bridge potential proton donor and acceptor groups. Here, we address this issue by performing all-atom molecular dynamics simulations of wild-type and mutant photosystem II monomers, which we analyze using a new protocol designed to facilitate efficient analysis of hydrogen-bond networks. Our computations reveal that local protein/water hydrogen-bond networks can assemble transiently in photosystem II such that the reaction center connects to the lumen. The dynamics of the hydrogen-bond networks couple to the protonation state of specific carboxylate groups and are altered in a mutant with defective proton transfer. Simulations on photosystem II without its extrinsic PsbO subunit provide a molecular interpretation of the elusive functional role of this subunit.