Gas and light: triggers of c-di-GMP-mediated regulation.

The widespread bacterial second messenger c-di-GMP is responsible for regulating many important physiological functions such as biofilm formation, motility, cell differentiation, and virulence. The synthesis and degradation of c-di-GMP in bacterial cells depend, respectively, on diguanylate cyclases and c-di-GMP-specific phosphodiesterases. Since c-di-GMP metabolic enzymes (CMEs) are often fused to sensory domains, their activities are likely controlled by environmental signals, thereby altering cellular c-di-GMP levels and regulating bacterial adaptive behaviors. Previous studies on c-di-GMP-mediated regulation mainly focused on downstream signaling pathways, including the identification of CMEs, cellular c-di-GMP receptors, and c-di-GMP-regulated processes. The mechanisms of CME regulation by upstream signaling modules received less attention, resulting in a limited understanding of the c-di-GMP regulatory networks. We review here the diversity of sensory domains related to bacterial CME regulation. We specifically discuss those domains that are capable of sensing gaseous or light signals and the mechanisms they use for regulating cellular c-di-GMP levels. It is hoped that this review would help refine the complete c-di-GMP regulatory networks and improve our understanding of bacterial behaviors in changing environments. In practical terms, this may eventually provide a way to control c-di-GMP-mediated bacterial biofilm formation and pathogenesis in general.

All DACs in a Row: Domain Architectures of Bacterial and Archaeal Diadenylate Cyclases.

Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to various stresses. C-di-AMP is synthesized by diadenylate cyclases that contain the DAC (DisA_N) domain, which was originally characterized as the N-terminal domain in the DNA integrity scanning protein DisA. In other experimentally studied diadenylate cyclases, DAC domain is typically located at the protein C termini and its enzymatic activity is controlled by one or more N-terminal domains. As in other bacterial signal transduction proteins, these N-terminal modules appear to sense environmental or intracellular signals through ligand binding and/or protein-protein interactions. Studies of bacterial and archaeal diadenylate cyclases also revealed numerous sequences with uncharacterized N-terminal regions. This work provides a comprehensive review of the N-terminal domains of bacterial and archaeal diadenylate cyclases, including the description of five previously undefined domains and three PK_C-related domains of the DacZ_N superfamily. These data are used to classify diadenylate cyclases into 22 families, based on their conserved domain architectures and the phylogeny of their DAC domains. Although the nature of the regulatory signals remains obscure, the association of certain dac genes with anti-phage defense CBASS systems and other phage-resistance genes suggests that c-di-AMP might also be involved in the signaling of phage infection.

A decade of research on the second messenger c-di-AMP.

Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) is an emerging second messenger in bacteria and archaea that is synthesized from two molecules of ATP by diadenylate cyclases and degraded to pApA or two AMP molecules by c-di-AMP-specific phosphodiesterases. Through binding to specific protein- and riboswitch-type receptors, c-di-AMP regulates a wide variety of prokaryotic physiological functions, including maintaining the osmotic pressure, balancing central metabolism, monitoring DNA damage and controlling biofilm formation and sporulation. It mediates bacterial adaptation to a variety of environmental parameters and can also induce an immune response in host animal cells. In this review, we discuss the phylogenetic distribution of c-di-AMP-related enzymes and receptors and provide some insights into the various aspects of c-di-AMP signaling pathways based on more than a decade of research. We emphasize the key role of c-di-AMP in maintaining bacterial osmotic balance, especially in Gram-positive bacteria. In addition, we discuss the future direction and trends of c-di-AMP regulatory network, such as the likely existence of potential c-di-AMP transporter(s), the possibility of crosstalk between c-di-AMP signaling with other regulatory systems, and the effects of c-di-AMP compartmentalization. This review aims to cover the broad spectrum of research on the regulatory functions of c-di-AMP and c-di-AMP signaling pathways.

Cyclic di-AMP, a second messenger of primary importance: tertiary structures and binding mechanisms.

Cyclic diadenylate (c-di-AMP) is a widespread second messenger in bacteria and archaea that is involved in the maintenance of osmotic pressure, response to DNA damage, and control of central metabolism, biofilm formation, acid stress resistance, and other functions. The primary importance of c-di AMP stems from its essentiality for many bacteria under standard growth conditions and the ability of several eukaryotic proteins to sense its presence in the cell cytoplasm and trigger an immune response by the host cells. We review here the tertiary structures of the domains that regulate c-di-AMP synthesis and signaling, and the mechanisms of c-di-AMP binding, including the principal conformations of c-di-AMP, observed in various crystal structures. We discuss how these c-di-AMP molecules are bound to the protein and riboswitch receptors and what kinds of interactions account for the specific high-affinity binding of the c-di-AMP ligand. We describe seven kinds of non-covalent-π interactions between c-di-AMP and its receptor proteins, including π-π, C-H-π, cation-π, polar-π, hydrophobic-π, anion-π and the lone pair-π interactions. We also compare the mechanisms of c-di-AMP and c-di-GMP binding by the respective receptors that allow these two cyclic dinucleotides to control very different biological functions.

Evolution of cation binding in the active sites of P-loop nucleoside triphosphatases in relation to the basic catalytic mechanism.

The ubiquitous P-loop fold nucleoside triphosphatases (NTPases) are typically activated by an arginine or lysine 'finger'. Some of the apparently ancestral NTPases are, instead, activated by potassium ions. To clarify the activation mechanism, we combined comparative structure analysis with molecular dynamics (MD) simulations of Mg-ATP and Mg-GTP complexes in water and in the presence of potassium, sodium, or ammonium ions. In all analyzed structures of diverse P-loop NTPases, the conserved P-loop motif keeps the triphosphate chain of bound NTPs (or their analogs) in an extended, catalytically prone conformation, similar to that imposed on NTPs in water by potassium or ammonium ions. MD simulations of potassium-dependent GTPase MnmE showed that linking of alpha- and gamma phosphates by the activating potassium ion led to the rotation of the gamma-phosphate group yielding an almost eclipsed, catalytically productive conformation of the triphosphate chain, which could represent the basic mechanism of hydrolysis by P-loop NTPases.

Proposed Role for KaiC-Like ATPases as Major Signal Transduction Hubs in Archaea.

All organisms must adapt to ever-changing environmental conditions and accordingly have evolved diverse signal transduction systems. In bacteria, the most abundant networks are built around the two-component signal transduction systems that include histidine kinases and receiver domains. In contrast, eukaryotic signal transduction is dominated by serine/threonine/tyrosine protein kinases. Both of these systems are also found in archaea, but they are not as common and diversified as their bacterial and eukaryotic counterparts, suggesting the possibility that archaea have evolved other, still uncharacterized signal transduction networks. Here we propose a role for KaiC family ATPases, known to be key components of the circadian clock in cyanobacteria, in archaeal signal transduction. The KaiC family is notably expanded in most archaeal genomes, and although most of these ATPases remain poorly characterized, members of the KaiC family have been shown to control archaellum assembly and have been found to be a stable component of the gas vesicle system in Halobacteria Computational analyses described here suggest that KaiC-like ATPases and their homologues with inactivated ATPase domains are involved in many other archaeal signal transduction pathways and comprise major hubs of complex regulatory networks. We predict numerous input and output domains that are linked to KaiC-like proteins, including putative homologues of eukaryotic DEATH domains that could function as adapters in archaeal signaling networks. We further address the relationships of the archaeal family of KaiC homologues to the bona fide KaiC of cyanobacteria and implications for the existence of a KaiC-based circadian clock apparatus in archaea.IMPORTANCE Little is currently known about signal transduction pathways in Archaea Recent studies indicate that KaiC-like ATPases, known as key components of the circadian clock apparatus in cyanobacteria, are involved in the regulation of archaellum assembly and, likely, type IV pili and the gas vesicle system in Archaea We performed comprehensive comparative genomic analyses of the KaiC family. A vast protein interaction network was revealed, with KaiC family proteins as hubs for numerous input and output components, many of which are shared with two-component signal transduction systems. Putative KaiC-based signal transduction systems are predicted to regulate the activities of membrane-associated complexes and individual proteins, such as signal recognition particle and membrane transporters, and also could be important for oxidative stress response regulation. KaiC-centered signal transduction networks are predicted to play major roles in archaeal physiology, and this work is expected to stimulate their experimental characterization.

The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes.

This year's Database Issue of Nucleic Acids Research contains 152 papers that include descriptions of 54 new databases and update papers on 98 databases, of which 16 have not been previously featured in NAR As always, these databases cover a broad range of molecular biology subjects, including genome structure, gene expression and its regulation, proteins, protein domains, and protein-protein interactions. Following the recent trend, an increasing number of new and established databases deal with the issues of human health, from cancer-causing mutations to drugs and drug targets. In accordance with this trend, three recently compiled databases that have been selected by NAR reviewers and editors as 'breakthrough' contributions, denovo-db, the Monarch Initiative, and Open Targets, cover human de novo gene variants, disease-related phenotypes in model organisms, and a bioinformatics platform for therapeutic target identification and validation, respectively. We expect these databases to attract the attention of numerous researchers working in various areas of genetics and genomics. Looking back at the past 12 years, we present here the 'golden set' of databases that have consistently served as authoritative, comprehensive, and convenient data resources widely used by the entire community and offer some lessons on what makes a successful database. The Database Issue is freely available online at the https://academic.oup.com/nar web site. An updated version of the NAR Molecular Biology Database Collection is available at http://www.oxfordjournals.org/nar/database/a/.

Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 µM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date.

The 2016 database issue of Nucleic Acids Research and an updated molecular biology database collection.

The 2016 Database Issue of Nucleic Acids Research starts with overviews of the resources provided by three major bioinformatics centers, the U.S. National Center for Biotechnology Information (NCBI), the European Bioinformatics Institute (EMBL-EBI) and Swiss Institute for Bioinformatics (SIB). Also included are descriptions of 62 new databases and updates on 95 databases that have been previously featured in NAR plus 17 previously described elsewhere. A number of papers in this issue deal with resources on nucleic acids, including various kinds of non-coding RNAs and their interactions, molecular dynamics simulations of nucleic acid structure, and two databases of super-enhancers. The protein database section features important updates on the EBI's Pfam, PDBe and PRIDE databases, as well as a variety of resources on pathways, metabolomics and metabolic modeling. This issue also includes updates on popular metagenomics resources, such as MG-RAST, EBI Metagenomics, and probeBASE, as well as a newly compiled Human Pan-Microbe Communities database. A significant fraction of the new and updated databases are dedicated to the genetic basis of disease, primarily cancer, and various aspects of drug research, including resources for patented drugs, their side effects, withdrawn drugs, and potential drug targets. A further six papers present updated databases of various antimicrobial and anticancer peptides. The entire Database Issue is freely available online on the Nucleic Acids Research website (http://nar.oxfordjournals.org/). The NAR online Molecular Biology Database Collection, http://www.oxfordjournals.org/nar/database/c/, has been updated with the addition of 88 new resources and removal of 23 obsolete websites, which brought the current listing to 1685 databases.

Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP.

The 2015 Nucleic Acids Research Database Issue and molecular biology database collection.

The 2015 Nucleic Acids Research Database Issue contains 172 papers that include descriptions of 56 new molecular biology databases, and updates on 115 databases whose descriptions have been previously published in NAR or other journals. Following the classification that has been introduced last year in order to simplify navigation of the entire issue, these articles are divided into eight subject categories. This year's highlights include RNAcentral, an international community portal to various databases on noncoding RNA; ValidatorDB, a validation database for protein structures and their ligands; SASBDB, a primary repository for small-angle scattering data of various macromolecular complexes; MoonProt, a database of 'moonlighting' proteins, and two new databases of protein-protein and other macromolecular complexes, ComPPI and the Complex Portal. This issue also includes an unusually high number of cancer-related databases and other databases dedicated to genomic basics of disease and potential drugs and drug targets. The size of NAR online Molecular Biology Database Collection, http://www.oxfordjournals.org/nar/database/a/, remained approximately the same, following the addition of 74 new resources and removal of 77 obsolete web sites. The entire Database Issue is freely available online on the Nucleic Acids Research web site (http://nar.oxfordjournals.org/).

How many signal peptides are there in bacteria?

Over the last 5 years proteogenomics (using mass spectroscopy to identify proteins predicted from genomic sequences) has emerged as a promising approach to the high-throughput identification of protein N-termini, which remains a problem in genome annotation. Comparison of the experimentally determined N-termini with those predicted by sequence analysis tools allows identification of the signal peptides and therefore conclusions on the cytoplasmic or extracytoplasmic (periplasmic or extracellular) localization of the respective proteins. We present here the results of a proteogenomic study of the signal peptides in Escherichia coli K-12 and compare its results with the available experimental data and predictions by such software tools as SignalP and Phobius. A single proteogenomics experiment recovered more than a third of all signal peptides that had been experimentally determined during the past three decades and confirmed at least 31 additional signal peptides, mostly in the known exported proteins, which had been previously predicted but not validated. The filtering of putative signal peptides for the peptide length and the presence of an eight-residue hydrophobic patch and a typical signal peptidase cleavage site proved sufficient to eliminate the false-positive hits. Surprisingly, the results of this proteogenomics study, as well as a re-analysis of the E. coli genome with the latest version of SignalP program, show that the fraction of proteins containing signal peptides is only about 10%, or half of previous estimates.

The role of energy in the emergence of biology from chemistry.

Any scenario of the transition from chemistry to biology should include an "energy module" because life can exist only when supported by energy flow(s). We addressed the problem of primordial energetics by combining physico-chemical considerations with phylogenomic analysis. We propose that the first replicators could use abiotically formed, exceptionally photostable activated cyclic nucleotides both as building blocks and as the main energy source. Nucleoside triphosphates could replace cyclic nucleotides as the principal energy-rich compounds at the stage of the first cells, presumably because the metal chelates of nucleoside triphosphates penetrated membranes much better than the respective metal complexes of nucleoside monophosphates. The ability to exploit natural energy flows for biogenic production of energy-rich molecules could evolve only gradually, after the emergence of sophisticated enzymes and ion-tight membranes. We argue that, in the course of evolution, sodium-dependent membrane energetics preceded the proton-based energetics which evolved independently in bacteria and archaea.

The 2011 Nucleic Acids Research Database Issue and the online Molecular Biology Database Collection.

The current 18th Database Issue of Nucleic Acids Research features descriptions of 96 new and 83 updated online databases covering various areas of molecular biology. It includes two editorials, one that discusses COMBREX, a new exciting project aimed at figuring out the functions of the 'conserved hypothetical' proteins, and one concerning BioDBcore, a proposed description of the 'minimal information about a biological database'. Papers from the members of the International Nucleotide Sequence Database collaboration (INSDC) describe each of the participating databases, DDBJ, ENA and GenBank, principles of data exchange within the collaboration, and the recently established Sequence Read Archive. A testament to the longevity of databases, this issue includes updates on the RNA modification database, Definition of Secondary Structure of Proteins (DSSP) and Homology-derived Secondary Structure of Proteins (HSSP) databases, which have not been featured here in >12 years. There is also a block of papers describing recent progress in protein structure databases, such as Protein DataBank (PDB), PDB in Europe (PDBe), CATH, SUPERFAMILY and others, as well as databases on protein structure modeling, protein-protein interactions and the organization of inter-protein contact sites. Other highlights include updates of the popular gene expression databases, GEO and ArrayExpress, several cancer gene databases and a detailed description of the UK PubMed Central project. The Nucleic Acids Research online Database Collection, available at: http://www.oxfordjournals.org/nar/database/a/, now lists 1330 carefully selected molecular biology databases. The full content of the Database Issue is freely available online at the Nucleic Acids Research web site (http://nar.oxfordjournals.org/).

Characterization of the N-ATPase, a distinct, laterally transferred Na+-translocating form of the bacterial F-type membrane ATPase.

UNLABELLED: An analysis of the distribution of the Na(+)-translocating ATPases/ATP synthases among microbial genomes identified an atypical form of the F(1)F(o)-type ATPase that is present in the archaea Methanosarcina barkeri and M. acetivorans, in a number of phylogenetically diverse marine and halotolerant bacteria and in pathogens Burkholderia spp. In complete genomes, representatives of this form (referred to here as N-ATPase) are always present as second copies, in addition to the typical proton-translocating ATP synthases. The N-ATPase is encoded by a highly conserved atpDCQRBEFAG operon and its subunits cluster separately from the equivalent subunits of the typical F-type ATPases. N-ATPase c subunits carry a full set of sodium-binding residues, indicating that most of these enzymes are Na(+)-translocating ATPases that likely confer on their hosts the ability to extrude Na(+) ions. Other distinctive properties of the N-ATPase operons include the absence of the delta subunit from its cytoplasmic sector and the presence of two additional membrane subunits, AtpQ (formerly gene 1) and AtpR (formerly gene X). We argue that N-ATPases are an early-diverging branch of membrane ATPases that, similarly to the eukaryotic V-type ATPases, do not synthesize ATP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Co-evolution of primordial membranes and membrane proteins.

Studies of the past several decades have provided major insights into the structural organization of biological membranes and mechanisms of many membrane molecular machines. However, the origin(s) of the membrane(s) and membrane proteins remains enigmatic. We discuss different concepts of the origin and early evolution of membranes with a focus on the evolution of the (im)permeability to charged molecules such as proteins, nucleic acids and small ions. Reconstruction of the evolution of F-type and A/V-type membrane ATPases (ATP synthases), which are either proton- or sodium-dependent, might help us to understand not only the origin of membrane bioenergetics but also of membranes themselves. We argue that evolution of biological membranes occurred as a process of co-evolution of lipid bilayers, membrane proteins and membrane bioenergetics.

The past and present of sodium energetics: may the sodium-motive force be with you.

All living cells routinely expel Na(+) ions, maintaining lower concentration of Na(+) in the cytoplasm than in the surrounding milieu. In the vast majority of bacteria, as well as in mitochondria and chloroplasts, export of Na(+) occurs at the expense of the proton-motive force. Some bacteria, however, possess primary generators of the transmembrane electrochemical gradient of Na(+) (sodium-motive force). These primary Na(+) pumps have been traditionally seen as adaptations to high external pH or to high temperature. Subsequent studies revealed, however, the mechanisms for primary sodium pumping in a variety of non-extremophiles, such as marine bacteria and certain bacterial pathogens. Further, many alkaliphiles and hyperthermophiles were shown to rely on H(+), not Na(+), as the coupling ion. We review here the recent progress in understanding the role of sodium-motive force, including (i) the conclusion on evolutionary primacy of the sodium-motive force as energy intermediate, (ii) the mechanisms, evolutionary advantages and limitations of switching from Na(+) to H(+) as the coupling ion, and (iii) the possible reasons why certain pathogenic bacteria still rely on the sodium-motive force.

Evolutionary primacy of sodium bioenergetics.

BACKGROUND: The F- and V-type ATPases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of ATP. Both the F-type (found in most bacteria and eukaryotic mitochondria and chloroplasts) and V-type (found in archaea, some bacteria, and eukaryotic vacuoles) ATPases can translocate either protons or sodium ions. The prevalent proton-dependent ATPases are generally viewed as the primary form of the enzyme whereas the sodium-translocating ATPases of some prokaryotes are usually construed as an exotic adaptation to survival in extreme environments. RESULTS: We combine structural and phylogenetic analyses to clarify the evolutionary relation between the proton- and sodium-translocating ATPases. A comparison of the structures of the membrane-embedded oligomeric proteolipid rings of sodium-dependent F- and V-ATPases reveals nearly identical sets of amino acids involved in sodium binding. We show that the sodium-dependent ATPases are scattered among proton-dependent ATPases in both the F- and the V-branches of the phylogenetic tree. CONCLUSION: Barring convergent emergence of the same set of ligands in several lineages, these findings indicate that the use of sodium gradient for ATP synthesis is the ancestral modality of membrane bioenergetics. Thus, a primitive, sodium-impermeable but proton-permeable cell membrane that harboured a set of sodium-transporting enzymes appears to have been the evolutionary predecessor of the more structurally demanding proton-tight membranes. The use of proton as the coupling ion appears to be a later innovation that emerged on several independent occasions.

Inventing the dynamo machine: the evolution of the F-type and V-type ATPases.

The rotary proton- and sodium-translocating ATPases are reversible molecular machines present in all cellular life forms that couple ion movement across membranes with ATP hydrolysis or synthesis. Sequence and structural comparisons of F- and V-type ATPases have revealed homology between their catalytic and membrane subunits, but not between the subunits of the central stalk that connects the catalytic and membrane components. Based on this pattern of homology, we propose that these ATPases originated from membrane protein translocases, which, themselves, evolved from RNA translocases. We suggest that in these ancestral translocases, the position of the central stalk was occupied by the translocated polymer.