RESUMEN
Plasmids are mobile genetic elements found in many clades of Archaea and Bacteria. They drive horizontal gene transfer, impacting ecological and evolutionary processes within microbial communities, and hold substantial importance in human health and biotechnology. To support plasmid research and provide scientists with data of an unprecedented diversity of plasmid sequences, we introduce the IMG/PR database, a new resource encompassing 699 973 plasmid sequences derived from genomes, metagenomes and metatranscriptomes. IMG/PR is the first database to provide data of plasmid that were systematically identified from diverse microbiome samples. IMG/PR plasmids are associated with rich metadata that includes geographical and ecosystem information, host taxonomy, similarity to other plasmids, functional annotation, presence of genes involved in conjugation and antibiotic resistance. The database offers diverse methods for exploring its extensive plasmid collection, enabling users to navigate plasmids through metadata-centric queries, plasmid comparisons and BLAST searches. The web interface for IMG/PR is accessible at https://img.jgi.doe.gov/pr. Plasmid metadata and sequences can be downloaded from https://genome.jgi.doe.gov/portal/IMG_PR.
Asunto(s)
Metagenoma , Microbiota , Humanos , Metadatos , Programas Informáticos , Bases de Datos Genéticas , Plásmidos/genéticaRESUMEN
Genetic interventions on microbiomes, for clinical or biotechnological purposes, remain challenging. Conjugation-based delivery of genetic cargo is still unspecific and limited by low conjugation rates. Here we report an approach to overcome these problems, based on a synthetic bacterial adhesion system. Mating assemblers consist on a synthetic adhesion formed by the expression on the surface of donor and target cells of specific nanobodies (Nb) and their cognate antigen (Ag). The Nb-Ag bridge increased 1-3 logs transfer of a variety of plasmids, especially in liquid media, confirming that cell-cell docking is a main determinant limiting mating efficiency. Synthetic cell-to-cell adhesion allows efficient conjugation to targeted recipients, enhancing delivery of desired genes to a predefined subset of prey species, or even specific pathogenic strains such as enterohemorrhagic Escherichia coli (EHEC), within a bacterial community. The synthetic conjugation enhancer presented here optimizes plasmid delivery by selecting the target hosts with high selectivity.
Asunto(s)
Conjugación Genética , Técnicas Genéticas , Microbiota , Adhesión Celular , Conjugación Genética/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Plásmidos/genética , Biotecnología/métodos , Microbiota/genéticaRESUMEN
Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.
Asunto(s)
ADN , Factores de Transcripción , Factores de Transcripción/metabolismo , Secuencia de Bases , Secuencia de Aminoácidos , Secuencias Hélice-Giro-Hélice , ADN/química , Inversión de Secuencia , Sitios de UniónRESUMEN
Conjugation drives the horizontal transfer of adaptive traits across prokaryotes. One-fourth of the plasmids encode the functions necessary to conjugate autonomously, the others being eventually mobilizable by conjugation. To understand the evolution of plasmid mobility, we studied plasmid size, gene repertoires, and conjugation-related genes. Plasmid gene repertoires were found to vary rapidly in relation to the evolutionary rate of relaxases, for example, most pairs of plasmids with 95% identical relaxases have fewer than 50% of homologs. Among 249 recent transitions of mobility type, we observed a clear excess of plasmids losing the capacity to conjugate. These transitions are associated with even greater changes in gene repertoires, possibly mediated by transposable elements, including pseudogenization of the conjugation locus, exchange of replicases reducing the problem of incompatibility, and extensive loss of other genes. At the microevolutionary scale of plasmid taxonomy, transitions of mobility type sometimes result in the creation of novel taxonomic units. Interestingly, most transitions from conjugative to mobilizable plasmids seem to be lost in the long term. This suggests a source-sink dynamic, where conjugative plasmids generate nonconjugative plasmids that tend to be poorly adapted and are frequently lost. Still, in some cases, these relaxases seem to have evolved to become efficient at plasmid mobilization in trans, possibly by hijacking multiple conjugative systems. This resulted in specialized relaxases of mobilizable plasmids. In conclusion, the evolution of plasmid mobility is frequent, shapes the patterns of gene flow in bacteria, the dynamics of gene repertoires, and the ecology of plasmids.
Asunto(s)
Conjugación Genética , Elementos Transponibles de ADN , Bacterias/genética , Bacterias/metabolismo , Elementos Transponibles de ADN/genética , Transferencia de Gen Horizontal , Plásmidos/genética , Células ProcariotasRESUMEN
Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid Biology derive from the efforts to classify plasmids. Here, we review the main plasmid classification systems, starting by phenotype-based methods, such as fertility inhibition and incompatibility, followed by schemes based on a single gene (replicon type and MOB class), and finishing with recently developed approaches that use genetic distances between whole plasmid sequences. A comparison of the latter highlights significant differences between them. We further discuss the need for an operational definition of plasmid species that reveals their biological features, akin to plasmid taxonomic units (PTUs).
Asunto(s)
Antibacterianos , Bacterias , Plásmidos/genética , Bacterias/genética , Antibacterianos/farmacología , Transferencia de Gen HorizontalRESUMEN
Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.
Asunto(s)
Conjugación Genética , Proteínas Virales/química , Dominio Catalítico , Cristalografía por Rayos X , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Especificidad del Huésped , Magnesio/química , Metaloproteasas/química , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. RESULTS: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. CONCLUSIONS: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.
Asunto(s)
Transferencia de Gen Horizontal , Genómica , Farmacorresistencia Microbiana , Plásmidos/genética , Factores de VirulenciaRESUMEN
Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, often pose a serious risk not only when delivered in the bloodstream but also in air, the environment and several industrial fields such as pharmaceutics or food. LPS is constituted of three regions; the O-specific chain, the core region and the lipid A, which is the responsible segment of the toxicity. Previous literature dealt with the study of lipid A, its potential ligands as well as the mechanisms of Lipid A interactions that, among other applications, establish the basis for detection methods such as Limulus Amebocyte Lysate (LAL) assays and emerging biosensoring techniques. However, quantifying LPS binding affinity is an urgent need that still requires thorough studies. In this context, this work reviews the molecules that bind LPS, highlighting quantitative affinity parameters. Moreover, state of the art methods to analyze the affinity and kinetics of lipid-ligand interactions are also reviewed and different techniques have been briefly described. Thus, first, we review existing information on LPS ligands, classifying them into three main groups and targeting the comparison of molecules in terms of their interaction affinities and, second, we establish the basis for further research aimed at the development of effective methods for LPS detection and removal.
Asunto(s)
Proteínas Portadoras/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/metabolismo , Bacterias Gramnegativas/metabolismo , Humanos , Sistema Inmunológico/metabolismo , Lípido A/metabolismo , Conformación ProteicaRESUMEN
Bacterial conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and bacterial conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of bacterial conjugation, which may be pivotal for combating multidrug-resistant bacteria.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Alquinos/farmacología , Antibacterianos/farmacología , Conjugación Genética/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos Insaturados/farmacología , Ácido Palmítico/farmacología , Alquinos/química , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Simulación del Acoplamiento MolecularRESUMEN
Self-transmissible mobile genetic elements drive horizontal gene transfer between prokaryotes. Some of these elements integrate in the chromosome, whereas others replicate autonomously as plasmids. Recent works showed the existence of few differences, and occasional interconversion, between the two types of elements. Here, we enquired on why evolutionary processes have maintained the two types of mobile genetic elements by comparing integrative and conjugative elements (ICE) with extrachromosomal ones (conjugative plasmids) of the highly abundant MPFT conjugative type. We observed that plasmids encode more replicases, partition systems, and antibiotic resistance genes, whereas ICEs encode more integrases and metabolism-associated genes. ICEs and plasmids have similar average sizes, but plasmids are much more variable, have more DNA repeats, and exchange genes more frequently. On the other hand, we found that ICEs are more frequently transferred between distant taxa. We propose a model where the different genetic plasticity and amplitude of host range between elements explain the co-occurrence of integrative and extrachromosomal elements in microbial populations. In particular, the conversion from ICE to plasmid allows ICE to be more plastic, while the conversion from plasmid to ICE allows the expansion of the element's host range.
Asunto(s)
Conjugación Genética , Secuencias Repetitivas Esparcidas , Plásmidos , Proteobacteria/genéticaRESUMEN
AcCNET (Accessory genome Constellation Network) is a Perl application that aims to compare accessory genomes of a large number of genomic units, both at qualitative and quantitative levels. Using the proteomes extracted from the analysed genomes, AcCNET creates a bipartite network compatible with standard network analysis platforms. AcCNET allows merging phylogenetic and functional information about the concerned genomes, thus improving the capability of current methods of network analysis. The AcCNET bipartite network opens a new perspective to explore the pangenome of bacterial species, focusing on the accessory genome behind the idiosyncrasy of a particular strain and/or population. AVAILABILITY AND IMPLEMENTATION: AcCNET is available under GNU General Public License version 3.0 (GPLv3) from http://sourceforge.net/projects/accnet CONTACT: : valfernandez.vf@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Genoma Bacteriano , Genómica/métodos , Metagenoma , Filogenia , Programas Informáticos , Bacterias/genética , ProteomaRESUMEN
SUMMARY: PLACNET is a graph-based tool for reconstruction of plasmids from next generation sequence pair-end datasets. PLACNET graphs contain two types of nodes (assembled contigs and reference genomes) and two types of edges (scaffold links and homology to references). Manual pruning of the graphs is a necessary requirement in PLACNET, but this is difficult for users without solid bioinformatic background. PLACNETw, a webtool based on PLACNET, provides an interactive graphic interface, automates BLAST searches, and extracts the relevant information for decision making. It allows a user with domain expertise to visualize the scaffold graphs and related information of contigs as well as reference sequences, so that the pruning operations can be done interactively from a personal computer without the need for additional tools. After successful pruning, each plasmid becomes a separate connected component subgraph. The resulting data are automatically downloaded by the user. AVAILABILITY AND IMPLEMENTATION: PLACNETw is freely available at https://castillo.dicom.unican.es/upload/. CONTACT: delacruz@unican.es. SUPPLEMENTARY INFORMATION: A tutorial video and several solved examples are available at https://castillo.dicom.unican.es/placnetw_video/ and https://castillo.dicom.unican.es/examples/.
Asunto(s)
Bacterias/genética , Transferencia de Gen Horizontal , Genoma Bacteriano , Plásmidos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Genómica/métodos , InternetRESUMEN
All plasmids that spread by conjugative transfer encode a relaxase. That includes plasmids that encode the type IV secretion machinery necessary to mediate cell to cell transfer, as well as mobilizable plasmids that exploit the existence of other plasmids' type IV secretion machinery to enable their own lateral spread. Relaxases perform key functions in plasmid transfer by first binding to their cognate plasmid as part of a multiprotein complex called the relaxosome, which is then specifically recognized by a receptor protein at the opening of the secretion channel. Relaxases catalyze a site- and DNA-strand-specific cleavage reaction on the plasmid then pilot the single strand of plasmid DNA through the membrane-spanning type IV secretion channel as a nucleoprotein complex. In the recipient cell, relaxases help terminate the transfer process efficiently and stabilize the incoming plasmid DNA. Here, we review the well-studied MOBF family of relaxases to describe the biochemistry of these versatile enzymes and integrate current knowledge into a mechanistic model of plasmid transfer in Gram-negative bacteria.
Asunto(s)
Bacterias Gramnegativas , Proteínas Bacterianas , Conjugación Genética , ADN Bacteriano , PlásmidosRESUMEN
BACKGROUND: Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to find genetic or metabolic conditions to increase DHA production in E. coli. RESULTS: First, we analysed the effect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations affecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most effective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is affected in its first catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA. CONCLUSIONS: DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in different organisms.
Asunto(s)
Alanina/análogos & derivados , Aminoácidos/antagonistas & inhibidores , Ácidos Borónicos/antagonistas & inhibidores , Cerulenina/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Organofosfonatos/metabolismo , Alanina/metabolismo , Expresión GénicaRESUMEN
Conjugative transfer of the broad-host-range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC-nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC-nic operon. A 7-bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3 was delineated to a 47-bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM . This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.
Asunto(s)
Endodesoxirribonucleasas/genética , Plásmidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Conjugación Genética , ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Transferencia de Gen , Transferencia de Gen Horizontal , Operón , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Conjugación Genética/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Ácidos Grasos Insaturados/farmacología , Ácido Linoleico/farmacología , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/química , Ácidos Grasos Insaturados/síntesis química , Cinética , Simulación del Acoplamiento Molecular , PlásmidosRESUMEN
Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening-closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.
Asunto(s)
Conjugación Genética/genética , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Plásmidos/genética , Proteínas Represoras/genética , Sistemas de Secreción Tipo IV/genética , Adenosina Trifosfatasas/metabolismo , ADN Bacteriano/genética , Lisina/genética , Translocación Genética/genéticaRESUMEN
BACKGROUND: Rhodococcus jostii RHA1 and other actinobacteria accumulate triglycerides (TAG) under nutrient starvation. This property has an important biotechnological potential in the production of sustainable oils. RESULTS: To gain insight into the metabolic pathways involved in TAG accumulation, we analysed the transcriptome of R jostii RHA1 under nutrient-limiting conditions. We correlate these physiological conditions with significant changes in cell physiology. The main consequence was a global switch from catabolic to anabolic pathways. Interestingly, the Entner-Doudoroff (ED) pathway was upregulated in detriment of the glycolysis or pentose phosphate pathways. ED induction was independent of the carbon source (either gluconate or glucose). Some of the diacylglycerol acyltransferase genes involved in the last step of the Kennedy pathway were also upregulated. A common feature of the promoter region of most upregulated genes was the presence of a consensus binding sequence for the cAMP-dependent CRP regulator. CONCLUSION: This is the first experimental observation of an ED shift under nutrient starvation conditions. Knowledge of this switch could help in the design of metabolomic approaches to optimize carbon derivation for single cell oil production.
Asunto(s)
Redes y Vías Metabólicas , Rhodococcus/metabolismo , Triglicéridos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Ácidos Grasos/metabolismo , Perfilación de la Expresión Génica , Glucosa/metabolismo , Glucólisis , Redes y Vías Metabólicas/genética , Vía de Pentosa Fosfato/genética , Rhodococcus/genéticaRESUMEN
Horizontal gene transfer (HGT) is a major force driving bacterial evolution. Because of their ability to cross inter-species barriers, bacterial plasmids are essential agents for HGT. This ability, however, poses specific requisites on plasmid physiology, in particular the need to overcome a multilevel selection process with opposing demands. We analyzed the transcriptional network of plasmid R388, one of the most promiscuous plasmids in Proteobacteria. Transcriptional analysis by fluorescence expression profiling and quantitative PCR revealed a regulatory network controlled by six transcriptional repressors. The regulatory network relied on strong promoters, which were tightly repressed in negative feedback loops. Computational simulations and theoretical analysis indicated that this architecture would show a transcriptional burst after plasmid conjugation, linking the magnitude of the feedback gain with the intensity of the transcriptional burst. Experimental analysis showed that transcriptional overshooting occurred when the plasmid invaded a new population of susceptible cells. We propose that transcriptional overshooting allows genome rebooting after horizontal gene transfer, and might have an adaptive role in overcoming the opposing demands of multilevel selection.
Asunto(s)
Transferencia de Gen Horizontal , Plásmidos/genética , Proteobacteria/genética , Selección Genética/genética , Simulación por Computador , Evolución Molecular , Redes Reguladoras de Genes , Genoma BacterianoRESUMEN
To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA-PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria.