Negative feedback increases information transmission, enabling bacteria to discriminate sublethal antibiotic concentrations

In the cell, noise constrains information transmission through signaling pathways and regulatory networks. There is growing evidence that the channel capacity of cellular pathways is limited to a few bits, questioning whether cells quantify external stimuli or rely on threshold detection and binary on/off decisions. Here, using fluorescence microscopy and information theory, we analyzed the ability of the transcriptional regulator TetR to sense and quantify the antibiotic tetracycline. The results showed that noise filtering by negative feedback increased information transmission up to 2 bits, generating a graded response able to discriminate different antibiotic concentrations. This response matched the antibiotic subinhibitory selection window, suggesting that information transmission through TetR is optimized to quantify sublethal antibiotic levels. Noise filtering by negative feedback may thus boost the discriminative power of cellular sensors, enabling signal quantification.This work was funded by grants BFU2017-86378-P and BFU2014-62190-EXP from the Spanish MINECO and by the European Seventh Framework Program [projects 612146/FP7-ICT-2013-10 (PLASWIRES) and 289326/KBBE-2011-5 (ST-FLOW)]

Conjugation Inhibitors and Their Potential Use to Prevent Dissemination of Antibiotic Resistance Genes in Bacteria

Antibiotic resistance has become one of the most challenging problems in health care. Bacteria conjugation is one of the main mechanisms whereby bacteria become resistant to antibiotics. Therefore, the search for specific conjugation inhibitors (COINs) is of interest in the fight against the spread of antibiotic resistances in a variety of laboratory and natural environments. Several compounds, discovered as COINs, are promising candidates in the fight against plasmid dissemination. In this review, we survey the effectiveness and toxicity of the most relevant compounds. Particular emphasis has been placed on unsaturated fatty acid derivatives, as they have been shown to be efficient in preventing plasmid invasiveness in bacterial populations. Biochemical and structural studies have provided insights concerning their potential molecular targets and inhibitory mechanisms. These findings open a new avenue in the search of new and more effective synthetic inhibitors. In this pursuit, the use of structure-based drug design methods will be of great importance for the screening of ligands and binding sites of putative targets.

Cis-Acting Relaxases Guarantee Independent Mobilization of MOBQ4 Plasmids

Plasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. In this work, we studied a group of plasmids from enterobacteria that encode phylogenetically related mobilization functions that populate the previously non-described MOBQ4 relaxase family. These plasmids encode two transfer genes: mobA coding for the MOBQ4 relaxase; and mobC, which is non-essential but enhances the plasmid mobilization frequency. The origin of transfer is located between these two divergently transcribed mob genes. We found that MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative dissemination among clinically relevant enterobacteria. While highly similar in their mobilization module, two sub-groups with unrelated replicons (Rep_3 and ColE2) can be distinguished in this plasmid family. These subgroups can stably coexist (are compatible) and transfer independently, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such a strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.Funding: This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2017-86378-P, AEI/FEDER, UE, to FC) and Consejo Superior de Investigaciones Científicas (201820I143 to MG-B). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

A high security double lock and key mechanism in HUH relaxases controls oriT-processing for plasmid conjugation

Relaxases act as DNA selection sieves in conjugative plasmid transfer. Most plasmid relaxases belong to the HUH endonuclease family. TrwC, the relaxase of plasmid R388, is the prototype of the HUH relaxase family, which also includes TraI of plasmid F. In this article we demonstrate that TrwC processes its target nic-site by means of a highly secure double lock and key mechanism. It is controlled both by TrwC-DNA intermolecular interactions and by intramolecular DNA interactions between several nic nucleotides. The sequence specificity map of the interaction between TrwC and DNA was determined by systematic mutagenesis using degenerate oligonucleotide libraries. The specificity map reveals the minimal nic sequence requirements for R388-based conjugation. Some nic-site sequence variants were still able to form the U-turn shape at the nic-site necessary for TrwC processing, as observed by X-ray crystallography. Moreover, purified TrwC relaxase effectively cleaved ssDNA as well as dsDNA substrates containing these mutant sequences. Since TrwC is able to catalyze DNA integration in a nic-site-containing DNA molecule, characterization of nic-site functionally active sequence variants should improve the search quality of potential target sequences for relaxase-mediated integration in any target genome

Negative feedback and transcriptional overshooting in a regulatory network for horizontal gene transfer

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

Key components of the eight classes of type IV secretion systems involved in bacterial conjugation or protein secretion

Conjugation of DNA through a type IV secretion system (T4SS) drives horizontal gene transfer. Yet little is known on the diversity of these nanomachines. We previously found that T4SS can be divided in eight classes based on the phylogeny of the only ubiquitous protein of T4SS (VirB4). Here, we use an ab initio approach to identify protein families systematically and specifically associated with VirB4 in each class. We built profiles for these proteins and used them to scan 2262 genomes for the presence of T4SS. Our analysis led to the identification of thousands of occurrences of 116 protein families for a total of 1623 T4SS. Importantly, we could identify almost always in our profiles the essential genes of well-studied T4SS. This allowed us to build a database with the largest number of T4SS described to date. Using profile-profile alignments, we reveal many new cases of homology between components of distant classes of T4SS. We mapped these similarities on the T4SS phylogenetic tree and thus obtained the patterns of acquisition and loss of these protein families in the history of T4SS. The identification of the key VirB4-associated proteins paves the way toward experimental analysis of poorly characterized T4SS classes.Funding. Spanish Ministry of Economy [BFU2011-26608]; European Seventh Framework Program [282004/FP7-HEALTH.2011, 612146/FP7-ICT-2013]; European Research Council Grant [EVOMOBILOME no. 281605]. Source of open access funding: European Research Council grant to the PI

Plasmid conjugation from Proteobacteria as evidence for the origin of xenologous genes in Cyanobacteria

Comparative genomics showed that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of inter-phylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64 and F) to transfer DNA from E. coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host range plasmids, such as RP4 and R388, but also narrower host-range plasmids, such as pKM101, all encoding MPFT-type IV secretion systems, were able to transfer plasmid DNA from E. coli to S. elongatus by conjugation. Neither MPFF, nor MPFI could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in E. coli by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between Proteobacteria and Cyanobacteria and point to MPFT-based inter-phylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of S. elongatus xenologous genes

Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPaseTrwD, providing a mechanism to inhibit bacterial conjugation

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.This work was supported by Spanish Ministerio de Economia y Competitividad (MINECO) Grants BFU2016-78521-R (to E. C. and I. A.) and BFU2014-55534 (to F. d. l. C.) and by Grant P20GM103475-16 from the National Center for Research Resources and NIGMS, National Institutes of Health (to D. S. R.). The authors declare that they have no conflicts of interest withthe contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Triacilglicerol sintasas termoestables y usos de las mismas

ABSTRACT: The invention concerns: an isolated polynucleotide having at least 75% homology with SEQ ID NO. 1; a polypeptide coded by the above polynucleotide and having triacylglycerol synthase activity; a vector comprising the above polynucleotide; a host cell transformed by said vector; a method for obtaining TAGs based on the expression of the polypeptide coded by the above polynucleotide by culture of the above transformed host cell in a medium comprising industrial waste or residues as carbon source; and the use of the TAGs obtained by this method as biofuel or as starter material for obtaining biofuel.RESUMEN: Polinucleótido aislado que presenta al menos un 75% de homología con la secuencia SEQ ID NO 1. Polipéptido codificado por el polinucleótido anterior, que presenta actividad triacilglicerol sintasa. Vector que comprende el polinucleótido anterior. Célula hospedadora transformada con dicho vector. Método para la obtención de TAGs basado en la expresión del polipéptido codificado por el polinucleótido anterior mediante el cultivo de la célula hospedadora transformada anterior en un medio que comprende residuos o desechos industriales como fuente de carbono. Empleo de los TAGs obtenidos en dicho método como biocombustible o como material d partida para la obtención de biocombustible.Solicitud: PCT/ES2013/000211 (23.09.2013)Nº de Publicación: WO2014/049180A1 (03.04.2014

Análisis automático de la posición y del número de plásmidos en una célula bacteriana

Plasmids are small chromosomes with the ability to pass from one cell to another. Recent advances in the study of its behavior shows that one of the main ways to assure its long-term survival is to adapt themselves to the cellular division mechanism of the host cell, assuring that at least one copy of the plasmid remains on each of the replicas generated in the cell-division process. Usually these studies have been made by hand by analyzing the images of a microscope and measuring the distance between each plasmid and the centre of the cell in which it is immersed. However, this manual process is slow and tedious, and reduces the applicability of the method to large image data sets. This communication shows an automated tool for cell segmentation based on a pre-emphasis filtering and parametric curve fitting that is able to accurately segment cells in microscopic images, count the plasmids inside it and extract its positions inside the cell