Characterization of the histidine quinase protein CbrA and the role of the CbrX in P. putida KT2442

Motivation: The g-proteobacteria Pseudomonas, is present in a wide ecological niches due to metabolic, physiological and genetic versatility. It bears a very high number of regulatory systems that may allow them to adapt to many environmental conditions. Within this genus, P. putida KT2440 serves as a model microorganism of biotechnological interest. One of these regulatory two-components systems, unique in the pseudomonads, is CbrAB where CbrA is a histidine kinase sensor protein and CbrB a transcriptional activator of σN-dependent promoter, many involved in the assimilation of different C sources [1,3]. In this project, we will characterise the role of CbrA in the reception of the environmental signal to activate Cbr system when there is limited in C availability. In addition, we will study the role of an open reading frame upstream and overlapping with cbrA, called cbrX, and its involvement in transcriptional/translational regulation of CbrA.Methods: A deletion mutant of cbrA and cbrX (∆cbrXA) has been constructed (MPO494) in P. putida KT2442, and the phenotypic characterization of its ability to grow in a minimal medium using different C sources (succinate, citrate, histidine, glucose) has been evaluated. Also the transcriptional activation of three different targets of the Cbr regulatory system has been studied by analysis of the β-galactosidase activity of a transcriptional fusion to the promoter regions of crcZ, crcY, PP2810 [2]. Complementation of the mutant at the Tn7 integration site with the complete sequence cbrXA, and different constructs bearing cbrA or cbrX have also been constructed and their phenotypes analysed. Finally, a truncated form of CbrA expressed from heterologous Ptac promoter, which lacks 13 transmembrane domains, that is presumably not anchored to the inner membrane has also been constructed.Results: The ∆cbrXA deletion mutant MPO494 shows a longer lag phase when growing in succinate and glucose as C source, and even longer when growing on citrate medium, when compared to the wild-type strain KT2442. MPO494 is not able to use histidine as C source. Complementation of the MPO494 with cbrXA sequence fully recovers the wild-type phenotype. The activation of crcZ, crcY and PP2810 genes is 26 to 20 fold lower in a medium containing succinate or oxalacetate as C source in a mutant background compared to the KT2442, but it is fully complemented when the cbrXA sequence is supported in trans. The effects of cbrX on the CbrA expression/activity is currently being analysed.

A new element involved in the regulation of tetralin degradation genes in Sphingopyxis granuli strain TFA

Motivation: Sphingopyxis granuli strain TFA is a gram-negative bacterium able to grow on the organic solvent tetralin as the sole carbon and energy source. Tetralin is a bicyclic molecule, composed of an aromatic and an alicyclic moiety, which is toxic to bacterial cells as it makes the membrane permeable for ions (protons) and inhibits the respiratory enzymes (Sikkema et al., 1992). In our lab, the metabolic pathway and the specific regulation of genes involved on tetralin degradation (thn genes) has been deeply characterized (López-Sánchez et al., 2010 and references therein, Rivas-Marín et al., 2016 and references therein). Regarding the regulation, it is known that structural thn genes are induced in the presence of tetralin by ThnR, a LysR-like transcriptional regulator, and ThnY, a ThnR co-activator. Besides, the expression of thn genes is under carbon catabolite repression (CCR) by preferential carbon sources, such as β-hydroxybutyrate (β-HB) or sebacic acid. However, not very much is known about the regulatory elements involved in this repression. Synthesis of the carbon storage granule PHB is indirectly involved in CCR on thn genes.Methods: Comparison of the global gene expression in tetralin- and β-HB-grown cells revealed the presence of a small non-coding RNA (sRNA), annotated by Infernal Software 1.1, preferentially expressed in β-HB. Northern Blot analysis and β-galactosidase assays of a chromosomally integrated suhB::lacZ transcriptional fusion confirmed the differential expression of this sRNA. Expression of thn genes under CCR conditions in a mutant lacking the sRNA was evaluated using a chromosomally integrated thnC::lacZ translational fusion. Furthermore, putative targets of the sRNA were detected in vitro by IntaRNA software and the predicted interaction was experimentally validated by RNA-RNA EMSA.Results: A differentially expressed sRNA has been identified in TFA as belonging to the Rfam family RF00519 (SuhB) (García-Romero et al., 2016). It is a highly conserved sRNA in α-proteobacteria. Under CCR conditions, thn genes are partially de-repressed in a mutant lacking SuhB. Furthermore, the 5' UTR of thnR mRNA has been identified in silico as a target of SuhB. Direct interaction of SuhB at the thnR ribosomal binding site has been demonstrated. The high level of ThnR in the SuhB mutant indicates a negative role of SuhB on ThnR translation.Conclusions: The available data so far indicate that SuhB is one of the elements involved in CCR of thn genes in Sphingopyxis granuli strain TFA, by blocking the translation of the regulator ThnR.    

A new generation of vectors with increased induction ratios by overimposing a second regulatory level by attenuation

A major drawback of regulated gene expression from vectors bearing strong promoters is the associated high basal expression level. Simple regulatory systems have an intrinsic limitation in the range of induction, and attempts to mutate promoters to reduce basal expression usually result in concomitant reduction of induced levels. We have explored the possibility of reducing basal levels of gene expression while keeping induced levels intact by incorporating an additional regulatory circuit controlling a different step of the expression process. We have integrated the nasFEDCBA transcriptional attenuation system of Klebsiella oxytoca into a cascade expression circuit based on different regulatory elements of Pseudomonas putida, and also into a system based on the tac promoter, to expand their regulatory capacity. Basal expression from the promoters of these circuits was reduced by more than 10-fold by the nasF attenuator sequence while keeping the induced levels intact in the presence of the antiterminator protein, thus increasing the induction ratio by up to 1700-fold. In addition, using different combinations of regulatory elements and inducing conditions, we were able to obtain a broad range of expression levels. These vectors and the concept of their design will be very useful in regulating overproduction of heterologous proteins both at laboratory and industrial scales

Regulation of gene expression and control of protein synthesis in different biotechnological process: Theory and Reality

The impressive biodiversity of microorganisms on the Earth represents an endless source of genetic elements that can be rationally combined by synthetic biology to solve different biotechnological issues.In our laboratory, we have contributed to the development of a protein expression system, which responds to permissive concentrations of different inducers such as salicylate, acetyl salicylic acid or 3-methyl benzoate. The system involves two transcriptional regulators working in cascade, whereas the first one controls the expression of the second that finally activates the expression of heterologous genes cloned under the control of an inducible promoter. In addition to conventional processes of bioproduction developed in bioreactors, this cascade system has been adapted to its use in bacteria inside higher organisms.The study of the interactions between micro-organisms and their hosts presents certain limitations during the infection process as bacterial tracking inside higher organisms. The restricted number of tools that allow the control of genes involved in bacteria-host interactions hampers the ability to activate or inactivate these genes at the time or place desired during the course of the infection. These elements come from different bacteria, and have shown their effectiveness in the production of heterologous proteins in various organisms pathogenic for animals (Salmonella) or plant symbionts (Sinorhizobium). One of the interesting features of this system is that inducers freely diffuse between the different tissues of the host without toxicity at tested concentrations. This feature in combination with a good system to monitoring the infection process in vivo, allows to switch on the expression of genes of interest at the desired “time and place”, what could help in the understanding of its role during the infection process

Development of Genetic Tools for the Manipulation of the Planctomycetes

Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system. Studies of the cellular and molecular biology of these infrequent characteristics are currently limited due to the lack of genetic tools for most of the species. So far, genetic manipulation in Planctomycetes has been described in Planctopirus limnophila only. Here, we show a simple approach that allows mutagenesis by homologous recombination in three different planctomycetes species (i.e., Gemmata obscuriglobus, Gimesia maris, and Blastopirellula marina), in addition to P. limnophila, thus extending the repertoire of genetically modifiable organisms in this superphylum. Although the Planctomycetes show high resistance to most antibiotics, we have used kanamycin resistance genes in G. obscuriglobus, P. limnophila, and G. maris, and tetracycline resistance genes in B. marina, as markers for mutant selection. In all cases, plasmids were introduced in the strains by mating or electroporation, and the genetic modification was verified by Southern Blotting analysis. In addition, we show that the green fluorescent protein (gfp) is expressed in all four backgrounds from an Escherichia coli promoter. The genetic manipulation achievement in four phylogenetically diverse planctomycetes will enable molecular studies in these strains, and opens the door to developing genetic approaches not only in other planctomycetes but also other species of the superphylum, such as the Lentisphaerae.ER-M and DPD are supported by the Spanish Ministry of Economy and Competitivity (Grant BFU2013-40866-P) and the Junta de Andalucía (CEIC Grant C2A program to DPD). IC and ES are supported by the Spanish Ministry of Economy and Competitivity (Grant BIO2014-57545-R).Peer reviewedPeer Reviewe

The CBRB regulon: Promoter dissection reveals novel insights into the CbrAB expression network in Pseudomonas putida

CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed in vivo binding analysis of CbrB in P. putida and determined that it directly controls the expression of at least 61 genes; 20% involved in regulatory functions, including the previously identified CrcZ and CrcY small regulatory RNAs. The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN—depen-dent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multi-merisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.Ministerio de Economía y Competitividad BIO2014-57545-

Engineered Salmonella allows real-time heterologous gene expression monitoring within infected zebrafish embryos

Short communication.Microbial host–pathogen interactions have been traditionally well studied at genetic and physiological levels, but cell-resolution analyses have been particularly scarce. This has been especially remarkable for intracellular parasites for two major reasons: first, the inherent loss of bacteria traceability once infects its hosts; second and more important, the limited availability of genetic tools that allow a tight regulated expression of bacterial virulence genes once inside the host tissues. Here we present novel data supporting the use of zebrafish embryos to monitor Salmonella enterica serovar Thyphimurium infection. Intravenous infection of Salmonella can be easily monitored using in vivo fluorescence that allows the visualization of free-swimming bacteria through the circulatory system. Moreover, we have engineered Salmonella to voluntarily activate heterologous gene expression at any point during infection once inside the zebrafish macrophages using a salicylate-based expression system. This approach allows real-time cell-resolution in vivo monitoring of the infection. All together, this approach paves the road to cell-based resolution experiments that would be harder to mimic in other vertebrate infection models.his work was funded by grants BFU2010-14839, CSD2007-00008, CSD2007-00005 and Proyectos de Excelencia CVI-3488 and P07-CVI-02518 from the Spanish and Andalusian Governments, respectively. Jose Luis Royo holds a JAE DOC contract from the Spanish National Research Council (CSIC).Peer reviewe

Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB

This is an open access article under the terms of the Creative Commons Attribution License.Rhodococcus sp. strain TFB is a metabolic versatile bacterium able to grow on naphthalene as the only carbon and energy source. Applying proteomic, genetic and biochemical approaches, we propose in this paper that, at least, three coordinated but independently regulated set of genes are combined to degrade naphthalene in TFB. First, proteins involved in tetralin degradation are also induced by naphthalene and may carry out its conversion to salicylaldehyde. This is the only part of the naphthalene degradation pathway showing glucose catabolite repression. Second, a salicylaldehyde dehydrogenase activity that converts salicylaldehyde to salicylate is detected in naphthalene-grown cells but not in tetralin- or salicylate-grown cells. Finally, we describe the chromosomally located nag genes, encoding the gentisate pathway for salicylate conversion into fumarate and pyruvate, which are only induced by salicylate and not by naphthalene. This work shows how biodegradation pathways in Rhodococcus sp. strain TFB could be assembled using elements from different pathways mainly because of the laxity of the regulatory systems and the broad specificity of the catabolic enzymes.Work in the authors laboratory was supported by the Spanish Ministry of Economy and Competitivity, grants BIO2011-24003 and CSD2007-00005, and by the Andalusian Government, grants P05-CVI-131 and P07-CVI-2518.Peer Reviewe

Caracterización de la proteína Hfq en la bacteria TFA

Las bacterias detectan diferentes señales procedentes del entorno y responden a ellas específicamente variando sus perfiles de expresión génica con el objetivo de adaptarse mejor a los cambios ambientales. Dichos mecanismos de regulación de la expresión génica pueden operar tanto a nivel transcripcional como a nivel post-transcripcional y responden a señales específicas y/o globales para asegurar una correcta expresión génica. Esta regulación post-transcripcional de la expresión génica puede estar mediada por sRNAs (smallRNAs en inglés). Estos sRNAs tienen un papel crucial en el control de las funciones celulares tales como, por ejemplo, el metabolismo central o la virulencia en algunas bacterias patógenas (1).Se ha descrito que la proteína Hfq juega un papel esencial en la actividad de algunos de estos sRNAs con los que interacciona en numerosas bacterias (1, 2). Hfq fue descrita por primera vez en la bacteria E. coli como un factor esencial para la replicación del fago Qβ, un virus de ARN (3). Hfq se engloba dentro de la familia de proteínas Sm-Sm_like que se caracteriza por tener una estructura cuaternaria en forma de anillo hexamérico que le permite la interacción con otras macromoléculas, como serían los sRNAs y los ARN mensajeros diana de los anteriores (1, 2). La bacteria Sphingopyxis macrogolitabida estirpe TFA es una alfa-proteobacteria perteneciente a la familia Sphingomonadaceae capaz de degradar el contaminante ambiental tetralina y utilizarlo como fuente de carbono y energía (4). En dicha bacteria, objeto de estudio en este trabajo, se ha detectado un gen que codifica para una proteína reconocible como hfq pero con una estructura primaria diferente a las descritas hasta el momento en otras bacterias. estas diferencias encontradas a nivel estructural en la proteína junto al hecho de que no haya estudios previos sobre esta proteína en esta familia de bacterias hacen muy interesante su caracterización con objeto de determinar si su mecanismo de actuación en la célula es diferente o no al descrito previamente en otras bacterias