Characterization of the anaerobic metabolism of Sphingopyxis macrogoltabida TFA strain

Motivation: Sphingopyxis macrogoltabida TFA strain is an alphaproteobacteria which is able to degrade the contaminant compound tetralin, found in multiple substances such as coal tar and petroleum (1). Tetralin biodegradation has been characterized under aerobic conditions, however no anaerobic growth has ever been observed in Sphingomonadaceae family. Many nitrate-respiring bacteria are able to transform nitrate to nitrite using nar genes, and eventually to gas nitrogen in a process called denitrification (2). Computer analysis of the genome sequence of TFA has shown different genes whose encoded proteins might be involved in anaerobic respiration pathways in TFA: three fixLJ pairs of genes, an fnrL gene and a fixK gene. It would be interesting to determine whether TFA is able to grow with no oxygen and to characterize this growth. Therefore, the objectives of this project are the characterization of the anaerobic metabolism of TFA and to determine growth rate under anaerobic conditions.Aims: In order to determine whether TFA is able to grow under anaerobic conditions we will use nitrate, nitrite, fumarate and DMSO as final electron acceptors at different concentrations. We will design two insertion mutants in fnrL and fixK genes in order to test their anaerobic growth. The phenotype of some fixLJ mutants will be determined.Results: We have successfully observed anaerobic growth of TFA reaching OD600 of 0.9 with some terminal electron acceptors. No anaerobic growth was observed using nitrite, fumarate or DMSO as sole electron acceptors. None of the mutants in fixLJ1 and fixLJ2 showed significant differences in their growth compared to the wild type strain

Characterization of flagellar-specific sigma factor FliA in Sphingopyxis granuli TFA

Motivation: Sigma factors are RNA polymerase subunits which play a crucial role in the transcriptional regulation of bacterial gene expression. These dissociable proteins bind to RNA polymerase controlling promoter recognition and thus, gene expression (Francez-charlot et al., 2015). Previous studies in Alphaproteobacteria identified a sigma factor FliA as a key transcriptional regulator of chemotaxis and flagella biogenesis by controlling the expression of genes involved in flagellin biosynthesis, an essential structural protein which forms flagella filament (Maruyama et al., 2015). Sphingopyxis granuli TFA is a Gram-Negative Alphaproteobacteria very interesting since is one of the few strains able to grow on the organic solvent tetralin as a sole carbon and energy source and able to grow respiring nitrate under anaerobic conditions (García-Romero et al., 2016). The aim of this study is to construct and phenotypically characterize a fliA deletion mutant that help us to elucidate the function of FliA sigma factor and thus, the hierarchy of transcriptional regulation of flagellar genes, in Sphingopyxis granuli TFA.Methods: A fliA deletion mutant is being constructed using a DNA-recombination method based on a double-strand break caused by SceI nuclease. Firstly, flanking regions of fliA gene must be cloned in a multiple cloning site (MCS) of a non-replicative vector. At the same time, this MCS is flanked by two SceI target sites. Once this integrative vector is integrated into the chromosome and selected in wild-type TFA, a broad host range vector including SceI gene downstream of an inducible promoter must be introduced. A double-strand break is caused in the chromosome after the induction of SceI nuclease by 3-methylbenzoate and DNA will be repaired causing FliA gene deletion.Once fliA deletion mutant is constructed, a phenotypic characterization will be performed. In addition, both semi-quantitative PCR and Q-PCR assays will be used in order to confirm which genes are regulated by FliA in Sphingopyxis granuli TFA

Role of the ECF sigma factor EcfG in the general stress response of Sphingopyxis granuli TFA

Motivation: One of the main mechanisms bacteria use to face ever-changing environmental conditions is the swapping of sigma factors. These are small proteins which direct the core RNA polymerase in its target promoter recognition (Staron et al., 2009). On the other hand, Sphingopyxis granuli TFA is an alphaproteobacterium capable of growing using the organic solvent tetralin (1,2,3,4-tetrahydronaphtalene) as a sole carbon source (Moreno-Ruiz et al., 2003). Preliminar RNA-seq data showed that the alternative sigma factor EcfG, a homolog of the general stress response master regulator in alphaproteobacteria, is strongly overexpressed in the presence of tetralin, among other conditions. In this project, an ecfG deletion mutant was constructed and its phenotype partially characterized, regarding its capability to respond to a variety of stresses and to activate the expression of tetralin degradation genes.Methods: The ecfG deletion mutant was constructed using molecular biology techniques and adapting the SceI-based genomes editing platform to TFA (Martínez-García & de Lorenzo, 2011). This method consists of two plasmids: one with a narrow host range origin of replication and a MCS flanked by two SceI targets and other one with a broad host range origin of replication and the SceI nuclease ORF downstream an inducible promoter. Flanking regions of the target gene must be cloned within the MCS of the first plasmid and its integration in the chromosome, selected. Upon introduction of the second plasmid and induction of the nuclease, a double-strand break is caused in the chromosome. The final repair of this break results with a high frequency in the deletion of the target gene (ecfG in our case).A bioinformatic analysis of TFA promoter sequences was performed in order to search for putative EcfG-regulated genes and thus, testable phenotypes.Growth inhibition assays were performed in the presence of different stress agents and different culture media, either in liquid culture or disk diffusion assays. EcfG and tetralin degradation genes expression was measured by means of β-galactosidase activity assays using a lacZ translational fusion to ecfG and thnC promoters.Results: The ecfG deletion mutant was not affected either in the ability to use tetralin as carbon source nor in the capability to activate the expression of the tetralin degradation genes compared to the wild type.The mutant showed a slightly increased sensitivity to certain oxidative agents and antibiotics. However, it did not show a different behavior from the wild type strain in the presence of other compounds predicted to trigger the general stress response in this bacterium, according to the bioinformatic analysis, such as copper and fusaric acid.In the case of its expression pattern, the ecfG mutant showed different activity levels compared with the wild type in all the conditions tested, such as presence of tetralin, stationary phase and anaerobiosis

Entrevista a Miguel Arévalo de Biomedal S.L.

Biomedal es una empresa biotecnológica que dedica su actividad al desarrollo y comercialización de productos, servicios y tecnologías de especial interés en la investigación, el diagnóstico y los procesos industriales.Miguel Arévalo Rodríguez, director de una parte del desarrollo y la investigación en Biomedal, nos explica muy amablemente su experiencia dentro de la empresa y su punto de vista sobre el emprendimiento

Elaboración de un producto biofertilizante y biocontrol con bacterias promotoras del crecimiento vegetal (PGPRs) del género Azospirillum.

Desde hace tiempo,  se habla de la necesidad de que la producción agrícola a nivel global, se convierta en una agricultura sostenible, necesaria para poder abastecer a toda la población. Actualmente el uso de bacterias PGPRs (del inglés, plant growth promoting rhizobacteria) se percibe como una de las herramientas más importantes para la consecución de una agricultura sostenible así como para la recuperación de ecosistemas degradados. Las bacterias PGPRs comprenden un grupo de microorganismos que ejercen un efecto beneficioso sobre las plantas debido a su capacidad de colonizar la superficie de la raíz, la rizosfera , la filosfera y el tejido interno de la planta.  Pueden estimular el crecimiento vegetal por medio de varios procesos, incluyendo : fijación biológica de nitrógeno, producción de sideróforos, solubilización de fosfatos, producción de reguladores del crecimiento vegetal (síntesis de hormonas como auxinas, citoquininas, etileno, giberelinas) y ejercer de control biológico de patógenos. Una de las aplicaciones de estas bacterias entre otras es, actuar como biofertilizantes, agentes biocontrol, así como agentes de inducción de resistencia sistémica a enfermedades. Las bacterias del género Azospirillum, son PGPRs,  fijan nitrógeno, producen sideróforos y producen sustancias reguladoras del crecimiento (ácido indolacético, ácido giberélico, citoquininas y vitaminas). Por esta razón y otras, este proyecto se ha centrado en dicho género, en el aislamiento de algunas especies que sean capaces de actuar como biofertilizante (bajo costo, eficaz y fuente renovable de nutrientes de las plantas ) y biocontrol. Para ello se ha desarrollado una serie de métodos, como cuantificación de aia (ácido indolacético), compatibilidad entre las distintas azospirullum, producción de sideróforos (éstos, además de mejorar la nutrición de la planta, dificulta el crecimiento de patógenos), entre otros, para obtener un producto que ayude a mejorar el rendimiento de la planta y conseguir así una agricultura sostenible

Study of the expression of ecfG1 and ecfG2, two extracytoplasmic function sigma factors (ECFs) in Sphingopyxis granuli estirpe TFA

Motivation: In bacteria, the initiation of transcription requires a specific multi-domain subunits of RNA polymerase (RNAP) called sigma (σ) factors that binds to its core that play critical roles, including the recognition and opening of promoters for the RNA synthesis (Paget, 2015). One type of sigma factors are extracytoplasmic function sigma factors (ECF) which provide a means of regulating gene expression in response to a wide range of environmental changes (Feklistov et al., 2014). Sphingopyxis granuli TFA is a Gram-negative Alphaproteobacteria that is one of the few strains able to grow on the organic solvent tetralin as a sole carbon and energy source and able to grow respiring nitrate under anaerobic conditions (Gonzalez-Flores et al., 2016). In Sphingopyxis granuli TFA two ECF σ factors have been described, EcfG1 and EcfG2, that have a critcal biological role in the General Stress Response (GSR) in this bacterium (de Dios et al., 2020)Methods: With the aim of studying the transcriptional and postranscriptional regulation of each ecfG genes, a recombinant protein was built in which each EcfG have a FLAG-tag fused which allowed us to quantified the amount of each sigma factor by Western Blot studies in different growth conditions. This protein was constructed using a DNA-recombination method based on a double-strand break caused by SceI nuclease. Flanking regions of each ecfG genes were cloned in a multiple cloning site (MCS) of a non-replicative vector, this MCS is flanked by two SceI target sites. When this integrative vector is integrated into the chromosome of TFA, a broad host range vector including SceI gene downstream of an inducible promoter must be introduced and a double-strand break is caused in the chromosome. The final repair of this break results with a high frequency in the deletion of the target gene (ecfG). Thus we only get the gene with the FLAG-tagResults: In this work we analyzed the levels of expression of each sigma factors both at the transcriptional by RNA-seq and at translational level by quantifying the levels of each protein through Western Blot studies under different growth condition

Entrevista Gennova Scientific S.L.

Gennova Scientific S.L. es una empresa internacional de biotecnología ubicada en San José de la Rinconada (Sevilla). Enmarcada en el ámbito del diagnóstico in vitro, fue constituida a principios de 2009 con el objetivo de desarrollar técnicas y productos de laboratorio que pudieran aplicarse en análisis clínicos, biológicos y patológicos, tanto a nivel de investigación como en la producción a gran escala y el comercio internacional. Actualmente, la empresa cuenta con más de quince especialistas en biomedicina, bioquímica, biotecnología, medio ambiente, marketing y comercio exterior..

Caracterización fisiológica, molecular y genética de una cepa de Magnetospirillum aislada de un bioreactor con tolueno.

Motivación: El tolueno está presente en la gasolina en un 5 a 7% por lo que es común encontrarlo en aguas subterráneas contaminadas por derrames de gasolina. El tolueno está catalogado como uno de los principales contaminantes debido a que  se ha comprobado que deprime el sistema nervioso central y potencia carcinogénesis en la piel (2). Debido a su poder contaminante se han estudiado distintas medidas para degradarlo in/ex situ mediante biorremediación. En este trabajo se propuso caracterizar a nivel fisiológico y genético una cepa de Magnetospirillum aislada de un reactor y evaluar la degradación de tolueno a partir de ésta. Métodos: Para cultivar la cepa, así como para ensayar la degradación de ciertos compuestos aromáticos como tolueno y benzoato de sodio y evaluar el crecimiento bacteriano en distintas fuentes de carbono (etanol y glucosa) se utilizó el medio Shinoda como base (1). Para todos los casos, el medio Shinoda se ajustó a pH 7.6, la incubación se realizó a 30°C y en agitación constante. A excepción del tolueno todos los ensayos, se llevaron a cabo en condiciones de aerobiosis y anaerobiosis. De los ensayos degradación de tolueno y benzoato, se evaluó la degradación mediante un cromatografo de gases y HPLC respectivamente. El crecimiento se midió en un espectrofotometro a 610 nm. Paralelamente, se procedió a identificar los genes implicados en los procesos de degradación (bss y bcr) y construir un árbol filogenético a partir de ésta información. Resultados: Durante 128 días, se evaluó el crecimiento de Magnetospirillum y degradación de tolueno. A lo largo de los primeros 38 días, no se observó crecimiento significativo de la bacteria, ni degradación significativa de tolueno manteniendose siempre alrededor de 45 mg/L. Sin embargo, después de 128 días, la concentración de tolueno era de 25,25 mg/L, degradandose casi la mitad de la concentración inicial. En los ensayos de degradación de benzoato, se observó una degradación de benzoato y crecimiento de la bacteria significante creciendo tanto en condiciones de anaerobiosis como aerobiosis. A partir  de los analisis filogenéticos se determinó que la cepa estaba emparentada a la especie M. magnetotacticum. Conclusiones: En este trabajo se aisló desde un microcosmos una cepa de Magnetospirillum candidata para degradar compuestos tóxicos como tolueno entre otros, ideal para el tratamiento de suelos contaminados tanto en tecnicas in situ como ex situ

Involvement of sensor histidine kinases in general stress response of Sphingopyxis granuli strain TFA.

Motivation: In their natural habitat, bacteria face constantly changing conditions that can portraiy a challenge to their survival. To overcome this problem, bacteria are able to react to their medium and adapt accordingly to it. One of these responses is the so-called General Stress Response (GSR), an unspecific response to a myriad of different stress signals.Sphingopyxis granuli strain TFA is an alphaproteobacteria with the ability to use tetralin, an organic solvent with industrial applications, as a carbon and energy source. It is also the first facultative anaerobe described within its genus. These characteristics made TFA an interesting strain from a biotechnological point of view.In alphaproteobacterial, the regulatory network of the GSR is composed at its most basic level by a sigma factor (EcfG), an anti-sigma factor (NepR), an anti-anti-sigma factor (PhyR) and a sensor histidine kinase that targets the anti-anti-sigma factor. In TFA, EcfG, NepR and PhyR are duplicated and a total of four putative sensor histidine kinases have been found within TFA genome. Previous works by de Dios et al have partially described the regulatory network of the GSR at to the point of the histidine kinases. Therefore, in this work we aim to unravel role that two of these histidine kinases, SGRAN_1655 and SGRAN_2544, have in the regulation of the GSR in TFA.Methods: The experimental approach for this work consist in the obtention of deletion mutants in the SGRAN_2544 and SGRAN_1165 by the method described by Martínez-García and de Lorenzo. Once the mutants were obtained, We examined their response to several stresses, like high sodium chloride concentration, high heavy metals concentration, hydrogen peroxide treatment and desiccation, by both checking the effect on their growth and quantifying the activity of lacZ fusions to a GSR reporter gene, such as nepR2.Results: We have been able to succesfully obtain a deletion mutant of one of the two target genes, SGRAN_2544 gene

Role of FnrS small regulatory RNA in the anaerobic response of Sphingopyxis granuli TFA.

Motivation: Sphingopyxis granuli strain TFA is a Gram-negative α-Proteobacteria, that belongs to the Sphigomonadaceae family and that was isolated from the Rhine river in Germany (Dorn, E. et al., 1974). TFA is the first of its genus which exhibits the capability to grow under anaerobic conditions using nitrate as a terminal electron acceptor (García-Romero, I. et al.,2016). Non-coding RNAs (ncRNA) are critical regulators of bacterial responses to changes in the environment and achieve refined regulation through base pairing with mRNAs, modulating their stability and/or translation. These potential ncRNAs and their interaction targets have been identified in TFA by RIL-seq. Among them, a sRNA called FnrS is of particular interest since it seems to be induced under anaerobic conditions. The aim of this project is to perform a general characterisation of this sRNA, including its regulatory mechanism and its function under anaerobic conditions.Methods: First of all, we looked into the growth alteration by microbial culturing and we measured the O.D. (600nm) by spectrophotometry. For this purpose, we compared the growth of wild-type TFA and the fnrS-depleted mutant, in the presence and absence of oxygen. In addition, the level of expression of fnrS and its Fnr dependence, a global regulator of anaerobiosis, were carried out by quantitative PCR.Results: Both variants of TFA can growth likewise, with a generation time of four hours in the presence of oxygen, and ten hours in anaerobic condition. Concerning the quantitative PCR experiment, fnrS is more expressed in anaerobiosis than in aerobiosis in wild-type TFA, whereas its expression tends to zero in the fnrS-depleted mutant. Moreover, fnrS is expressed 121-fold less in the fnr-depleted mutant than the wild-type TFA.Conclusions: The sRNA fnrS engages in anaerobic respiration but it is not essential for growth under this condition. FnrS is drastically more expressed under anaerobic condition, and its expression is conditioned by the regulatory proteins Fn