ECF sigma factor SigX: caracterization and transcriptional regulation in Pseudomonas putida.

Motivation: Extracytoplasmatic function (ECF) sigma factors play a key role in bacteria response to the environment. ECF σ factors are commonly inactivated by an anti-σ factor, which can detect an external signal and release the σ factor. Then, the activated σ factor can bind the RNA polymerase and redirect transcription toward specific response promotors. Pseudomonas putida is a ubiquitous Gram-negative bacterium capable of surviving in a broad range of natural environments which has 19 different ECF σ factors. SigX is an ECF σ factor without an identified anti-σ factor, but there is a putative upstream anti-σ factor (CfrX). In this study we made a characterization of SigX, investigating if there is read-through transcription from cfrX-cmpX unit and analysing the regulation of sigX expression from three different promoters (P1, P2 and P3), also testing if CbrAB two-component system has a control over sigX transcription.Methods: The read-through transcription of sigX has been studied by RT-PCR by amplification of the intergenic region between cmpX and sigX. Growth of a sigX insertion mutant has been studied in LB with different salt concentrations (from 0-500 mM NaCl) and in M9 medium with succinate. Expression was measured by β-galactosidase assays or by fluorescence in transcriptional fusions of the three putative promoters P1, P2 and P3 to lacZ and gfp, respectively.Results: There is read-through transcription from cfrX-cmpX unit into sigX. The growth of a SigX mutant is not affected in the absence of salt in comparison with KT2442, but the promoters P2 and P3 are downregulated in a sigX background, thus suggesting they may be activated by SigX. P1 does not respond to SigX. Also, P1, P2 and P3 seem to be activated by the CbrAB control system in a medium containing succinate as carbon source. The expression of P3 is always higher than P2 suggesting there might be a regulatory element between them.Conclusions: sigX is transcribed from its own promoter and also from an upstream promoter containing cfrX-cmpX, which may contain a regulatory element of the sigma factor. SigX is involved in the response to osmotic stress and is also controled by the CbrAB regulatory system. There is a regulatory element upstream P2 which increases the expression of sigX

Characterization of the ECF sigma factor PP_0865 of Pseudomonas putida KT2442

Motivation: Pseudomonas putida is a well characterized environmental bacterium capable of removing toxic components, including heavy metals and xenobiotics from different provenance including contaminated soils or water. Also, P. putida has been successfully shown to efficiently work as cell factories, for the synthesis of different products of biotechnological interest (1). We are interested in the characterization of an alternative extracituplasmic sigma factor (PP_0865) that may contribute to this detoxification ability of KT2442 (2). Methods: We used the following methods to characterize ECF sigma factor PP_0865:- RT-qPCR: Expression determination of PP_0865 in different mediums with different carbon availability; i.e. LB, succinate and oxaloacetate. - β- galactosidase activity: Expression determination using a transcriptional fusion of the promoter region of PP0865 to lacZ. We assayed its activity in LB medium and a minimal medium containing succinate in the wild type background and in a cbrB mutant, which is a global control regulator which may regulate the ECF expression.- Growth curve of the wild type KT2442 strains and a deletion mutant of the ECF sigma factor (MPO526) or the anti-sigma element (MPO527) in a minimal medium containing succinate as a carbon source in the presence of an excess of iron (34mM FeCl3) and in the total absence of iron (3). - Metal ions screening: We analysed the tolerance of strains KT2442, MPO526 and MPO527 to copper, zinc and cobalt in LB medium with a supplement of 2 mM CuCl2, 3 mM ZnCl2 and LB 0,6 mM CoCl2.Results:- PP_0865 seems to be overexpressed in LB compared to succinate and oxaloacetate, both by RT-qPCR and β-Galactosidase analysis. - There are no significant differences in the growth of KT2442, MPO526 or MPO527 strains in the presence or absence of iron.- Mutant strain MPO527 seems to be more tolerant to copper than KT2442 and MPO526.Conclusions: Preliminary data indicate that the ECF sigma factor system PP_0865-PP_0867 may be involved in the tolerance to metal ions such as copper

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.

Characterization of plant growth promoting bacteria isolated from red fruits. Studies on growth promotion and fruit quality in strawberries plants

Microoganisms associated to the rizosphere of cultivated plants used for human consumption are scarcely analyzed. However, nowadays, organic farming, where the use of microbial inoculants is essential, has arisen as an emergent alternative with great commercial interest. In this study, a collection of bacterial strains isolated from strawberry and blueberry rhizosphere (healthy and infected with Macrophomina phaseolina) as well as from the inside of stolons of strawberries plants (endophytic bacteria) has been constructed and characterized by their PGP and biocontrol properties. Three PGP properties have been determinate: auxin and siderophores production and phosphate solubilization. Regarding biocontrol activities, the presence of five enzymatic activities have been determined: protease, chitinase, cellulose, amylase and β-Glucosidase. On the other hand, the ability of the isolated strains to inhibit under in vitro conditions the growth of two pathogenic fungi of rump fruits, TOR 102 and TOR 872 (both belonging to the specie M. phaseolina) was tested. Strains reaching the better results were sequenced and identified as: Cupriavidius metalliduras, Bacillus proteolyticus, Arthrobacter pascens, Bacillus amyloliquefaciens, Raoultella planticola, Enterobacter roggernkampii, Bacillus megaterium, Pseudomonas multiresinivorans, Bacillus invictae, Pseudomonas aeruginosa, Chryseobacterium cucumelis, Klebsiella pneumonia, Achromobacter denitrificans, Bacillus velezenvelezensis, Burkholdelia contaminans, Bacillus niacin, Pantoea annatis, and Bacillus frigoritolerans. After that, a strawberry growth promotion assay was performed under controlled conditions. Strawberries plants were inoculated with three bacterial strains previously characterized by its high level of auxin production, namely Enterobacter rooggenkampii (AC8), Chryseobacterium cucumelis (ACH2) and Klebsiella pneumoniae (ACH7t). A greenhouse assay was carried out, with 6 replicates per treatment, including three strains as well as an uninoculated control. Biometric parameters (flowering precocity, number and weight or fruits, root and shoot dry weight), as well as quality ones (fruit size and Brix degrees) were determined at the end of the assay. Results showed that strains ACH7t was significantly superior in flowering precocity and number of fruits, while strains AC8 and ACH7t showed Brix values significantly different than the other treatments

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

Biodegradation of anti-inflammatory drugs and plastics. Identification of microbial activities by metagenomics

Motivation: The rise of plastic and its subsequent accumulation in the environment has led us to face great challenges in our society today. One of these huge challenges is to avoid plastic debris, especially microplastics as well as other pollutants such as ibuprofen, naproxen and diclofenac, from reaching our seas or rivers. Currently wastewater treatment plants (WWTP) aren´t capable of preventing this contamination [1]. Therefore, we decided to identify bacteria or consortium of bacteria present in these WWTPs, prioritizing the consortium and thus increasing the possibility of finding biodegradation routes for more complex compounds such as plastic. On the other hand, we have also tried to determine the presence of genes or biodegradation pathways which allow these bacteria to use these pollutants as a carbon source.Methods: For the isolation of these microorganism, we used two samples from the WWTPs of Ubeda and Seville. Samples were taken from the secondary treatment (sludge before decantation) from both WWTPs. Enrichment cultures were then prepared in a minimal medium with the pollutants used as carbon source [2]. When differences in growth were observed between the control Erlenmeyer flask and the different flasks with samples, different strategies were used such as drop seeding, a subsequent liquid passage, etc. Once a bacteria or microbial consortium capable of degrading a compound has been obtained, a preliminary morphological or biochemical characterisation is made to identify the microorganisms responsible for this degradation. Moreover, we confirm, where possible, whether they possess the genes necessary to degrade the compound.Results: To date, a differential growth has been obtained for some samples with ibuprofen, naproxen and diclofenac which could contain microorganisms capable of growing using said pollutants as a carbon source. Moreover, a phylogenetic analysis of the 16S rRNA sequences has been peformed to determine the family affiliation. In the plastic sample, biomass accumulation has been observed around the plastic, although the turbidity of the culture is not yet significant.Conclusions: A significant number of enrichments cultures have been obtained from WWTP samples, able to grow on ibuprofen, diclofenac, naproxen. Some of the culture members have been characterised biochemically and genetically. The results obtained so far are highly promising for defining new biodegradation routes for these compounds

Estudio del papel de la proteína PII en el control por carbono mediado por el sistema regulador CbrAB

Motivación: Pseudomonas es un género de bacilos Gram negativos quimiorganótrofos aerobios pertenecientes al grupo de las gamma-proteobacterias que engloba un gran número de especies, algunas de gran interés biotecnológico, medioambiental o biosanitario como  P. putida o P. aeruginosa, consideradas excelentes sistemas modelo por gran versatilidad y su fácil manipulación. La caracterización del metabolismo de Pseudomonas es de gran utilidad para muchas aplicaciones biotecnológicas, por lo que se profundizará en la regulación del metabolismo del carbono mediado por el sistema regulador CbrAB, y su posible interacción el metabolismo del nitrógeno. Estudios del metaboloma de P. putida realizados en el grupo indican que, ante de limitación de carbono, existen niveles elevados de α-cetoglutarato, molécula inductora del sistema de control por nitrógeno NtrBC, mediado por la proteína PII (M. García- Mauriño, 2013). Se pretende determinar si la proteína PII del sistema Ntr, está implicada en la activación del sistema Cbr. Para ello se estudiará si la activación de las dianas crcZ y PP2810, controladas por CbrB, está también bajo el control de la proteína PII.Método: Estudio de la expresión génica mediante ensayo de la actividad β-galactosidasa (Miller, 1992) de una fusión transcripcional al promotor de crcZ  y del gen PP2810 en un fondo silvestre y un fondo mutante PII (ΔglnK), en distintas condiciones de disponibilidad de carbono/Nitrógeno. Como control llevaremos una fusión nifL::lacZ, controlada por PII. Los ensayos se realizarán a partir de pre-inóculos en medio mínimo con succinato para el fondo silvestre y en LB para el fondo mutante PII como fuente de carbono y amonio como fuente de nitrógeno, y se realizarán inducciones en  medio rico LB y otros medios con distinta disponibilidad de carbono, como son Succ/NH4, Succ /Ser, Succ/Gln, OAA/ NH4, Pir/ NH4 y Arg/ NH4, todos ellos con antibiótico, y se incubarán hasta una fase exponencial media (A600= 0,3-0,5),tras las cuales se realizará el ensayo.Resultados: Los valores de actividad observados en succinato no difieren significativamente de los valores en oxalacetato, estas diferencias si son significativas para PP2810, para el cual obtenemos  diferencias de inducción en los medios LB, succinato y oxalacetato, piruvato y arginina, aunque la inducción es mucho menor que la observada para CrcZ.Conclusión: En base a los datos preliminares obtenidos CrcZ no parece estar regulado por pII en las condiciones ensayadas

Study of water quality through microbiological and physico-chemical analysis of the Guadaira river.

Water is one of the most abundant and used resources in the world since ancient times, necessary for life on Earth (Díaz Peña, D. 2019). Its main purpose is to cover the basic needs of all living beings, and it is sometimes thought that this resource is unlimited, but it must be remembered that we are dealing with a limited and valuable resource that must be managed efficiently and prudently (Pardo, C.F. 2004). The problem of water and wastewater management is a global problem that has been around for a long time. (Satyendra, et al. 2023). The problem of water and wastewater management is a long-standing global problem, and with population growth and human modifications causing pollution of natural waters, water quality is getting worse and worse (Prato-Moreno, J.G., et al. 2020). The Guadaira River is a local river that runs through important towns in the province of Seville, such as Morón, Alcalá de Guadaira and Seville itself. The high number of inhabitants in these towns means that the wastewater treatment plants are sometimes very stressed and their sewage treatment capacity is limited. We have performed microbiological and physicochemical analyses at two critical locations in the Guadaira river to study water quality. The first sampling point is upstream of the treatment bridge at the discharge of the Dos Hermanas ETAP and the other downstream in the Arroyo Culebras in Bellavista. Samples collected in plastic bottles with thiosulphate were plated on different culture media depending on the microorganism to be studied. In addition, physicochemical analyses were carried out on samples collected in unsterilised plastic jars for further analysis in the laboratory equipment. The microbiological analysis was carried out in accordance with Royal Decree 817/2015, and showed high levels of contamination, both from a microbiological point of view and in terms of ammonium and phosphate levels in the water. The study concludes that the river Guadaíra is polluted but within the limits of the legislation, so that the problem of pollution of the river could be solved by means of a filtration mesh at the water outlet of the ETAP or by avoiding the dumping of solid urban waste into the river. We can include that the sampling and analysis of the waters of the Guadaíra river can be carried out again after the rainy season, in this way, we check if the level of contamination increases or decreases

Caracterización del operón PP2810-13 en Pseudomonas putida

Motivación: El género Pseudomonas se encuentra en una gran diversidad de nichos ecológicos, debido a su gran versatilidad metabólica, es capaz de degradar contaminantes del tipo  compuestos orgánicos, etc. Su estudio nos puede abrir las puertas hacia el desarrollo de estrategias con aplicación ambiental, industrial, etc. Nosotros nos centraremos en Pseudomonas putida, su genoma está secuenciado, y es inocuo (GRAS). Aproximadamente un 10% de sus genes participan en la transducción de señales y la regulación de la expresión; algunos de éstos pertenecen a sistemas de dos componentes(1) permitiendo  acoplar un estímulo-respuesta haciendo que el organismo pueda responder ante cambios en el ambiente, formando una compleja red de regulación. Uno de los sistemas de dos componentes, exclusivo de Pseudomonas es el sistema CbrAB, donde CbrA es la proteína sensora y CbrB es un regulador transcripcional dependiente de σ54. En estudios anteriores se observó mediante ChIP- Seq(no publicado)  que el gen PP2810 era candidato a estar regulado por este sistema de dos componentes, y su región promotora tiene tres posibles sitios de unión para CbrB.Métodos: Se analizará la expresión como medida de la actividad β-galactosidasa para fusiones  transcripcionales de la región promotora  del  gen PP2810 a lacZ  tanto silvestre como con modificaciones en los sitios de unión  en dos  fondos: silvestre y mutante carente de cbrB. Además se realizarán ensayos de unión  de la proteína CbrB a la región promotora silvestre y  mutante. También se construirá un  mutante por inserción en el primer gen del operón de un casete de Kanamicina y se realizará un análisis fenotípico en  medio mínimo con distintas  fuentes de carbono, antibióticos y metales(3).Resultados: Los resultados muetran que CbrB activa transcripcionalmente el gen PP2810 y que la contribución de los subsitios es diferente para cada uno de ellos. En los ensayos de unión se apreció diferencias que correlacionan con los datos obtenidos mediante los ensayos de actividad β-galactosidasa.  No se han obtenido diferencias significativas  todavía del mutante de inserción en los ensayos realizados, aunque esta parte se encuentra en desarrollo.Conclusiones: El operón PP2810-13 parece está regulado por CbrB, pero hay que seguir trabajando en la búsqueda de un fenotipo asociado a este operón

Biotecnología Ambiental

Guía docente de la asignatura Biotecnología Ambiental.Módulo fundamentalmente práctico en el que se abordan las aplicaciones biotecnológicas de la microbiología, ecología y genética.Peer reviewe