Group photo. From left to right: Standing, Marina Rebollo, Yolanda E. González, Rubén de Dios Barranco, Francisca Reyes, Carlos Medina, Amando Flores, Inés Canosa and Eduardo Santero. Crouching, Inmaculada García, Guadalupe Martín, Eva Camacho and Belén Floriano.

Scientific activities of the group so far (where we come from)

The use of bacteria for the biological degradation of organic compounds is of great interest due to: (i) the gradual increase in the problem of contamination in the environment due to industrial activity and, (iii) the enormous catabolic versatility of bacteria. However, bacterial biodegradation is often limited by tightly regulated degradation gene expression, rendering the pathways inactive under most environmental conditions. Thus, the challenge to design efficient biodegradation processes is to understand the cellular behavior of bacteria and their adaptation to environmental conditions as a whole, that is, as a system. For this type of study, it is necessary to apply technologies that generate data on a large scale (genomics, transcriptomics, proteomics and metabolomics) and integrate them to globally understand bacterial behavior, predict it and modify it. In addition, the characterization of the molecular mechanism of regulation allows the design of more efficient heterologous expression systems and application in a greater number of hosts.

The main topics of interest of our research group in this field have been:

  1. Complete biochemical and genetic characterization of the tetralin degradation pathway in bacteria. This characterization has been carried out in two bacteria, Sphingopyxis macrogolitabida TFA lineage (Gram negative) (López-Sánchez et al, 2010) y Rhodococcus sp. TFB lineage (Gram positive) (Tomás-Gallardo et al, 2009). In TFB, the degradation of phthalate and naphthalene have also been studied (Tomás-Gallardo et alRamos-Morales, 2013). In addition, transcriptomic analysis was performed on HeLa cells stably transfected with the SteA effector gene. The expression of SteA in these epithelial cells, through the modification of different signal transduction pathways, resulted in an alteration of cell morphology and a decrease in the rate of spontaneous cell death, intercellular junctions and migration speed. cell phone (Cardinal-Muñoz
  2. Elucidation of the molecular mechanisms that regulate tetralin (thn) degradation genes. The implication of tetralin as an inducing molecule and the ThnR and ThnY proteins in the specific regulation of genes has been demonstrated. thn in TFA (López-Sánchez et al, 2009) and a two-component system (ThnSR) in TFB (Tomás-Gallardo et al, 2009, 2012). In addition, a novel mechanism has been described in TFA by which the degradation pathway communicates with the regulatory system to prevent the free expression of genes. thn in the presence of molecules similar to tetralin but which are not substrates of the pathway (Ledesma et al, 2011, 2013). Regarding global regulation, tetralin degradation genes are subject to catabolic repression (mechanism that prioritizes the use of carbon sources). In TFA, the connection between the accumulation of reserve granules inside the cell and the expression capacity of these genes in the presence of preferential carbon sources has been studied (Martín-Cabello et al, 2011). In TFB, the involvement of a regulator of the CRP/Fnr family in said regulation has been proposed (Tomás-Gallardo et al, 2012).
  3. We have also studied the global regulation of nitrogen metabolism in Pseudomonas putida identifying NtrC_as the global regulator of the assimilation of different nitrogen sources. NtrC not only acts by activating routes of assimilation of alternative nitrogen sources, when this element is limiting in the environment, but it is also capable of directly repressing the expression of other genes (Hervás et al, 2009, 2010). In addition, the two-component system CbrAB has been studied, which acts in a coordinated manner with NtrC but which, in addition, controls very important aspects in the relationship of the bacterium with the environment, such as chemotaxis and resistance to toxins (Amador et al, 2010). In addition, CbrB controls the expression of small regulatory RNAs (CrcZ and CrcY) involved in catabolic repression (Garcia-Mauriño et al, 2013). Through a metabolomics approach we have described the key changes that reveal the metabolic adaptation of P. putida under carbon-limited conditions (Valentini et al, 2014).
  4. The use of the elements that direct the gene expression of degradation genes has materialized in the successful development of a collection of expression vectors inducible by acetylsalicylic acid (aspirin) in attenuated intracellular pathogenic bacteria (Medina et al, 2011) for their use as therapeutic agents (Mesa-Pereira et al, 2013, 2014). In this way, by infecting a host with these bacteria, we can control the expression of different proteins with therapeutic amounts of aspirin. It is the most applied part of our science that has given rise to two patents.
  5. In turn, we have developed a patented expression system that allows heterologously expressing genes from meta-libraries located in large fragments, which solves the main problem of functional metagenomic analysis. (Terrón-González et al, 2013, 2014).

Scientific activities of the group in the future (where are we going)

In the last two years of activity, we have been applying -omic techniques to understand the behavior of bacteria globally. We intend to continue advancing in the functional characterization of Sphingopyxis macrogolitabida TFA, which includes transcriptomic studies using dRNAseq under different conditions, reconstruction of its aerobic and anaerobic metabolism and regulation by small RNAs and Hfq proteins, as well as by sigma factors with extracytoplasmic function. In turn, we intend to complete the characterization of the CbrAB regulon of Pseudomonas putida.

In addition, we work with companies (R+D+i)

In recent years we have developed R&D contracts with companies such as Biomedal, Canagrosa and Abengoa Research. The works of greater application have resulted in 3 patents.

Representative bibliography

Lover , CI , Canosa , I , Govants , F , Santero , E . (2010). Lack of CbrB in Pseudomonas putida affects not only amino acids metabolism but also different stress responses and biofilm development. Environmental Microbiology, 12: 1748-1761.

Hervás AB, Canosa I, Little R, Dixon R, Santero E. (2009). NtrC-dependent regulatory network for nitrogen assimilation in Pseudomonas putida. Journal of Bacteriology,191: 6123-6135.

Hervas AB, Canosa I, Santero E. (2010). Regulation of glutamate dehydrogenase expression in Pseudomonas putida results from its direct repression by NtrC under nitrogen-limiting conditions. Mol. Microbiol., 78: 305-319.

Ledesma-Garcia, L. Rivas-Marin, E., Floriano, B., Bernhardt, R., Ewen, M., Reyes-Ramirez, F., Santero, E. (2011). ThnY is a ferredoxin reductase-like iron-sulfur flavoprotein that has evolved to function as a regulator of tetralin biodegradation genes expression. J. Biol. Chem., 286: 1709-1718.

Ledesma-García L, Reyes-Ramírez F, Santero E. (2013). The ferredoxin ThnA3 negatively regulates tetralin biodegradation gene expression via ThnY, a ferredoxin reductase that functions as a regulator of the catabolic pathway. PLoS One, 8:e73910. doi: 10.1371/journal. pone.0073910.

López-Sánchez, A. E. Rivas-Marín, O. Martínez-Pérez, B. Floriano & Santero. (2009). Co-ordinated regulation of two divergent pro. moters through higher-order complex formation by the LysR-type regulator ThnR. Mol. Microbiol., 73: 1086-1100.

López-Sánchez, A., Floriano, B., Andújar, E., Hernáez, M.J., Santero, E. (2010). Tetralin-induced and ThnR regulated aldehyde dehydrogenase and beta-oxidation genes in Sphingomonas macrogolitabida strain TFA. Appl. Environ. Microbiol., 76: 110-118.

Martín-Cabello, G., Moreno-Ruiz, E., Morales, V., Floriano, B., Santero, E. (2011). Involvement of poly(3-hydroxybutyrate) synthesis in catabolite repression of tetralin biodegradation genes in Sphingomonas macrogolitabida strain TFA. Environ. Microbiol. Reports, 3: 627-631.

Medina, C., Camacho, E.M., Flores, A., Mesa-Pereira, B., Santero, E. (2011). Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells. PLOSone, 6: e23055.

Mesa-Pereira B, Medina C, Camacho EM, Flores A, Santero E. (2014). Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells. Microb. Biotechnol DOI: 10.1111/1751-7915.12153.

Mesa-Pereira B, Medina C, Camacho EM, Flores A, Santero E. (2013). Novel tools to analyze the function of Salmonella effectors show that svpB ectopic expression induces cell cycle arrest in tumor cells. PLoS One, 8:e78458. doi: 10.1371/journal.pone.0078458.

García-Mauriño SM, Pérez-Martínez I, Amador CI, Canosa I, Santero (2013). Transcriptional activation of the CrcZ and CrcY regulatory RNAs by the CbrB response regulator in Pseudomonas putida. Mol Microbiol., 89: 189-205.

Terrón-González, L., Medina, C., Limón-Mortés, MC., Santero, E.(2013). Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries. Sci. Reports, 3: 1107.

Terrón-González, L., Genilloud, O., Santero, E. (2014). Potential and limitations of metagenomic functional analyses. Chapter 1. Pp. 1-43. In: Metagenomics, Methods, Applications and Perspectives. Benedetti, C. (Ed.). Nova Publishers, New York. ISBN: 978-63321-698-3 (eBook).

Tomas, L., Santero, E., Floriano, B., (2009). Molecular and biochemical characterization of the tetralin degradation pathway in Rhodococcus sp. strain TFB. Microb. Biotechnol., 2: 262- 273

Tomas-Gallardo, L., Santero, E., Floriano, B. (2012). Involvement of a putative cyclic amp receptor protein (CRP)-like binding sequence and a CRP-like protein in glucose-mediated catabolite repression of thn genes in Rhodococcus sp. strain TFB. Appl. Environ. Microbiol. 78(15): 5460-2

Tomás-Gallardo L, Gómez-Álvarez H, Santero, E, Floriano B. (2014). Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB. Microb. Biotechnol., 7: 100-103

Valentini M, Garcia-Mauriño SM, Pérez-Martinez I, Santero E, CanosaI, Lapouge K. (2014). Hierarchical management of carbon sources is regulated similarly by the CbrA/B system in Pseudomonas aeruginosa and Pseudomonas putida. Microbiology, DOI: 10.1099/mic.0.078873-0.