We are interested to study both the denitrification and nitrogen fixation processes using as model microorganism the soybean endosymbiont Bradyrhizobium diazoefficiens (formely B. japonicum). B. diazoefficiens is not only able to establish nitrogen-fixing symbiosis with leguminous plants (soybean, mungbean, cowpea, siratro) but also to grow under oxygen-limiting conditions with nitrate, through the denitrification pathway. Further, B. diazoefficiens is the only rhizobial species that has been identified as being able to denitrify under both free-living and symbiotic conditions.
In B. diazoefficiens, a sophisticated regulatory network consisting of two linked regulatory cascades which responds to different oxygen concentrations in the gas phase, FixLJ-FixK2 and RegSR-NifA, coordinates the expression of genes required for microoxic, anoxic and symbiotic growth (Fig. 1; Sciotti et al., 2003; Torres et al., 2011). The FixLJ two-component regulatory system activates the expression of several genes, including the transcription factor gene fixK2, at a concentration equal or below 5% O2 in the gas phase. In turn, FixK2 activates the expression of more than 300 genes, among them some additional regulators, such as NnrR, which is required for the maximal expression of denitrification genes in response to nitrogen oxides (NOx) (Fig. 1; Mesa et al., 2003).
Fig. 1. Two interconnected cascades control genes required for microoxic, anoxic and symbiotic growth in B. diazoefficiens.
Our research is mainly focused on the identification and characterization of relevant genes and novel regulatory mechanisms underlying both denitrification and nitrogen fixation, as well as on the elucidation of possible relationships between these two antagonistic processes. We specifically aim to help expand the understanding of the molecular mechanism of the transcriptional regulatory proteins FixK2 and NnrR. These two proteins are members of the CRP/FNR family of bacterial transcription factors, ubiquitous proteins which respond to a broad range of metabolic and environmental signals (Körner et al., 2003).
The FixK2 protein not only has a central role in the complex regulatory network specified above, but also it is involved in the oxidative stress response (Mesa et al., 2009). FixK2 is also subjected to proteolytic control, by specific cleavage, and by general degradation mediated by the ClpAP1S1 chaperone-proteases system in vitro (Bonnet et al., 2013b). The role of this proteolytic system in vivo (degradation of FixK2 in B. diazoefficiens cells, identification of other substrates, response to stresses, etc) is currently being investigated.
We were also able to solved FixK2 stucture in complex with DNA (Fig. 2; Bonnet et al., 2013a), which is the first known structure of such oxygen-sensitive CRP/FNR-type proteins. The functional characterization of FixK2 based on its structure might serve as model to understand the molecular mechanism of other members of this family and therefore, further expand the spectrum of possibilities how activity of CRP/FNR-like transcription factors can be regulated. In this regard, we are also interested in studying the NOx sensing mechanism of NnrR as well as its molecular determinants to specifically activate the expression of the norCBQD genes coding for the denitrifying nitric oxide reductase (Bueno et al., 2017).
Fig. 2. Ribbon representation of X-ray crystal structure of FixK2 bound to DNA. The two monomers are shown in red and blue (Bonnet et al., 2013a).
Lately, we reported on a novel interplay between FixK2 and the synthesis and accumulation of the carbon reserve polymer polyhydroxybutyrate (PHB) in B. diazoefficiens. In particular, the global carbon metabolism regulator PhaR is not only involved in controlling PHB granule formation by regulating the expression of PHB biosynthetic and accumulation proteins but it also downregulates the expression of the fixK2 gene (Quelas et al., 2016).
Altogether, with our research, we aim to contribute to the better understanding of the regulatory mechanisms which govern B. diazofficiens microoxic free-living and in symbiotic lifestyles to advance in the current knowledge about Rhizobium-leguminous plants interaction. At long-term, our goal is giving a foundation of scientifically supported decision-making to design more friendly precise formulations and practices for a sustainable Agriculture.
LITERATURE
Bonnet M, Kurz M, Mesa S, Briand C, Hennecke H, Grütter MG (2013a) Structure of Bradyrhizobium japonicum FixK2, a transcription factor for microoxic life in symbiosis, unveils sites of DNA binding and oxidation. J Biol Chem 288:14238-14246
Bonnet M, Stegmann M, Maglica Z, Stiegeler E, Weber-Ban E, Hennecke H, Mesa S (2013b) FixK2, a key regulator in Bradyrhizobium japonicum, is a substrate for the protease ClpAP in vitro. FEBS Lett 587: 88-93
Bueno E, Robles EF, Torres MJ, Krell T, Bedmar EJ, Delgado MJ, Mesa S (2017) Disparate response to microoxia and nitrogen oxides of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes. Nitric oxide-Biol Chem 68:137-149.
Körner H, Sofia HJ, Zumft WG (2003) Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs. FEMS Microbiol Rev 27: 559-592
Mesa S, Bedmar EJ, Chanfon A, Hennecke H, Fischer HM (2003). Bradyrhizobium japonicum NnrR, a denitrification regulator, expands the FixLJ-FixK2 regulatory cascade. J Bacteriol 185:3978-3982
Mesa S, Reutimann L, Fischer HM, Hennecke H (2009) Posttranslational control of transcription factor FixK2, a key regulator for the Bradyrhizobium japonicum-soybean symbiosis. Proc Natl Acad Sci USA 106: 21860-21865
Sciotti MA, Chanfon A, Hennecke H, Fischer HM (2003) Disparate oxygen responsiveness of two regulatory cascades that control expression of symbiotic genes in Bradyrhizobium japonicum. J Bacteriol 185: 5639-5642
Quelas JI, Mesa S, Jendrossek J, Lodeiro AR (2016) Regulation of polyhydroxybutyrate synthesis in the soil bacterium Bradyrhizobium diazoefficiens. App. Env. Microbiol 82:4299-4308.
Torres MJ, Bueno E, Mesa S, Bedmar EJ, Delgado MJ (2011) Emerging complexity in the denitrification regulatory network of Bradyrhizobium japonicum. Biochem Soc Trans 39: 284-288
COLLABORATIONS
Regulation underlying B. diazoefficiens microoxic metabolism
Prof. Leo Eberl (University of Zurich, Switzerland)
Dr. Gabriella Pessi (University of Zurich, Switzerland)
Dr. Christian H. Ahrens (Agroscope and Swiss Institute of Bioinformatics, Switzerland)
Prof. Hans-Martin Fischer (ETH-Zurich, Switzerland)
Functional Genomics Center (Zurich, Switzerland)
Structure-function studies on FixK2
Dra. Laura Tomás-Gallardo (Andalusian Centre for Developmental Biology, CSIC-Pablo de Olavide University, Seville, Spain)
FixK2-PhaR relationship
Dr. Juan I. Quelas (Instituto de Biotecnología y Biología Molecular [IBBM] Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Buenos Aires, Argentina)
Dr. Anibal R. Lodeiro (Instituto de Biotecnología y Biología Molecular [IBBM] Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Buenos Aires, Argentina)
Molecular mechanism of NnrR
Prof. David J. Richardson (University of East Anglia, United Kingdom)
Dr. Andrew. J. Gates (University of East Anglia, United Kingdom)
Small RNAs in B. diazoefficiens
Prof. Elena Evguenieva-Hackenberg (Justus-Liebig-University of Giessen, Germany)
Socorro Mesa laboratory 