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Bueno, Emilio
Publications (5 of 5) Show all publications
Jiménez-Leiva, A., Cabrera, J. J., Bueno, E., Torres, M. J., Salazar, S., Bedmar, E. J., . . . Mesa, S. (2019). Expanding the Regulon of the Bradyrhizobium diazoefficiens NnrR Transcription Factor: New Insights Into the Denitrification Pathway. Frontiers in Microbiology, 10, Article ID 1926.
Open this publication in new window or tab >>Expanding the Regulon of the Bradyrhizobium diazoefficiens NnrR Transcription Factor: New Insights Into the Denitrification Pathway
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2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1926Article in journal (Refereed) Published
Abstract [en]

Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK(2)-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK(2) and NnrR exert disparate control on expression of core denitrifying systems encoded by napEDABC, nirK, norCBQD, and nosRZDFYLX genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in B. diazoefficiens, we compared the transcriptional profile of an nnrR mutant with that of the wild type, both grown under anoxic denitrifying conditions. This approach revealed more than 170 genes were simultaneously induced in the wild type and under the positive control of NnrR. Among them, we found the cycA gene which codes for the c(550) soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc(1) complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in cycA neither affected nosZ gene expression nor NosZ protein steady-state levels. Furthermore, cycA, nnrR and its proximal divergently oriented nnrS gene, are direct targets for FixK(2) as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK(2) and NnrR in anoxic denitrifying conditions was validated at transcriptional level, determined by quantitative reverse transcription PCR, and at the level of protein by performing heme c-staining of soluble cytochromes. Thus, this study expands the regulon of NnrR and demonstrates the role of CycA in the activity of the nitrous oxide reductase, the key enzyme for nitrous oxide mitigation.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
CRP/FNR proteins, in vitro transcription, microoxia, nitrogen oxides, Rhizobia, transcriptomics
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-162844 (URN)10.3389/fmicb.2019.01926 (DOI)000481765700001 ()31481951 (PubMedID)
Available from: 2019-09-09 Created: 2019-09-09 Last updated: 2019-09-09Bibliographically approved
Bueno, E., Sit, B., Waldor, M. K. & Cava, F. (2018). Anaerobic nitrate reduction divergently governs population expansion of the enteropathogen Vibrio cholerae [Letter to the editor]. Nature Microbiology, 3(12), 1346-1353
Open this publication in new window or tab >>Anaerobic nitrate reduction divergently governs population expansion of the enteropathogen Vibrio cholerae
2018 (English)In: Nature Microbiology, E-ISSN 2058-5276, Vol. 3, no 12, p. 1346-1353Article in journal, Letter (Refereed) Published
Abstract [en]

To survive and proliferate in the absence of oxygen, many enteric pathogens can undergo anaerobic respiration within the host by using nitrate (NO3-) as an electron acceptor(1,2). In these bacteria, NO3- is typically reduced by a nitrate reductase to nitrite (NO2-), a toxic intermediate that is further reduced by a nitrite reductase(3). However, Vibrio cholerae, the intestinal pathogen that causes cholera, lacks a nitrite reductase, leading to NO2- accumulation during nitrate reduction 4(.) Thus, V. cholerae is thought to be unable to undergo NO3-(-)dependent anaerobic respiration(4). Here, we show that during hypoxic growth, NO3- reduction in V. cholerae divergently affects bacterial fitness in a manner dependent on environmental pH. Remarkably, in alkaline conditions, V. cholerae can reduce NO3- to support population growth. Conversely, in acidic conditions, accumulation of NO2- from NO3- reduction simultaneously limits population expansion and preserves cell viability by lowering fermentative acid production. Interestingly, other bacterial species such as Salmonella typhimurium, enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium also reproduced this pH-dependent response, suggesting that this mechanism might be conserved within enteric pathogens. Our findings explain how a bacterial pathogen can use a single redox reaction to divergently regulate population expansion depending on the fluctuating environmental pH.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-154339 (URN)10.1038/s41564-018-0253-0 (DOI)000451259600007 ()30275512 (PubMedID)
Available from: 2018-12-18 Created: 2018-12-18 Last updated: 2018-12-18Bibliographically approved
Bueno, E., Robles, E. F., Torres, M. J., Krell, T., Bedmar, E. J., Delgado, M. J. & Mesa, S. (2017). Disparate response to microoxia and nitrogen oxides of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes. Nitric oxide, 68, 137-149
Open this publication in new window or tab >>Disparate response to microoxia and nitrogen oxides of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes
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2017 (English)In: Nitric oxide, ISSN 1089-8603, E-ISSN 1089-8611, Vol. 68, p. 137-149Article in journal (Refereed) Published
Abstract [en]

Expression of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes requires low oxygen (O-2) tension and nitrate (NO3), through a regulatory network comprised of two coordinated cascades, FixLJ-FixK(2)-NnrR and RegSR-NifA. To precisely understand how these signals are integrated in the FixLJ-FixK(2)-NnrR circuit, we analyzed beta-Galactosidase activities from napE-lacZ, nirK-lacZ and norC-lacZ fusions, and performed analyses of NapC and NorC levels as well as periplasmic nitrate reductase (Nap) activity, in B. japonicum wildtype and fixK(2) and nnrR mutant backgrounds. While microoxic conditions (2% O-2 at headspace) were sufficient to induce expression of napEDABC and nirK genes and this control depends on FixK(2), norCBQD expression requires, in addition to microoxia, nitric oxide gas (NO) and both FixK(2) and NnrR transcription factors. Purified FixK(2) protein directly interacted and activated transcription in collaboration with B. japonicum RNA polymerase (RNAP) from the napEDABC and nirK promoters, but not from the norCBQD promoter. Further, recombinant NnrR protein bound exclusively to the norCBQD promoter in an O-2-sensitive manner. Our work suggest a disparate regulation of B. japonicum denitrifying genes expression with regard to their dependency to microoxia, nitrogen oxides (NOx), and the regulatory proteins FixK(2) and NnrR. In this control, expression of napEDABC and nirK genes requires microoxic conditions and directly depends on FixK2, while expression of norCBQD genes relies on NO, being NnrR the candidate which directly interacts with the norCBQD promoter. 

Place, publisher, year, edition, pages
Elsevier, 2017
Keywords
CRP/FNR transcription factors, FixK(2)-like box, NnrR, Promoter, Rhizobia, Signal molecule
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-138212 (URN)10.1016/j.niox.2017.02.002 (DOI)000405978000015 ()
Available from: 2017-08-18 Created: 2017-08-18 Last updated: 2018-06-09Bibliographically approved
Torres, M. J., Bueno, E., Jimenez-Leiva, A., Cabrera, J. J., Bedmar, E. J., Mesa, S. & Delgado, M. J. (2017). FixK(2) Is the Main Transcriptional Activator of Bradyrhizobium diazoefficiens nosRZDYFLX Genes in Response to Low Oxygen. Frontiers in Microbiology, 8, Article ID 1621.
Open this publication in new window or tab >>FixK(2) Is the Main Transcriptional Activator of Bradyrhizobium diazoefficiens nosRZDYFLX Genes in Response to Low Oxygen
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2017 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 1621Article in journal (Refereed) Published
Abstract [en]

The powerful greenhouse gas, nitrous oxide (N2O) has a strong potential to drive climate change. Soils are the major source of N2O and microbial nitrification and denitrification the main processes involved. The soybean endosymbiont Bradyrhizobium diazoefficiens is considered a model to study rhizobial denitrification, which depends on the napEDABC, nirK, norCBQD, and nosRZDYFLX genes. In this bacterium, the role of the regulatory cascade FixLJ-FixK(2)-NnrR in the expression of napEDABC, nirK, and norCBQD genes involved in N2O synthesis has been previously unraveled. However, much remains to be discovered regarding the regulation of the respiratory N2O reductase (N2OR), the key enzyme that mitigates N2O emissions. In this work, we have demonstrated that nosRZDYFLX genes constitute an operon which is transcribed from a major promoter located upstream of the nosR gene. Low oxygen was shown to be the main inducer of expression of nosRZDYFLX genes and N2OR activity, FixK(2) being the regulatory protein involved in such control. Further, by using an in vitro transcription assay with purified FixK(2) protein and B. diazoefficiens RNA polymerase we were able to show that the nosRZDYFLX genes are direct targets of FixK(2).

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2017
Keywords
climate change, denitrification, greenhouse gas, nitrous oxide, nitrous oxide reductase, regulation
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-142877 (URN)10.3389/fmicb.2017.01621 (DOI)000408652100001 ()28912756 (PubMedID)
Available from: 2017-12-13 Created: 2017-12-13 Last updated: 2018-06-09Bibliographically approved
Felgner, S., Frahm, M., Kocijancic, D., Rohde, M., Eckweiler, D., Bielecka, A., . . . Weiss, S. (2016). aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant. mBio, 7(5), Article ID e01220-16.
Open this publication in new window or tab >>aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant
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2016 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 5, article id e01220-16Article in journal (Refereed) Published
Abstract [en]

Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that Delta aroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, Delta aroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of Delta aroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. 

IMPORTANCE Recombinant attenuated bacterial vector systems based on genetically engineered Salmonella have been developed as highly potent vaccines. Due to the pathogenic properties of Salmonella, efficient attenuation is required for clinical applications. Since the hallmark study by Hoiseth and Stocker in 1981 (S. K. Hoiseth and B. A. D. Stocker, Nature 291:238-239, 1981, http://dx.doi.org/10.1038/291238a0), the auxotrophic Delta aroA mutation has been generally considered safe and universally used to attenuate bacterial strains. Here, we are presenting the remarkable finding that a deletion of aroA leads to pronounced alterations of gene expression, metabolism, and cellular physiology, which resulted in increased immunogenicity, virulence, and adjuvant potential of Salmonella. These results suggest that the enhanced immunogenicity of aroA-deficient Salmonella strains might be advantageous for optimizing bacterial vaccine carriers and immunotherapy. Accordingly, we demonstrate a superior performance of Delta aroA Salmonella in bacterium-mediated tumor therapy. In addition, the present study highlights the importance of a functional shikimate pathway to sustain bacterial physiology and metabolism.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-128275 (URN)10.1128/mBio.01220-16 (DOI)000390132900014 ()
Available from: 2016-12-01 Created: 2016-12-01 Last updated: 2018-06-09Bibliographically approved
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