Publications by authors named "Eulogio J Bedmar"

66 Publications

Dissection of FixK protein-DNA interaction unveils new insights into Bradyrhizobium diazoefficiens lifestyles control.

Environ Microbiol 2021 Jul 5. Epub 2021 Jul 5.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Granada, 18008, Spain.

The FixK protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen-fixing lifestyles in Bradyrhizobium diazoefficiens. Among the microoxic-responsive FixK -activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric-oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK is a CRP/FNR-type transcription factor, which recognizes a 14 bp-palindrome (FixK box) at the regulated promoters through three residues (L195, E196, and R200) within a C-terminal helix-turn-helix motif. Here, we mapped the determinants for discriminatory FixK -mediated regulation. While R200 was essential for DNA binding and activity of FixK , L195 was involved in protein-DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK box at the fixNOQP promoter impaired transcription activation by FixK , which was residual when a second mutation affecting the box palindromy was introduced. The substitution of nucleotide 11 within the NnrR box at the norCBQD promoter allowed FixK -mediated activation in response to microoxia. Thus, position 11 within the FixK /NnrR boxes constitutes a key element that changes FixK targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.
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http://dx.doi.org/10.1111/1462-2920.15661DOI Listing
July 2021

Bacterial nitric oxide metabolism: Recent insights in rhizobia.

Adv Microb Physiol 2021 7;78:259-315. Epub 2021 Jun 7.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain. Electronic address:

Nitric oxide (NO) is a reactive gaseous molecule that has several functions in biological systems depending on its concentration. At low concentrations, NO acts as a signaling molecule, while at high concentrations, it becomes very toxic due to its ability to react with multiple cellular targets. Soil bacteria, commonly known as rhizobia, have the capacity to establish a N-fixing symbiosis with legumes inducing the formation of nodules in their roots. Several reports have shown NO production in the nodules where this gas acts either as a signaling molecule which regulates gene expression, or as a potent inhibitor of nitrogenase and other plant and bacteria enzymes. A better understanding of the sinks and sources of NO in rhizobia is essential to protect symbiotic nitrogen fixation from nitrosative stress. In nodules, both the plant and the microsymbiont contribute to the production of NO. From the bacterial perspective, the main source of NO reported in rhizobia is the denitrification pathway that varies significantly depending on the species. In addition to denitrification, nitrate assimilation is emerging as a new source of NO in rhizobia. To control NO accumulation in the nodules, in addition to plant haemoglobins, bacteroids also contribute to NO detoxification through the expression of a NorBC-type nitric oxide reductase as well as rhizobial haemoglobins. In the present review, updated knowledge about the NO metabolism in legume-associated endosymbiotic bacteria is summarized.
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http://dx.doi.org/10.1016/bs.ampbs.2021.05.001DOI Listing
June 2021

Involvement of the metabolically active bacteria in the organic matter degradation during olive mill waste composting.

Sci Total Environ 2021 Oct 25;789:147975. Epub 2021 May 25.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín (EEZ), Agencia Estatal CSIC, c/ Profesor Albareda, 1, 18008 Granada, Spain.

RNA-based high-throughput sequencing is a valuable tool in the discernment of the implication of metabolically active bacteria during composting. In this study, "alperujo" composting was used as microbial model for the elucidation of structure-function relationships with physicochemical transformation of the organic matter. DNA and RNA, subsequently retrotranscribed into cDNA, were isolated at the mesophilic, thermophilic and maturation phases. 16S rRNA gene was amplified by quantitative PCR (qPCR) and Illumina MiSeq platform to assess bacterial abundance and diversity, respectively. The results showed that the abundance of active bacteria assessed by qPCR was maximum at thermophilic phase, which confirm it as the most active stage of the process. Concerning diversity, Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the main phyla presented in composts. Concomitantly, three different behaviours were observed for bacterial dynamics: some genera decreased during the whole process meanwhile others proliferated only at thermophilic or maturation phase. Statistical correlation between physicochemical transformations of the organic matter and bacterial diversity revealed bacterial specialisation. This result indicated that specific groups of bacteria were only involved in the organic matter degradation during bio-oxidative phase or humification at maturation. Metabolic functions predictions confirmed that active bacteria were mainly involved in carbon (C) and nitrogen (N) cycles transformations, and pathogen reduction.
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http://dx.doi.org/10.1016/j.scitotenv.2021.147975DOI Listing
October 2021

Characterization of Retama sphaerocarpa microsymbionts in Zaida lead mine tailings in the Moroccan middle Atlas.

Syst Appl Microbiol 2021 May 18;44(3):126207. Epub 2021 Apr 18.

Centre de Biotechnologies végétales et microbiennes, Biodiversité et Environnement, Faculty of Sciences, Mohammed V University in Rabat, 4 Avenue Ibn Battouta, Agdal, Rabat, Morocco. Electronic address:

In the Moroccan Middle Atlas, the tailings rich in lead and other metal residues, in the abandoned Zaida mining district, represent a real threat to environment and the neighboring villages' inhabitants' health. In this semi-arid to arid area, phytostabilisation would be the best choice to limit the transfer of heavy metals to populations and groundwater. The aim of this work was to characterize the bacteria that nodulate Retama sphaerocarpa, spontaneous nitrogen fixing shrubby legume, native to the Zaida mining area, with great potential to develop for phytostabilisation. Forty-three bacteria isolated from root nodules of the plant were characterized. Based on REP-PCR and ARDRA, four strains were selected for further molecular analyzes. The 16S rRNA gene sequences analysis revealed that the isolated strains are members of the genus Bradyrhizobium, and the phylogenetic analysis of the housekeeping genes glnII, atpD, gyrB, rpoB, recA and dnaK individual sequences and their concatenation showed that the strains are close to B. algeriense RST89 and B. valentinum LmjM3 with similarity percentages of 89.07% to 95.66% which suggest that the newly isolated strains from this mining site may belong to a potential novel species. The phylogeny of the nodA and nodC genes showed that the strains belong to the symbiovar retamae of the genus Bradyrhizobium. These strains nodulate also R. monosperma, R. dasycarpa and Lupinus luteus.
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http://dx.doi.org/10.1016/j.syapm.2021.126207DOI Listing
May 2021

The endemic Chamaecytisus albidus is nodulated by symbiovar genistearum of Bradyrhizobium in the Moroccan Maamora Forest.

Syst Appl Microbiol 2021 May 26;44(3):126197. Epub 2021 Mar 26.

Centre de Biotechnologies Végétale et Microbienne, Biodiversité et Environnement, Faculté des Sciences, Université Mohammed V de Rabat, 4, Avenue Ibn Battouta, Rabat, Morocco. Electronic address:

Out of 54 isolates from root nodules of the Moroccan-endemic Chamaecytisus albidus plants growing in soils from the Maamora cork oak forest, 44 isolates formed nodules when used to infect their original host plant. A phenotypic analysis showed the metabolic diversity of the strains that used different carbohydrates and amino acids as sole carbon and nitrogen sources. The isolates grew on media with pH values ranging from 6 to 8. However, they did not tolerate high temperatures or drought and they did not grow on media with salt concentrations higher than 85 mM. REP-PCR fingerprinting grouped the strains into 12 clusters, of which representative strains were selected for ARDRA and rrs analyses. The rrs gene sequence analysis indicated that all 12 strains were members of the genus Bradyrhizobium and their phylogeny showed that they were grouped into two different clusters. Two strains from each group were selected for multilocus sequence analysis (MLSA) using atpD, recA, gyrB and glnII housekeeping genes. The inferred phylogenetic trees confirmed that the strains clustered into two divergent clusters. Strains CM55 and CM57 were affiliated to the B. canariense/B. lupini group, whereas strains CM61 and CM64 were regrouped within the B. cytisi/B. rifense lineage. The analysis of the nodC symbiotic gene affiliated the strains to the symbiovar genistearum. The strains were also able to nodulate Retama monosperma, Lupinus luteus and Cytisus monspessulanus, but not Phaseolus vulgaris or Glycine max. Inoculation tests with C. albidus showed that some strains could be exploited as efficient inocula that could be used to improve plant growth in the Maamora forest.
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http://dx.doi.org/10.1016/j.syapm.2021.126197DOI Listing
May 2021

Bradyrhizobium sp. sv. retamae nodulates Retama monosperma grown in a lead and zinc mine tailings in Eastern Morocco.

Braz J Microbiol 2021 Jun 14;52(2):639-649. Epub 2021 Jan 14.

Centre de Biotechnologies végétales et microbiennes, Biodiversité et Environnement, Faculty of Sciences, Mohammed V University, 4 Avenue Ibn Battouta, Agdal, Rabat, Morocco.

The aim of this work was to characterize and identify some bacteria isolated from the root nodules of Retama monosperma grown in Sidi Boubker lead and zinc mine tailings. Very few root nodules were obtained on the root nodules of R. monosperma grown in these soils. The three bacteria isolated from the root nodules were tolerant in vitro to different concentrations of heavy metals, including lead and zinc. The rep-PCR experiments showed that the three isolates have different molecular fingerprints and were considered as three different strains. The analysis of their 16S rRNA gene sequences proved their affiliation to the genus Bradyrhizobium. The analysis and phylogeny of the housekeeping genes atpD, glnII, gyrB, recA, and rpoB confirmed that the closest species was B. valentinum with similarity percentages of 95.61 to 95.82%. The three isolates recovered from the root nodules were slow-growing rhizobia capable to renodulate their original host plant in the presence of Pb-acetate. They were able to nodulate R. sphaerocarpa and Lupinus luteus also but not Glycine max or Phaseolus vulgaris. The phylogeny of the nodA and nodC nodulation genes as well as the nifH gene of the three strains showed that they belong to the symbiovar retamae of the genus Bradyrhizobium. The three strains isolated could be considered for use as inoculum for Retama plants before use in phytoremediation experiments.
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http://dx.doi.org/10.1007/s42770-021-00420-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105474PMC
June 2021

Agronomical parameters of host and non-host legumes inoculated with Melilotus indicus-isolated rhizobial strains in desert unreclaimed soil.

Arch Microbiol 2020 Sep 25;202(7):1929-1938. Epub 2020 May 25.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, CSIC, 419, Granada, Spain.

In a search for identification of rhizobial strains with superior N-fixation efficiency and improved plant agronomic characteristics upon inoculation, four strains, 4.21, 9.17, 11.2 and 14.1, isolated from root nodules of wild-grown Melilotus indicus have been used to inoculate field-grown common bean, pea, cowpea and fenugreek plants. Uninoculated plants and those inoculated with host-specific commercial inoculants were used as a control. The root length, shoot height, shoot dry weight and root dry weight and the grain yield of the plants were determined after harvest. The content of N, organic C and carbohydrates content of the grain were also recorded. The inoculation with the strains 4.21 and 14.1 increased the grain yield of the fenugreek compared both with the uninoculated plants and those inoculated with the commercial strain ARC-1. The grain yield of the common bean treated with the strains 9.17 and 14.1 was also higher than that of the uninoculated and the commercial strains ARC-301. In contrast, none of the strains increased the grain yield of the pea and cowpea plants compared to the commercial strains ARC-201 and ARC-169, respectively. Significant increases of some agronomical parameters were observed in some plant-bacterium couples, albeit nodulation was not observed. It is possible that the positive effects of rhizobial inoculation on the agronomical parameters of the non-nodule forming legumes could be due to plant growth promotion characteristic of the strains used for inoculation. Analysis of the phylogeny of the almost complete 16S rRNA sequence of the rhizobial inoculants revealed that the strains 4.21 and 9.17 clustered together with R. skierniewicense and R. rosettiformans, respectively, and that the strains 11.2 and 14.1 grouped with E. meliloti. All the four strains produced IAA, and showed biocontrol activity against Rhizotocnia solani, Fusarium oxysporum, Pythium ultimum, Alternaria alternata and Sclerotonia rolsfi, albeit to a different extent.
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http://dx.doi.org/10.1007/s00203-020-01907-xDOI Listing
September 2020

Characterization of Pisum sativum and Vicia faba microsymbionts in Morocco and definition of symbiovar viciae in Rhizobium acidisoli.

Syst Appl Microbiol 2020 May 25;43(3):126084. Epub 2020 Apr 25.

Centre de Biotechnologies Végétales et Microbiennes, Biodiversité et Environnement, Faculty of Sciences, Mohammed V University in Rabat, Morocco.

In this work, we analyzed the diversity of seventy-six bacteria isolated from Pea and faba bean nodules in two regions of Morocco. The molecular diversity was realized using the analysis of the sequences of 16S rRNA and six housekeeping genes (recA, glnII, atpD, dnaK, rpoB and gyrB) and two symbiotic genes (nodA and nodC). The phylogeny of the 16S rRNA gene sequences revealed that all strains belong to the genus Rhizobium, being related to the type strains of R. leguminosarum, R. laguerreae, R. indigoferae, R. anhuiense and R. acidisoli. The housekeeping genes phylogenies showed that some strains formed a subclade distinct from the rhizobial species that usually nodulate Vicia faba and Pisum sativum which are closely related to R. acidisoli FH23 with sequence similarity of 98.3%. Analysis of the PGPR activities of the different isolates showed that the strains related to R. laguerreae were able to solubilize phosphates and to produce siderophores and auxin phytohormone. However, R. acidisoli strain F40D2 was unable to solubilize phosphates although they produce siderophores and IAA. The phylogenetic analysis of the nodA and nodC sequences showed that all isolated strains were closely related with the strains of symbiovar viciae. The nodulation tests confirmed the ability to nodulate V. faba and P. sativum but not Cicer arietinum or Phaseolus vulgaris. Hence, in Morocco P. sativum is nodulated by R. laguerreae; whereas V. faba is nodulated by R. laguerreae and the symbiovar viciae of R. acidisoli which has been not previously described in this species.
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http://dx.doi.org/10.1016/j.syapm.2020.126084DOI Listing
May 2020

The Hemoglobin Bjgb From Controls NO Homeostasis in Soybean Nodules to Protect Symbiotic Nitrogen Fixation.

Front Microbiol 2019 10;10:2915. Epub 2020 Jan 10.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

Legume-rhizobia symbiotic associations have beneficial effects on food security and nutrition, health and climate change. Hypoxia induced by flooding produces nitric oxide (NO) in nodules from soybean plants cultivated in nitrate-containing soils. As NO is a strong inhibitor of nitrogenase expression and activity, this negatively impacts symbiotic nitrogen fixation in soybean and limits crop production. In , denitrification is the main process involved in NO formation by soybean flooded nodules. In addition to denitrification, nitrate assimilation is another source of NO in free-living cells and a single domain hemoglobin (Bjgb) has been shown to have a role in NO detoxification during nitrate-dependent growth. However, the involvement of Bjgb in protecting nitrogenase against NO in soybean nodules remains unclear. In this work, we have investigated the effect of inoculation of soybean plants with a mutant on biological nitrogen fixation. By analyzing the proportion of N in shoots derived from N-fixation using the N isotope dilution technique, we found that plants inoculated with the mutant strain had higher tolerance to flooding than those inoculated with the parental strain. Similarly, reduction of nitrogenase activity and expression by flooding was less pronounced in than in WT nodules. These beneficial effects are probably due to the reduction of NO accumulation in flooded nodules compared to the wild-type nodules. This decrease is caused by an induction of expression and activity of the denitrifying NO reductase enzyme in bacteroids. As deficiency promotes NO-tolerance, the negative effect of NO on nitrogenase is partially prevented and thus demonstrates that inoculation of soybean plants with the mutant confers protection of symbiotic nitrogen fixation during flooding.
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http://dx.doi.org/10.3389/fmicb.2019.02915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965051PMC
January 2020

Assessment of the diversity and abundance of the total and active fungal population and its correlation with humification during two-phase olive mill waste (''alperujo") composting.

Bioresour Technol 2020 Jan 15;295:122267. Epub 2019 Oct 15.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín (EEZ), Agencia Estatal CSIC, c/Profesor Albareda, 1, 18008 Granada, Spain.

Metagenomic and transcriptomic techniques applied to composting could increase our understanding of the overall microbial ecology and could help us to optimise operational conditions which are directly related with economic interest. In this study, the fungal diversity and abundance of two-phase olive mill waste ("alperujo") composting was studied using Illumina MiSeq sequencing and quantitative PCR, respectively. The results showed an increase of the fungal diversity during the process, with Ascomycota being the predominant phylum. Penicillium was the main genera identified at the mesophilic and maturation phases, with Debaryomyces and Sarocladium at the thermophilic phase, respectively. The fungal abundance was increased during composting, which confirms their important role during thermophilic and maturation phases. Some Basidiomycota showed an increased during the process, which showed a positive correlation with the humification parameters. According to that, the genus Cystofilobasidium could be used as a potential fungal biomarker to assess alperujo compost maturation.
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http://dx.doi.org/10.1016/j.biortech.2019.122267DOI Listing
January 2020

Expanding the Regulon of the NnrR Transcription Factor: New Insights Into the Denitrification Pathway.

Front Microbiol 2019 20;10:1926. Epub 2019 Aug 20.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

Denitrification in the soybean endosymbiont is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK and NnrR exert disparate control on expression of core denitrifying systems encoded by , , , and genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in , we compared the transcriptional profile of an 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 gene which codes for the soluble cytochrome (CycA), previously identified as an intermediate electron donor between the complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in neither affected gene expression nor NosZ protein steady-state levels. Furthermore, , and its proximal divergently oriented gene, are direct targets for FixK as determined by transcription activation assays. The dependence of expression on FixK 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 -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.
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http://dx.doi.org/10.3389/fmicb.2019.01926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710368PMC
August 2019

Nodulation of by in an Abandonned Lead Mine Soils in Eastern Morocco.

Front Microbiol 2019 23;10:1456. Epub 2019 Jul 23.

Center for Biotechnology, Biodiversity and Environment, Faculty of Sciences, Mohammed V University, Rabat, Morocco.

Millions tons of lead and zinc wastes from the abandoned Touissit mine are stored in the open air as dikes in the vicinity of the villages in Eastern Morocco and pose a real danger to both the environment and local populations. To prevent the movement of minerals to the nearby villages and limit the damages to the environment and health, we proposed the nitrogen-fixing leguminous shrub , as a model plant to use for phytostabilization experimentations. This plant species is known by its ability to grow in hard climatic conditions and in heavy metals contaminated soils. The isolation of bacterial strains nodulating in the abandoned mine soils will permit the selection of rhizobia to inoculate young plant seedlings before their use for the phytostabilization of the mine tailings. In this work, 44 bacteria were isolated from the root nodules of grown in the Touissit abandoned mine. Twenty-four isolates were considered as true rhizobia as they possess a copy of the nodC symbiotic gene and were able to renodulate their original host. The phenotypic characterization showed that all the strains are tolerant to different concentrations of heavy metals. The analysis of the 16S rRNA sequences of two selected representative strains showed they were related to different strains of isolated from different legumes in three continents deserts. The , , and housekeeping genes analysis confirmed the affiliation of the strains to . Moreover, the phylogenic analysis of , , and symbiotic genes showed that the strains are more related to JNVUTP6 species isolated from root nodules in the Thar Desert in India. To our knowledge, this is the first report about the isolation of from root nodules.
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http://dx.doi.org/10.3389/fmicb.2019.01456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663986PMC
July 2019

Ensifer fredii symbiovar vachelliae nodulates endemic Vachellia gummifera in semiarid Moroccan areas.

Syst Appl Microbiol 2019 Sep 17;42(5):125999. Epub 2019 Jul 17.

Centre de Biotechnologie Végétale et Microbienne, Biodiversité et Environnement, Faculty of sciences, Mohammed V University, 4, Ibn Battouta Avenue, Rabat, Morocco. Electronic address:

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name "vachelliae" is proposed.
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http://dx.doi.org/10.1016/j.syapm.2019.06.004DOI Listing
September 2019

Produces Nitrous Oxide by Coupling the Assimilatory and Denitrification Pathways.

Front Microbiol 2019 7;10:980. Epub 2019 May 7.

Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

More than two-thirds of the powerful greenhouse gas nitrous oxide (NO) emissions from soils can be attributed to microbial denitrification and nitrification processes. Bacterial denitrification reactions are catalyzed by the periplasmic (Nap) or membrane-bound (Nar) nitrate reductases, nitrite reductases (NirK/ Nir), nitric oxide reductases (cNor, qNor/ CuNor), and nitrous oxide reductase (Nos) encoded by /, , and genes, respectively. CFN42, the microsymbiont of common bean, is unable to respire nitrate under anoxic conditions and to perform a complete denitrification pathway. This bacterium lacks the , and genes but contains genes encoding NirK and cNor. In this work, we demonstrated that is able to grow with nitrate as the sole nitrogen source under aerobic and microoxic conditions. Genetic and functional characterization of a gene located in the chromosome and annotated as demonstrated that growth under aerobic or microoxic conditions with nitrate as nitrogen source as well as nitrate reductase activity requires NarB. In addition to be involved in nitrate assimilation, NarB is also required for NO and NO production by NirK and cNor, respectively, in cells grown microoxically with nitrate as the only N source. Furthermore, β-glucuronidase activity from and fusions, as well as NorC expression and Nir and Nor activities revealed that expression of genes under microoxic conditions also depends on nitrate reduction by NarB. Our results suggest that nitrite produced by NarB from assimilatory nitrate reduction is detoxified by NirK and cNor denitrifying enzymes that convert nitrite into NO which in turn is reduced to NO, respectively.
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http://dx.doi.org/10.3389/fmicb.2019.00980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514139PMC
May 2019

An Integrated Systems Approach Unveils New Aspects of Microoxia-Mediated Regulation in .

Front Microbiol 2019 7;10:924. Epub 2019 May 7.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont USDA 110 (formerly USDA 110). As a first step, the complete genome of 1104, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110. Importantly, the deletion strain showed no significantly different phenotype during symbiosis with several host plants, reinforcing the value of previous OMICS studies. We next performed shotgun proteomics and detected 2,900 and 2,826 proteins in oxically and microoxically grown cells, respectively, largely expanding our knowledge about the inventory of rhizobial proteins expressed in microoxia. A set of 62 proteins was significantly induced under microoxic conditions, including the two nitrogenase subunits NifDK, the nitrogenase reductase NifH, and several subunits of the high-affinity terminal oxidase (FixNOQP) required for bacterial respiration inside nodules. Integration with the previously defined microoxia-induced transcriptome uncovered a set of 639 genes or proteins uniquely expressed in microoxia. Finally, besides providing proteogenomic evidence for novelties, we also identified proteins with a regulation similar to that of FixK: transcript levels of these protein-coding genes were significantly induced, while the corresponding protein abundance remained unchanged or was down-regulated. This suggested that, apart from , additional genes might be under microoxia-specific post-transcriptional control. This hypothesis was indeed confirmed for several targets (HemA, HemB, and ClpA) by immunoblot analysis.
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http://dx.doi.org/10.3389/fmicb.2019.00924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515984PMC
May 2019

Astragalus algarbiensis is nodulated by the genistearum symbiovar of Bradyrhizobium spp. in Morocco.

Syst Appl Microbiol 2019 Jul 25;42(4):440-447. Epub 2019 Mar 25.

Center for Biotechnology, Biodiversity and Environment, Faculty of Sciences, Mohammed V University, Rabat, Morocco. Electronic address:

Astragalus algarbiensis is a wild herbaceous legume growing in Maamora, the most important cork oak forest in northern Africa. It is a plant of great importance as fodder in silvopastoral systems, and in the restoration of poor and degraded soils. The purpose of this study was to describe the biodiversity of rhizobia nodulating this plant and determine their identity. Out of 80 bacterial isolates, 56 strains isolated from root nodules of A. algarbiensis were characterized. ERIC-PCR fingerprinting grouped the strains in two main clusters containing 29 and 27 isolates, respectively, and the amplified ribosomal DNA restriction analysis (ARDRA) generated two different ribotypes. Based on both the ERIC-PCR and ARDRA results, representative strains As21 and As36 were selected for further genetic studies. The nearly complete 16S rRNA gene sequences of As21 and As36 showed that they were closely related to Bradyrhizobium cytisi CTAW11 with similarity values of 99.84% and 99.77%, respectively. Concatenation of atpD, recA, gyrB and dnaK housekeeping gene sequences indicated that strains As21 and As36 had a 95.22% similarity but they showed values of 95.80% and 94.97% with B. cytisi CTAW11, respectively. The sequencing of the symbiotic nodC gene of the two strains revealed 97.20% and 97.76% identities, respectively, with that of B. cytisi CTAW11 isolated from Cytisus villosus growing in the Moroccan Rif Mountains. Furthermore, the phylogenic analysis showed that the strains isolated from A. algarbiensis clustered with B. cytisi and B. rifense within the bradyrhizobia genistearum symbiovar and may constitute two novel genospecies.
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http://dx.doi.org/10.1016/j.syapm.2019.03.004DOI Listing
July 2019

Phylogenetic diversity of Bradyrhizobium strains isolated from root nodules of Lupinus angustifolius grown wild in the North East of Algeria.

Syst Appl Microbiol 2019 May 4;42(3):397-402. Epub 2019 Feb 4.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Granada, Spain.

From a total of 80 bacterial strains isolated from root nodules of Lupinus angustifolius grown wild in the North-Eastern Algerian region of El Tarf, 64 plant host-nodulating strains clustered into 17 random amplified polymorphic DNA (RAPD) fingerprinting groups. The nearly complete 16S rRNA gene sequence from the representative strain of each group revealed they were closely related to members of the genus Bradyrhizobium of the Alphaproteobacteria, but their affiliation at the species level was not clear. Sequencing of the housekeeping genes glnII and recA, and their concatenated phylogenetic analysis, showed that 12 strains belong to B. lupini, other 2 strains affiliated with B. diazoefficiens and that 1 strain was closely related to B. japonicum. The remaining two strains showed similarity values ≤95% with B. cytisi and could represent new lineages within the genus Bradyrhizobium. Sequencing of the symbiotic nodC gene from 4 selected bradyrhizobial strains showed they were all similar to those of the species included in symbiovar genistearum.
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http://dx.doi.org/10.1016/j.syapm.2019.01.003DOI Listing
May 2019

Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes.

J Exp Bot 2018 06;69(15):3703-3714

Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (CSIC), Apartado, Zaragoza, Spain.

Nitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants.
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http://dx.doi.org/10.1093/jxb/ery159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022593PMC
June 2018

Maize Endophytic Bacterial Diversity as Affected by Soil Cultivation History.

Front Microbiol 2018 16;9:484. Epub 2018 Mar 16.

Department of Agricultural Sciences, National University of Huancavelica, Huancavelica, Peru.

The bacterial endophytic communities residing within roots of maize ( L.) plants cultivated by a sustainable management in soils from the Quechua maize belt (Peruvian Andes) were examined using tags pyrosequencing spanning the V4 and V5 hypervariable regions of the 16S rRNA. Across four replicate libraries, two corresponding to sequences of endophytic bacteria from long time maize-cultivated soils and the other two obtained from fallow soils, 793 bacterial sequences were found that grouped into 188 bacterial operational taxonomic units (OTUs, 97% genetic similarity). The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from fallow soils. A mean of 30 genera were found in the fallow soil libraries and 47 were in those from the maize-cultivated soils. Both alpha and beta diversity indexes showed clear differences between bacterial endophytic populations from plants with different soil cultivation history and that the soils cultivated for long time requires a higher diversity of endophytes. The number of sequences corresponding to main genera and in the maize-cultivated libraries were statistically more abundant than those from the fallow soils. Sequences of genera and were significantly more abundant in the libraries from the fallow soils. Relative abundance of genera Glomeribacter, and were similar among libraries. A canonical correspondence analysis of the relative abundance of the main genera showed that the four libraries distributed in two clearly separated groups. Our results suggest that cultivation history is an important driver of endophytic colonization of maize and that after a long time of cultivation of the soil the maize plants need to increase the richness of the bacterial endophytes communities.
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http://dx.doi.org/10.3389/fmicb.2018.00484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890191PMC
March 2018

Overexpression of the periplasmic nitrate reductase supports anaerobic growth by Ensifer meliloti.

FEMS Microbiol Lett 2018 04;365(7)

Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, 18008 Granada, Spain.

The alfalfa endosymbiont Ensifer meliloti strain1021 is known to be an incomplete denitrifier due to its inability to grow anoxically using nitrate as respiratory substrate to produce ATP and grow under anoxic conditions. Although this bacterium contains and expresses the complete set of denitrification genes napEFDABC, nirK, norECBQD and nosRZDFYLX encoding the periplasmic nitrate reductase (Nap), Cu-containing nitrite reductase (NirK), c-type nitric oxide (cNor) and nitrous oxide reductase (Nos), respectively, the reasons of its inability to grow under anoxic conditions are still very poorly understood. In the present study, we have constructed an E. meliloti strain overexpressing napEFDABC genes (Nap+) and demonstrated that this strain is able to grow through anaerobic nitrate respiration. Furthermore, Nap+ showed increased NapC levels as well as Nap, Nir and cNor activities and higher capacity to produce NO and N2O compared to wild-type cells. These results suggest that the inability of E. meliloti to grow under anaerobic conditions using nitrate as electron acceptor is attributable to a limitation in the expression of the periplasmic nitrate reductase.
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http://dx.doi.org/10.1093/femsle/fny041DOI Listing
April 2018

Denitrification and Biodiversity of Denitrifiers in a High-Mountain Mediterranean Lake.

Front Microbiol 2017 6;8:1911. Epub 2017 Oct 6.

Instituto Universitario de Investigación del Agua, Universidad de Granada, Granada, Spain.

Wet deposition of reactive nitrogen (Nr) species is considered a main factor contributing to N inputs, of which nitrate ([Formula: see text]) is usually the major component in high-mountain lakes. The microbial group of denitrifiers are largely responsible for reduction of nitrate to molecular dinitrogen (N) in terrestrial and aquatic ecosystems, but the role of denitrification in removal of contaminant nitrates in high-mountain lakes is not well understood. We have used the oligotrophic, high-altitude La Caldera lake in the Sierra Nevada range (Spain) as a model to study the role of denitrification in nitrate removal. Dissolved inorganic Nr concentration in the water column of la Caldera, mainly nitrate, decreased over the ice-free season which was not associated with growth of microbial plankton or variations in the ultraviolet radiation. Denitrification activity, estimated as nitrous oxide (NO) production, was measured in the water column and in sediments of the lake, and had maximal values in the month of August. Relative abundance of denitrifying bacteria in sediments was studied by quantitative polymerase chain reaction of the 16S rRNA and the two phylogenetically distinct clades I and II genes encoding nitrous oxide reductases. Diversity of denitrifiers in sediments was assessed using a culture-dependent approach and after the construction of clone libraries employing the I gene as a molecular marker. In addition to genera , , , , , , and , which were present in the clone libraries, , , and were also detected in culture media that were not found in the clone libraries. Analysis of biological activities involved in the C, N, P, and S cycles from sediments revealed that nitrate was not a limiting nutrient in the lake, allowed NO production and determined denitrifiers' community structure. All these results indicate that denitrification could be a major biochemical process responsible for the N losses that occur in La Caldera lake.
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http://dx.doi.org/10.3389/fmicb.2017.01911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635049PMC
October 2017

FixK Is the Main Transcriptional Activator of Genes in Response to Low Oxygen.

Front Microbiol 2017 30;8:1621. Epub 2017 Aug 30.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones CientíficasGranada, Spain.

The powerful greenhouse gas, nitrous oxide (NO) has a strong potential to drive climate change. Soils are the major source of NO and microbial nitrification and denitrification the main processes involved. The soybean endosymbiont is considered a model to study rhizobial denitrification, which depends on the , and genes. In this bacterium, the role of the regulatory cascade FixLJ-FixK-NnrR in the expression of , and genes involved in NO synthesis has been previously unraveled. However, much remains to be discovered regarding the regulation of the respiratory NO reductase (NOR), the key enzyme that mitigates NO emissions. In this work, we have demonstrated that genes constitute an operon which is transcribed from a major promoter located upstream of the gene. Low oxygen was shown to be the main inducer of expression of genes and NOR activity, FixK being the regulatory protein involved in such control. Further, by using an transcription assay with purified FixK protein and RNA polymerase we were able to show that the genes are direct targets of FixK.
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http://dx.doi.org/10.3389/fmicb.2017.01621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582078PMC
August 2017

Spatio-Temporal Variations in the Abundance and Structure of Denitrifier Communities in Sediments Differing in Nitrate Content.

Curr Issues Mol Biol 2017 6;24:71-102. Epub 2017 Jul 6.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Granada, Spain.

Spatial and temporal variations related to hydric seasonality in abundance and diversity of denitrifier communities were examined in sediments taken from two sites differing in nitrate concentration along a stream Doñana National Park during a 3-year study. We found a positive relationship between the relative abundance of denitrifiers, determined as , , , and denitrification genes, and sediment nitrate content, with similar spatial and seasonal variations. However, we did not find association between denitrification activity and the community structure of denitrifiers. Because showed the strongest correlation with the content of nitrate in sediments, we used this gene as a molecular marker to construct eight genomic libraries. Analysis of these genomic libraries revealed that diversity of the -bearing communities was higher in the site with higher nitrate content. Regardless of nitrate concentration in the sediments, the Bradyrhizobiaceae and Rhodocyclaceae were the most abundant families. On the contrary, Rhizobiaceae was exclusively present in sediments with higher nitrate content. Results showed that differences in sediment nitrate concentration affect the composition and diversityof -bearing communities.
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http://dx.doi.org/10.21775/cimb.024.071DOI Listing
September 2018

Disparate response to microoxia and nitrogen oxides of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes.

Nitric Oxide 2017 Aug 3;68:137-149. Epub 2017 Feb 3.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), C/Profesor Albareda 1, E-18008, Granada, Spain. Electronic address:

Expression of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes requires low oxygen (O) tension and nitrate (NO), through a regulatory network comprised of two coordinated cascades, FixLJ-FixK-NnrR and RegSR-NifA. To precisely understand how these signals are integrated in the FixLJ-FixK-NnrR circuit, we analyzed β-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 and nnrR mutant backgrounds. While microoxic conditions (2% O at headspace) were sufficient to induce expression of napEDABC and nirK genes and this control depends on FixK, norCBQD expression requires, in addition to microoxia, nitric oxide gas (NO) and both FixK and NnrR transcription factors. Purified FixK 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-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 and NnrR. In this control, expression of napEDABC and nirK genes requires microoxic conditions and directly depends on FixK, while expression of norCBQD genes relies on NO, being NnrR the candidate which directly interacts with the norCBQD promoter.
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http://dx.doi.org/10.1016/j.niox.2017.02.002DOI Listing
August 2017

Evolution of bacterial diversity during two-phase olive mill waste ("alperujo") composting by 16S rRNA gene pyrosequencing.

Bioresour Technol 2017 Jan 29;224:101-111. Epub 2016 Nov 29.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín (EEZ), Agencia Estatal CSIC, E-419, 18080-Granada, Spain.

Microorganisms are the main contributing factor responsible for organic matter degradation during composting. In this research, the 454-pyrosequencing of the 16S rRNA gene was used to elucidate evolution of bacterial diversity during mesophilic, thermophilic and maturation composting stages of the two-phase olive mill waste ("alperujo"), the main by-product of the Spanish olive oil industry. Two similar piles were performance composting AL with sheep manure as bulking agent. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the main phyla found in genomic libraries from each composting phase. Shannon and Chao1 biodiversity indices showed a clear difference between the mesophilic/thermophilic and maturation phases, which was mainly due to detection of new genera. PCA analysis of the relative number of sequences confirmed maturation affected bacterial population structure, and Pearson correlation coefficients between physicochemical composting parameters and relative number of genera sequences suggest that Planomicrobium and Ohtaekwangia could be considered as biomarkers for AL composting maturation.
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http://dx.doi.org/10.1016/j.biortech.2016.11.098DOI Listing
January 2017

Bacterial Communities in the Rhizosphere of Amilaceous Maize (Zea mays L.) as Assessed by Pyrosequencing.

Front Plant Sci 2016 29;7:1016. Epub 2016 Jul 29.

Department of Agricultural Sciences, National University of Huancavelica Huancavelica, Peru.

Maize (Zea mays L.) is the staple diet of the native peasants in the Quechua region of the Peruvian Andes who continue growing it in small plots called chacras following ancestral traditions. The abundance and structure of bacterial communities associated with the roots of amilaceous maize has not been studied in Andean chacras. Accordingly, the main objective of this study was to describe the rhizospheric bacterial diversity of amilaceous maize grown either in the presence or the absence of bur clover cultivated in soils from the Quechua maize belt. Three 16S rRNA gene libraries, one corresponding to sequences of bacteria from bulk soil of a chacra maintained under fallow conditions, the second from the rhizosphere of maize-cultivated soils, and the third prepared from rhizospheric soil of maize cultivated in intercropping with bur clover were examined using pyrosequencing tags spanning the V4 and V5 hypervariable regions of the gene. A total of 26031 sequences were found that grouped into 5955 distinct operational taxonomic units which distributed in 309 genera. The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from bulk soil. One hundred ninety seven genera were found in the bulk soil library and 234 and 203 were in those from the maize and maize/bur clover-cultivated soils. Sixteen out of the 309 genera had a relative abundance higher than 0.5% and the were (in decreasing order of abundance) Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis. Genera Gp4 and Gp6 of the Acidobacteria, Gemmatimonas and Rhodoferax were the most abundant in bulk soil, whereas Flavobacterium, Dechloromonas and Ohtaekwangia were the main genera in the rhizosphere of maize intercropped with bur clover, and Gp4, Subdivision3 genera incertae sedis of phylum Verrucomicrobia, Gp6 and Rhodoferax were the main genera in the rhizosphere of maize plants. Taken together, our results suggest that bur clover produces specific changes in rhizospheric bacterial diversity of amilaceous maize plants.
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http://dx.doi.org/10.3389/fpls.2016.01016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966391PMC
August 2016

Isolation of N2 -fixing rhizobacteria from Lolium perenne and evaluating their plant growth promoting traits.

J Basic Microbiol 2016 Jan 11;56(1):85-91. Epub 2015 Sep 11.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Granada, Spain.

Twenty one dinitrogen (N2 )-fixing bacteria were isolated from the rhizosphere of Lolium perenne grown for more than 10 years without N-fertilization. The nearly complete sequence of the 16S rRNA gene of each strain and pairwise alignments among globally aligned sequences of the 16S rRNA genes clustered them into nine different groups. Out of the 21 strains, 11 were members of genus Bacillus, 3 belonged to each one of genera Paenibacillus and Pseudoxanthomonas, and the remaining 2 strains to each one of genera Burkholderia and Staphylococcus, respectively. A representative strain from each group contained the nifH gene and fixed atmospheric N2 as determined by the acetylene-dependent ethylene production assay (acetylene reduction activity, ARA). The nine selected strains were also examined to behave as plant growth promoting bacteria (PGPRs) including their ability to act as a biocontrol agent. The nine representative strains produced indol acetic acid (IAA) and solubilized calcium triphosphate, five of them, strains C2, C3, C12, C15, and C16, had ACC deaminase activity, and strains C2, C3, C4, C12, C16, and C17 produced siderophores. Strains C13, C16, and C17 had the capability to control growth of the pathogen Fusarium oxysporum mycelial growth in vitro. PCA analysis of determined PGPR properties showed that ARA, ACC deaminase activity, and siderophore production were the most valuable as they had the maximal contribution to the total variance.
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http://dx.doi.org/10.1002/jobm.201500247DOI Listing
January 2016

An integrated biochemical system for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum.

Biochem J 2016 Feb 12;473(3):297-309. Epub 2015 Nov 12.

Estación Experimental del Zaidín, CSIC, PO Box 419, Granada 18080, Spain

Rhizobia are recognized to establish N2-fixing symbiotic interactions with legume plants. Bradyrhizobium japonicum, the symbiont of soybeans, can denitrify and grow under free-living conditions with nitrate (NO3 (-)) or nitrite (NO2 (-)) as sole nitrogen source. Unlike related bacteria that assimilate NO3 (-), genes encoding the assimilatory NO3 (-) reductase (nasC) and NO2 (-) reductase (nirA) in B. japonicum are located at distinct chromosomal loci. The nasC gene is located with genes encoding an ABC-type NO3 (-) transporter, a major facilitator family NO3 (-)/NO2 (-) transporter (NarK), flavoprotein (Flp) and single-domain haemoglobin (termed Bjgb). However, nirA clusters with genes for a NO3 (-)/NO2 (-)-responsive regulator (NasS-NasT). In the present study, we demonstrate NasC and NirA are both key for NO3 (-) assimilation and that growth with NO3 (-), but not NO2 (-) requires flp, implying Flp may function as electron donor to NasC. In addition, bjgb and flp encode a nitric oxide (NO) detoxification system that functions to mitigate cytotoxic NO formed as a by-product of NO3 (-) assimilation. Additional experiments reveal NasT is required for NO3 (-)-responsive expression of the narK-bjgb-flp-nasC transcriptional unit and the nirA gene and that NasS is also involved in the regulatory control of this novel bipartite assimilatory NO3 (-)/NO2 (-) reductase pathway.
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http://dx.doi.org/10.1042/BJ20150880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724949PMC
February 2016

Anoxic growth of Ensifer meliloti 1021 by N2O-reduction, a potential mitigation strategy.

Front Microbiol 2015 27;6:537. Epub 2015 May 27.

Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Spanish Council for Scientific Research Granada, Spain.

Denitrification in agricultural soils is a major source of N2O. Legume crops enhance N2O emission by providing N-rich residues, thereby stimulating denitrification, both by free-living denitrifying bacteria and by the symbiont (rhizobium) within the nodules. However, there are limited data concerning N2O production and consumption by endosymbiotic bacteria associated with legume crops. It has been reported that the alfalfa endosymbiont Ensifer meliloti strain 1021, despite possessing and expressing the complete set of denitrification enzymes, is unable to grow via nitrate respiration under anoxic conditions. In the present study, we have demonstrated by using a robotized incubation system that this bacterium is able to grow through anaerobic respiration of N2O to N2. N2O reductase (N2OR) activity was not dependent on the presence of nitrogen oxyanions or NO, thus the expression could be induced by oxygen depletion alone. When incubated at pH 6, E. meliloti was unable to reduce N2O, corroborating previous observations found in both, extracted soil bacteria and Paracoccus denitrificans pure cultures, where expression of functional N2O reductase is difficult at low pH. Furthermore, the presence in the medium of highly reduced C-substrates, such as butyrate, negatively affected N2OR activity. The emission of N2O from soils can be lowered if legumes plants are inoculated with rhizobial strains overexpressing N2O reductase. This study demonstrates that strains like E. meliloti 1021, which do not produce N2O but are able to reduce the N2O emitted by other organisms, could act as even better N2O sinks.
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http://dx.doi.org/10.3389/fmicb.2015.00537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443521PMC
June 2015

Phenotypic and genetic characterization of rhizobia isolated from Hedysarum flexuosum in Northwest region of Morocco.

J Basic Microbiol 2015 Jul 27;55(7):830-7. Epub 2015 Feb 27.

Lab. Valorisation Biotechnologique des Microorganismes, Faculty of Sciences and Techniques, University Abdelmalek Essaadi, Tanger, Morocco.

Seventy bacterial strains were isolated from root nodules of the legume Hedysarum flexuosum grown wild in soils from Northwest Morocco. Repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) clustered the strains into 30 REP-PCR groups. The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR pattern showed that 17 strains were closely related to members of the genus Rhizobium of the family Rhizobiaceae of the Alphaproteobacteria. Pairwise alignments between globally aligned sequences of the 16S rRNA gene indicated that the strains from H. flexuosum had 99.75-100% identity with Rhizobium sullae type strain IS123(T). The phenotypic characteristics analyzed allowed description of a wide physiological diversity among the isolates, where the carbohydrate assimilation test was the most discriminating. Analysis of the 16S rRNA gene of a representative strains from the remaining 13 REP-PCR groups showed they belong to a wide variety of phylogenetic groups being closely related to species of genera Stenotrophomonas, Serratia, Massilia, Acinetobacter, Achromobacter, and Pseudomonas from the Beta- and Gammaproteobacteria. The R. sullae strains identified in this study produced effective symbiosis with their original host plant. None of the other bacterial strains could form nodules on H. flexuosum.
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http://dx.doi.org/10.1002/jobm.201400790DOI Listing
July 2015
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