Fine-Tuning Modulation of Oxidation-Mediated Posttranslational Control of Bradyrhizobium diazoefficiens FixK2 Transcription Factor.

FixK2 is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK2 gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK2 activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK2, we replaced C183 for aspartic acid. Purified C183D FixK2 protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK2, expression of a fixNOQP'-'lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK2 encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK2 gene expression levels and FixK2 protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK2 encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK2 in vivo.

Regulation of the Emissions of the Greenhouse Gas Nitrous Oxide by the Soybean Endosymbiont Bradyrhizobium diazoefficiens.

The greenhouse gas nitrous oxide (N2O) has strong potential to drive climate change. Soils are a major source of N2O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the napEDABC, nirK, norCBQD, and nosRZDYFLX genes, which sequentially reduce nitrate (NO3-) to nitrite (NO2-), nitric oxide (NO), N2O, and dinitrogen (N2). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK2 and NnrR transcriptional regulators. To understand the role of FixK2 and NnrR proteins in N2O turnover, we monitored real-time kinetics of NO3-, NO2-, NO, N2O, N2, and oxygen (O2) in a fixK2 and nnrR mutant using a robotized incubation system. We confirmed that FixK2 and NnrR are regulatory determinants essential for NO3- respiration and N2O reduction. Furthermore, we demonstrated that N2O reduction by B. diazoefficiens is independent of canonical inducers of denitrification, such as the nitrogen oxide NO3-, and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N2O turnover in B. diazoefficiens.

Agrobacterium leguminum sp. nov., isolated from nodules of Phaseolus vulgaris in Spain.

Two endophytic strains, coded MOVP5T and MOPV6, were isolated from nodules of Phaseolus vulgaris plants grown on agricultural soil in Southeastern Spain, and were characterized through a polyphasic taxonomy approach. Their 16S rRNA gene sequences showed 99.3 and 99.4 %, 98.9 and 99.6 %, and 99.0 and 98.7% similarity to 'A. deltaense' YIC 4121T, A. radiobacter LGM 140T, and A. pusense NRCPB10T, respectively. Multilocus sequence analysis based on sequences of recA and atpD genes suggested that these two strains could represent a new Agrobacterium species with less than 96.5 % similarity to their closest relatives. PCR amplification of the telA gene, involved in synthesis of protelomerase, confirmed the affiliation of strains MOPV5T and MOPV6 to the genus Agrobacterium. Whole genome average nucleotide identity and digital DNA-DNA hybridization average values were less than 95.1 and 66.7 %, respectively, with respect to its closest related species. Major fatty acids in strain MOPV5T were C18 : 1 ω7c/C18 : 1 ω6c in summed feature 8, C19 : 0 cyclo ω8c, C16 : 0 and C16 : 0 3-OH. Colonies were small to medium, pearl-white coloured on YMA at 28 °C and growth was observed at 10-42 °C, pH 5.0-10.0 and with 0.0-0.5 % (w/v) NaCl. The DNA G+C content was 59.9 mol%. These two strains differ from all other genomovars of Agrobacterium found so far, including those that have not yet given a Latin name. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strain MOPV5T as representing a novel species of Agrobacterium, for which the name Agrobacterium leguminum sp. nov. is proposed. The type strain is MOPV5T (=CECT 30096T=LMG 31779T).

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.

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.

Ensifer meliloti is the preferred symbiont of Medicago arborea in eastern Morocco soils.

Polyphasic characterization of 61 bacteria isolated from root nodules of Medicago arborea (Medic tree) plants growing in 4 arid soils of the arid eastern area of Morocco was studied. All the isolates characterized were fast growers. The phenotypic, symbiotic, and cultural characteristics analyzed allowed the description of a broad physiological diversity among the isolates. The results obtained suggest that the phenotype of these rhizobia might have evolved to adapt to the local conditions. The genetic characterization consisted of an analysis of the rep-PCR (repetitive extragenic palindromic polymerase chain reaction) fingerprints and a PCR-based RFLP (restriction fragment length polymorphism) of the 16S rDNA patterns. The diversity of the isolates was investigated by rep-PCR, giving a similarity of 62%, delineated into 3 clusters, 4 groups, and 6 subclusters. This wide diversity was also observed by a phenotypic approach, where the carbohydrate assimilation test was the most discriminating. The results show a relationship between rep-PCR fingerprinting and sugar assimilation, which are complementary in diversity investigation. The nearly complete 16S rRNA gene sequence from representative strains of each soil showed they are closely related to members of the genus Ensifer of the family Rhizobiaceae within the Alphaproteobacteria and shows the highest similitude values (99.93%/100%) with Ensifer meliloti LMG 6133(T) (X67222). Sequencing of the symbiotic nodC gene from 7 representative strains revealed they had 94.89% identity with the nodC sequence of the type strain E. meliloti LMG 6133(T) (EF428922). Therefore, the 61 M. arborea isolates from the 4 different soils have the same phylogenetic affiliation, which proves the restricted host specificity among M. arborea species.

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma.

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086(T), with 99.41% identity with respect to the strain Ro19(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19(T) and B. lablabi CCBAU 23086(T), with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086(T). Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19(T)=LMG 27393(T)=CECT 8261(T)). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name "retamae", that mainly contains nodulating strains isolated from Retama species in different continents.

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif.

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).

Bacterial adaptation of respiration from oxic to microoxic and anoxic conditions: redox control.

Under a shortage of oxygen, bacterial growth can be faced mainly by two ATP-generating mechanisms: (i) by synthesis of specific high-affinity terminal oxidases that allow bacteria to use traces of oxygen or (ii) by utilizing other substrates as final electron acceptors such as nitrate, which can be reduced to dinitrogen gas through denitrification or to ammonium. This bacterial respiratory shift from oxic to microoxic and anoxic conditions requires a regulatory strategy which ensures that cells can sense and respond to changes in oxygen tension and to the availability of other electron acceptors. Bacteria can sense oxygen by direct interaction of this molecule with a membrane protein receptor (e.g., FixL) or by interaction with a cytoplasmic transcriptional factor (e.g., Fnr). A third type of oxygen perception is based on sensing changes in redox state of molecules within the cell. Redox-responsive regulatory systems (e.g., ArcBA, RegBA/PrrBA, RoxSR, RegSR, ActSR, ResDE, and Rex) integrate the response to multiple signals (e.g., ubiquinone, menaquinone, redox active cysteine, electron transport to terminal oxidases, and NAD/NADH) and activate or repress target genes to coordinate the adaptation of bacterial respiration from oxic to anoxic conditions. Here, we provide a compilation of the current knowledge about proteins and regulatory networks involved in the redox control of the respiratory adaptation of different bacterial species to microxic and anoxic environments.

Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici.

Fifteen isolates from several nodulated tropical legumes from Puerto Rico (USA) were characterised by their phenotypic, molecular and symbiotic features. The identification of isolates was based on a polyphasic approach, including phenotypic characteristics, 16S rRNA sequencing, Low molecular weight (LMW) RNA profiles, Two Primers-RAPD patterns, and restriction patterns from 16S rDNA molecules. Despite of the variety of hosts included in this study the 15 isolates were separated into only two groups that corresponded to Rhizobium gallicum and Rhizobium tropici. This work shows that R. gallicum and R. tropici nodulate legume plants, such as Sesbania, Caliandra, Poitea, Piptadenia, Neptunia and Mimosa species, that were not previously considered as hosts for these rhizobia. Moreover, some of these host plants can be nodulated by both species. The results confirm the great promiscuity of R. tropici and also support the hypothesis that the species R. gallicum may be native from America or cosmopolitan and worldwide spread.