Solange Oliveira

Structure of a Dioxygen Reduction Enzyme from Desulfovibrio Gigas

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 Å resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-β-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.

STUDIES ON THE REDOX CENTERS OF THE TERMINAL OXIDASE FROM DESULFOVIBRIO GIGAS AND EVIDENCE FOR ITS INTERACTION WITH RUBREDOXIN

Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducerDesulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at ∼2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 ± 15 mV for the quinone/semiquinone couple and at −130 ± 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is −350 ± 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.

Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus?

The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

Aims:  To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains.

Desulfovibrio gigas Flavodiiron Protein Affords Protection against Nitrosative Stress In Vivo

Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O(2). Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas Deltaroo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O(2) reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.

Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species

Chickpea has been considered as a restrictive host for nodulation by rhizobia. However, recent studies have reported that several Mesorhizobium species may effectively nodulate chickpea. With the purpose of investigating the evolutionary relationships between these different species with the ability of nodulating the same host, we analysed 21 Portuguese chickpea rhizobial isolates. Symbiosis genes nifH and nodC were sequenced and used for phylogenetic studies. Symbiotic effectiveness was determined to evaluate its relationship with symbiosis genes. The comparison of 16S rRNA gene-based phylogeny with the phylogenies based on symbiosis genes revealed evidence of lateral transfer of symbiosis genes across different species. Chickpea is confirmed as a nonpromiscuous host. Although chickpea is nodulated by many different species, they share common symbiosis genes, suggesting recognition of only a few Nod factors by chickpea. Our results suggest that sequencing of nifH or nodC genes can be used for rapid detection of chickpea mesorhizobia.

High diversity of chickpea Mesorhizobium species isolated in a Portuguese agricultural region

Chickpea rhizobia isolated from Portuguese soils were assigned to the genus Mesorhizobium by 16S-rDNA sequencing. High species diversity was found within populations of an agricultural region in the south of Portugal. Besides the expected Mesorhizobium ciceri and M. mediterraneum, some isolates were close to M. loti or M. tianshanense and some formed a clade that may represent a new species. A new PCR-based approach, named direct amplified polymorphic DNA (DAPD) analysis, supported the 16S-based phylogeny. This suggests that this method could be used as a molecular tool to assess genetic relationships. Evaluation of genetic diversity by 16S-rDNA sequence, DAPD and protein profiles showed different levels of heterogeneity in natural populations.

Effect of heat and pH stress in the growth of chickpea mesorhizobia.

The development of rhizobial inoculants requires the selection of isolates that are symbiotically efficient as well as adapted to the local environmental conditions. Our aim was to find indigenous chickpea rhizobia tolerant to adverse environmental conditions, such as temperature and pH. Thirteen isolates of chickpea mesorhizobia from southern Portugal were examined. Tolerance to stress temperatures and pH was evaluated by quantification of bacterial growth at 20–37°C and pH 5–9, respectively. Tolerance to heat shocks was studied by submitting isolates to 46°C and 60°C. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis protein analysis revealed qualitative and quantitative differences when isolates were submitted to temperature stress. A 60-kDa protein was overproduced by all isolates under heat stress. Almost all isolates revealed to be more tolerant to 20°C than to 37°C. A positive correlation was found between the maximum growth pH and the isolate origin soil pH. Generally, isolates more tolerant to temperature stress showed a lower symbiotic efficiency.

Enhanced chickpea growth-promotion ability of a Mesorhizobium strain expressing an exogenous ACC deaminase gene

AimsThe main goal of the study reported herein was to assess the nodulation performance of a Mesorhizobium strain transformed with an exogenous ACC deaminase gene (acdS), and its subsequent ability to increase chickpea plant growth under normal and waterlogged conditions.MethodsThe Mesorhizobium ciceri strain LMS-1 was transformed with the acdS gene of Pseudomonas putida UW4 by triparental conjugation using plasmid pRKACC. A plant growth assay was conducted to verify the plant growth promotion ability of the LMS-1 (pRKACC) transformed strain under normal and waterlogging conditions. Bacterial ACC deaminase and nitrogenase activity was measured.ResultsBy expressing the exogenous acdS gene, the transformed strain LMS-1 showed a 127% increased ability to nodulate chickpea and a 125% promotion of the growth of chickpea compared to the wild-type strain, under normal conditions. Plants inoculated with the LMS-1 wild-type strain showed a higher nodule number under waterlogging stress than under control conditions, suggesting that waterlogging increases nodulation in chickpea. No significant relationship was found between ACC deaminase and nitrogenase activity.ConclusionsThe results obtained in this study show that the use of rhizobial strains with improved ACC deaminase activity might be very important for developing microbial inocula for agricultural purposes.

Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species

The genus Mesorhizobium includes species nodulating several legumes, such as chickpea, which has a high agronomic importance. Chickpea rhizobia were originally described as either Mesorhizobium ciceri or M. mediterraneum. However, rhizobia able to nodulate chickpea have been shown to belong to several different species within the genus Mesorhizobium. The present study used a multilocus sequence analysis approach to infer a high resolution phylogeny of the genus Mesorhizobium and to confirm the existence of a new chickpea nodulating genospecies. The phylogenetic structure of the Mesorhizobium clade was evaluated by sequence analysis of the 16S rRNA gene, ITS region and the five core genes atpD, dnaJ, glnA, gyrB, and recA. Phylogenies obtained with the different genes are in overall good agreement and a well-supported, almost fully resolved, phylogenetic tree was obtained using the combined data. Our phylogenetic analyses of core genes sequences and their comparison with the symbiosis gene nodC, corroborate the existence of one new chickpea Mesorhizobium genospecies and one new symbiovar, M. opportunistum sv. ciceri. Furthermore, our results show that symbiovar ciceri spreads over six species of mesorhizobia. To our knowledge this study shows the most complete Mesorhizobium multilocus phylogeny to date and contributes to the understanding of how a symbiovar may be present in different species.