The ‘Standard European Vector Architecture’ database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

Fur is a transcriptional regulator involved in iron-dependent control of gene expression in many bacteria. In this work we analyzed the phenotype of a fur mutant in Sinorhizobium meliloti, an ␣-proteobacterium that fixes N 2 in association with host plants. We demonstrated that some functions involved in high-affinity iron transport, siderophore production, and iron-regulated outer membrane protein expression respond to iron in a Fur-independent manner. However, manganese-dependent expression of the MntABCD manganese transport system was lost in a fur strain as discerned by constitutive expression of a mntA::gfp fusion reporter gene in the mutant. Thus, Fur directly or indirectly regulates a manganese-dependent function. The data indicate a novel function for a bacterial Fur protein in mediating manganese-dependent regulation of gene expression.

c Among the leguminous trees native to Uruguay, Parapiptadenia rigida (Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with P. rigida. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their nifH and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera Burkholderia, Cupriavidus, and Rhizobium, among which the Burkholderia genotypes were the predominant group. Phylogenetic studies of nifH, nodA, and nodC sequences from the Burkholderia and the Cupriavidus isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the Cupriavidus isolates were able to effectively nodulate Mimosa pudica, the Burkholderia isolates produced white and ineffective nodules on this host. Parapiptadenia rigida (Benth.) Brenan, which is also known by its vernacular names Angico, Angico vermelho, and Gurucaia, belongs to the tribe Mimoseae within the Mimosoideae subfamily of the Fabaceae (Leguminosae) (5,26,27,38). It is native to southern South America (south Brazil, Argentina, Paraguay, and Uruguay), where it can be found as one of the tallest species in the canopy of riverside forests, where it can reach heights of approximately 30 m and breast height diameters of from 30 to 120 cm. The wild tree is currently exploited by the locals owing to its economic value, although commercial cultivation of P. rigida has never been developed in Uruguay. Its main economic value is based on the excellence of its timber, which is appreciated for its high density (0.74 to 0.98 g/cm 3 ) and natural durability (26). It is mostly used for high-quality furniture, house construction, carpentry, and fire wood, and the reddish brown parquet floors built with its timber are deeply valued. Other reported uses are as a source of gums, tannins, and essential oils as well as for medicinal purposes (14, 37). Indeed, it is much appreciated by people in Brazil for its medicinal qualities and is duly included in the Brazilian Pharmacopeia.This heliophyte species is part of the forest succession during the first steps of recovery of degraded areas, as it can grow under adverse and low-soil-fertility conditions. Its ability to establish a nitrogen-fixing association with rhizobia is well documented (18)(19)(20)32), but information about the rhizobia associated with this leguminous...

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5-TTAAACGTTTCA-3 (K D ‫؍‬ 26.3 ؎ 3.1 nM) and that F1P binds the protein with an apparent stoichiometry of 1.06 ؎ 0.06 molecules per Cra monomer and a K D of 209 ؎ 20 nM. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.The central metabolism of bacteria is controlled through the interplay of global and specific regulators, the outcome of which depends on the nature of the carbon and energy sources and the particular culture conditions (1). The catabolite repressor/activator (Cra) protein (also known as FruR) is a pleiotropic regulatory protein that plays a key role in the control of carbon flow in Escherichia coli and Salmonella typhimurium (2). This factor was first identified as a repressor that inhibited expression of the fructose operon of these two bacteria when the sugar was not available, thereby the earlier name FruR (3). Later work, however, revealed that the same protein represses genes for many other enzymes of the central metabolism (pfkA, pykA, pykF, acnB, edd, eda, mtlADR, and gapB; and activates others (ppsA, fbp, pckA, acnA, icd, aceA, and aceB; Refs. 2 and 7-10), suggesting a dual character of Cra as both a transcriptional repressor and an activator.From a structural point of view, the Cra protein has been classified as a member of the GalR-LacI superfamily of DNAbinding transcriptional regulators (11,12). In E. coli, the Cra monomer is organized in two functional domains. The N-terminal helix-turn-helix module accounts for the binding of the protein to the cognate DNA operator sequence, whereas the C-terminal portion mediates the interactions between subunits and triggers changes in the protein upon effector binding (12).Earlier studies of the Cra proteins of E. coli and S. typhimurium suggested that the regulator is a tetramer that recognizes an imperfect palindromic DNA sequence to which it bin...

The large legume genus Mimosa is known to be associated with both alphaproteobacterial and betaproteobacterial symbionts, depending on environment and plant taxonomy, e.g., Brazilian species are preferentially nodulated by Burkholderia, whereas those in Mexico are associated with alphaproteobacterial symbionts. Little is known, however, about the symbiotic preferences of Mimosa spp. at the southern subtropical limits of the genus. In the present study, rhizobia were isolated from field-collected nodules from Mimosa species that are native to a region in southern Uruguay. Phylogenetic analyses of sequences of the 16S rRNA, recA, and gyrB core genome and the nifH and nodA symbiosis-essential loci confirmed that all the isolates belonged to the genus Cupriavidus. However, none were in the well-described symbiotic species C. taiwanensis, but instead they were closely related to other species, such as C. necator, and to species not previously known to be symbiotic (or diazotrophic), such as C. basilensis and C. pinatubonensis. Selection of these novel Cupriavidus symbionts by Uruguayan Mimosa spp. is most likely due to their geographical separation from their Brazilian cousins and to the characteristics of the soils in which they were found. IMPORTANCEWith the aim of exploring the diversity of rhizobia associated with native Mimosa species, symbionts were isolated from root nodules on five Mimosa species that are native to a region in southern Uruguay, Sierra del Abra de Zabaleta. In contrast to data obtained in the major centers of diversification of the genus Mimosa, Brazil and Mexico, where it is mainly associated with Burkholderia and Rhizobium/Ensifer, respectively, the present study has shown that all the isolated symbiotic bacteria belonged to the genus Cupriavidus. Interestingly, none of nodules contained bacteria belonging to the well-described symbiotic species C. taiwanensis, but instead they were related to other Cupriavidus species such as C. necator and C. pinatubonensis. These data suggest the existence of a higher diversity within beta-rhizobial Cupriavidus than was previously suspected, and that Mimosa spp. from Sierra del Abra de Zabaleta, may be natural reservoirs for novel rhizobia.

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

In Sinorhizobium meliloti, the Mur Sm protein, a homologue of the ferric uptake regulator (Fur), mediates manganese-dependent regulation of the MntABCD manganese uptake system. In this study, we analyzed Mur Sm binding to the promoter region of the S. meliloti mntA gene. We demonstrated that Mur Sm protein binds with high affinity to the promoter region of mntA gene in a manganese-responsive manner. Moreover, the results presented here indicate that two monomers, or one dimer, of Mur Sm binds the DNA. The binding region was identified by DNase I footprinting analysis and covers a region of about 30 bp long that contains a palindromic sequence. The Mur Sm binding site, present in the mntA promoter region, is similar to a Fur box; however, manganese-activated Mur Sm binds a canonical Fur box with very low affinity. Furthermore, the data obtained indicate that Mur Sm responds to physiological concentrations of manganese.

Rhizobia are soil bacteria that are able to establish symbiotic associations with leguminous hosts. In iron-limited environments these bacteria can use iron present in heme or heme compounds (hemoglobin, leghemoglobin). Here we report the presence in Sinorhizobium meliloti of an iron-regulated outer membrane protein that is able to bind hemin but not hemoglobin. Protein assignment was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Tryptic peptides correlated with the mass measurements obtained accounted for 54% of the translated sequence of a putative heme receptor gene present in the chromosome of S. meliloti 1021. The results which we obtained suggest that this protein (designated ShmR for Sinorhizobium heme receptor) is involved in high-affinity heme-mediated iron transport.