Raúl Platero

Sinorhizobium meliloti Fur-Like (Mur) Protein Binds a Fur Box-Like Sequence Present in the mntA Promoter in a Manganese-Responsive Manner

ABSTRACT In Sinorhizobium meliloti, the MurSm protein, a homologue of the ferric uptake regulator (Fur), mediates manganese-dependent regulation of the MntABCD manganese uptake system. In this study, we analyzed MurSm binding to the promoter region of the S. meliloti mntA gene. We demonstrated that MurSm 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 MurSm 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 MurSm binding site, present in the mntA promoter region, is similar to a Fur box; however, manganese-activated MurSm binds a canonical Fur box with very low affinity. Furthermore, the data obtained indicate that MurSm responds to physiological concentrations of manganese.

New betaproteobacterial Rhizobium strains able to efficiently nodulate Parapiptadenia rigida (Benth.) Brenan.

ABSTRACT 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.

Identification of an Iron-Regulated, Hemin-Binding Outer Membrane Protein in Sinorhizobium meliloti

ABSTRACT 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.

Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida

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′ (KD = 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 KD 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.

Novel Cupriavidus Strains Isolated from Root Nodules of Native Uruguayan Mimosa Species

ABSTRACT 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. IMPORTANCE With 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.

Intracellular Fe content influences nodulation competitiveness of Sinorhizobium meliloti strains as inocula of alfalfa

Abstract Rhizobia, as well as most soil bacteria, frequently face variable Fe conditions. The effects of Fe limitation or starvation upon rhizobia infectiveness are not fully understood. Our aim was to evaluate the effects of Fe limitation as well as the ability to acquire Fe in rhizobia competitiveness. Sinorhizobium meliloti 242 wild type strain and one of two-iron acquisition mutants (2.1 and 5.6) were co-inoculated at equal ratio onto alfalfa plants. Legumes were grown under gnotobiotic conditions in Fe-supplemented or Fe-chelated defined medium. Our results show that highly efficient Fe acquisition systems are involved in nodule competitiveness when Fe availability is low. Moreover, Fe-scarce inocula were out-competed by Fe-sufficient inocula.

The Crp regulator of Pseudomonas putida: evidence of an unusually high affinity for its physiological effector, cAMP

Although the genome of Pseudomonas putida KT2440 encodes an orthologue of the crp gene of Escherichia coli (encoding the cAMP receptor protein), the regulatory scope of this factor seems to be predominantly co-opted in this bacterium for controlling non-metabolic functions. In order to investigate the reasons for such a functional divergence in otherwise nearly identical proteins, the Crp regulator of P. putida (Crp(P. putida)) was purified to apparent homogeneity and subject to a battery of in vitro assays aimed at determining its principal physicochemical properties. Analytical ultracentrifugation indicated effector-free Crp(P. putida) to be a dimer in solution that undergoes a significant change in its hydrodynamic shape in the presence of cAMP. Such a conformational transition was confirmed by limited proteolysis of the protein in the absence or presence of the inducer. Thermodynamic parameters calculated by isothermal titration calorimetry revealed a tight cAMP-Crp(P. putida) association with an apparent K(D) of 22.5 ± 2.8 nM, i.e. much greater affinity than that reported for the E. colis counterpart. The regulator also bound cGMP, but with a K(D) = 2.6 ± 0.3 µM. An in vitro transcription system was then set up with purified P. putidas RNA polymerase for examining the preservation of the correct protein-protein architecture that makes Crp to activate target promoters. These results, along with cognate gel retardation assays indicated that all basic features of the reference Crp(E. coli) protein are kept in the P. putidas counterpart, albeit operating under a different set of parameters, the extraordinarily high affinity for cAMP being the most noticeable.

Draft Genome Sequence of Cupriavidus UYMMa02A, a Novel Beta-Rhizobium Species

ABSTRACT We present the draft genome of Cupriavidus UYMMa02A, a rhizobium strain isolated from root nodules of Mimosa magentea. The assembly has approximately 8.1 million bp with an average G+C of 64.1%. Symbiotic and metal-resistance genes were identified. The study of this genome will contribute to the understanding of rhizobial evolution.

Herbaspirillum seropedicae Differentially Expressed Genes in Response to Iron Availability

Herbaspirillum seropedicae Z67 is a nitrogen-fixing endophyte that colonizes many important crops. Like in almost all organisms, vital cellular processes of this endophyte are iron dependent. In order to efficiently acquire iron to fulfill its requirements, this bacterium produces the siderophores serobactins. However, the presence in its genome of many others iron acquisition genes suggests that serobactins are not the only strategy used by H. seropedicae to overcome metal deficiency. The aim of this work was to identify genes and proteins differentially expressed by cells growing in low iron conditions in order to describe H. seropedicae response to iron limitation stress. For this purpose, and by using a transcriptomic approach, we searched and identified a set of genes up-regulated when iron was scarce. One of them, Hsero_2337, codes for a TonB-dependent transporter/transducer present in the serobactins biosynthesis genomic locus, with an unknown function. Another TonB-dependent receptor, the one encoded by Hsero_1277, and an inner membrane ferrous iron permease, coded by Hsero_2720, were also detected. By using a proteomic approach focused in membrane proteins, we identified the specific receptor for iron-serobactin internalization SbtR and two non-characterized TonB-dependent receptors (coded by genes Hsero_1277 and Hsero_3255). We constructed mutants on some of the identified genes and characterized them by in vitro growth, biofilm formation, and interaction with rice plants. Characterization of mutants in gene Hsero_2337 showed that the TonB-dependent receptor coded by this gene has a regulatory role in the biosynthesis of serobactins, probably by interacting with the alternative sigma factor PfrI, coded by gene Hsero_2338. Plant colonization of the mutant strains was not affected, since the mutant strain normally colonize the root and aerial part of rice plants. These results suggest that the strategies used by H. seropedicae to acquire iron inside plants are far more diverse than the ones characterized in this work. In vivo expression studies or colonization competition experiments between the different mutant strains could help us in future works to determine the relative importance of the different iron acquisition systems in the interaction of H. seropedicae with rice plants.

The interplay of EIIANtr with C-source regulation of the Pu promoter of Pseudomonas putida mt-2: Role of PtsN in Pu promoter outcome

The presence of some sugars (e.g. glucose) downregulates the activity of the Pu promoter of plasmid pWW0 of Pseudomonas putida mt-2, which drives the upper TOL operon for biodegradation of m-xylene. Genetic evidence produced 20 years ago documented an effect of the EIIANtr (PtsN) protein of the nitrogen-related phosphoenolpyruvate-dependent phosphotransferase system (PTSNtr ) in such a C-source control of Pu activity. In this study, we have exploited the wealth of recent information on the PTS of P. putida as well as transcriptomic data available in the last few years on this bacterium to revisit this question - and the role of EIIANtr as such. To this end, we examined Pu output under physiological conditions known to either phosphorylate PTS proteins to saturation or to deplete them altogether from high-energy phosphate. The results showed that Pu activity is checked by EIIANtr regardless of its phosphorylation state. However, such inhibition is intensified during growth on glucose (which correlates with more phosphate-free EIIANtr ) and partially relieved in fructose, which triggers phosphorylation of PTS proteins. These data explain former inconsistencies on the Pu-PTSNtr interplay and provides a better understanding of the metabolic and regulatory retroactivity between the TOL plasmid and its host metabolism.