Antonio J. Palomares

In situ phytostabilisation of heavy metal polluted soils using Lupinus luteus inoculated with metal resistant plant-growth promoting rhizobacteria.

The aim of this work is the evaluation of metal phytostabilisation potential of Lupinus luteus inoculated with Bradyrhizobium sp. 750 and heavy metal resistant PGPRs (plant-growth promoting rhizobacteria), for in situ reclamation of multi-metal contaminated soil after a mine spill. Yellow lupines accumulated heavy metals mainly in roots (Cu, Cd and especially Pb were poorly translocated to shoots). This indicates a potential use of this plant in metal phytostabilisation. Furthermore, As accumulation was undetectable. On the other hand, zinc accumulation was 10-100 times higher than all other metals, both in roots and in shoots. Inoculation with Bradyrhizobium sp. 750 increased both biomass and nitrogen content, indicating that nitrogen fixation was effective in soils with moderate levels of contamination. Co-inoculation of lupines with a consortium of metal resistant PGPR (including Bradyrhizobium sp., Pseudomonas sp. and Ochrobactrum cytisi) produced an additional improvement of plant biomass. At the same time, a decrease in metal accumulation was observed, both in shoots and roots, which could be due to a protective effect exerted on plant rhizosphere. Our results indicate the usefulness of L. luteus inoculated with a bacterial consortium of metal resistant PGPRs as a method for in situ reclamation of metal polluted soils.

STRUCTURE AND ROLE IN SYMBIOSIS OF THE EXOB GENE OF RHIZOBIUM LEGUMINOSARUM BV TRIFOLII

Abstract The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifoliiexoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction.

The influence of several variables for nitrosoguanidine mutagenesis inLactobacillus plantarum

Lactobacillus plantarum is a lactic acid bacterium responsible for the lactic fermentation of Spanish-type green olives. N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) appears to be the most potent chemical mutagen and now is widely used by bacterial geneticists because it induces a high frequency of mutation. The efficiency of the MNNG treatment under a variety of conditions was examined, and it was found that the optimal effects are achieved by exposing a suspension of about 109 log-phase cells to 500μg MNNG in 1 ml Tris-maleate buffer, 0.2M, pH 5.5, for 30 min at 28°C in the dark and without shaking.

Correlation between extracellular polysaccharide composition and nodulating ability inRhizobium trifolii

SummaryTwo auxotrophic mutants ofRhizobium trifolii which are deficient in nodulating ability have been isolated. Both mutants (strain RS 164 His− and strain RS213 Leu−) appear to synthesize abnormal extracellular polysaccharides as compared with the wild type strain RS 55. Simultaneous recovery of nodulating ability and wild type polysaccharide composition has been found in a Leu+ revertant of strain RS 213.

Plasmid transfer detection in soil using the inducible λPr system fused to eukaryotic luciferase genes

We report a model system for plasmid transfer analysis using the regulated lambda phage right promoter, λPr, fused to luc and lucOR as repoter genes. We have demonstrated that the systems cI857-λPr::luc and cI857-λPr::lucOR are temperature-inducible in Escherichia coli but not in other Gram-negative bacteria analyzed, enabling detection of luminescence when plasmids were mobilized from E. coli to those Gram-negative backgrounds. Using light for the detection, we have observed plasmid transfer from E. coli harboring RK2 and R388 derived plasmids to Pseudomonas putida KT2440 (co-introduced with donors) and to indigenous microorganisms, in vitro and in nonsterile soil microcosms. The importance of nutrients for an efficient plasmid transfer in nonsterile soil microcosms has been confirmed. When plasmid transfer experiments were carried out into nonsterile soil microcosms, significant populations of indigenous transconjugants arose. This system provides efficient marker genes and avoids the use of antibiotics for the selection of transconjugants.

Analysis of the expression from Rhizobium meliloti fix-promoters in other Rhizobium backgrounds

Using translational fusions to lacZ, we have measured expression from the promoters of Rhizobium meliloti regulatory genes, nifA and fixK, and structural genes, nifH and fixA, in other fast-growing rhizobia whose nitrogen fixation regulation is less known. Neither nifA nor fixK promoters were activated under both free-living microaerobic and symbiotic conditions, except in R. tropici, where clear symbiotic activation of either nifA or fixK expression could be observed. Both nifH and fixA promoters showed strong heterologous activation during symbiosis and weak activation under free-living nitrogen starvation conditions. Only when the nifH promoter was in R. tropici and R. leguminosarum bv. phaseoli, was clear induction observed in the microaerobic free-living state. Deletion analysis of these promoters suggested that a NifA binding site (UAS) was needed for full heterologous activation of nifHp, either in microaerobiosis or symbiosis. In contrast, the UAS region seemed to be unnecessary for fixA activation. However, a region containing a potential integration host factor (IHF) binding site was observed to be needed for complete heterologous symbiotic induction from fixAp. The moderate induction observed in nitrogen-free medium only required the sigma 54 holoenzyme recognition sequence; this may be indicative of the existence of non-specific activation by NtrC-like proteins. Our results suggest possible common and different features in the control mechanisms of the nitrogen fixation gene expression among Rhizobium species.

Localization of his genes on the Rhizobium trifolii RS800 linkage map

SummaryWe have studied N-methyl-N′-nitro-N-nitrosoguanidine (NTG)- and Tn5-induced histidine auxotrophic mutants in Rhizobium trifolii. HisGE, hisD and hisH mutants have been characterized. Using the Kempers equation we have located them on the R. trifolii linkage map. The hisGE and hisD genes are clustered in the same region and are closely linked to the spectinomycin marker. The hisH gene is located in another region equidistant from the streptomycin and rifampicin markers. The two regions carrying his genes are separated by a distance approximately one-third of the length of the chromosome.

Firefly Luciferase Expression on Nitrogen Fixation with Non-Legumes

We have tested the firefly luciferase expression under Rhizobium meliloti symbiotic promoters control in nitrogen fixation bacteria non associated with legumes. Constitutive expression of luc gene was determined in the same bacteria using a fusion to λPR promoter. Luciferase activity was found in all nitrogen fixer backgrowns but it depended of promoter activation conditions. Bioluminiscence was high enough in bacteria containing λpr-luc fusion to be observed by a dark adapted eye and photographed.

Action ofN-methyl-N′-nitro-N-nitrosoguanidine inRhizobium trifolii

Rhizobium trifolii was highly resistant to the lethal effect ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but it was sensitive to the mutagenic action of this chemical. A concentration of 500μg/ml yields a survival of between 1% and 10%, which allows us to obtain a higher number of mutants than lower concentrations that yield higher survival rates. Lethal damage produced by nitrosoguanidine was repaired, and repair is inhibited by acriflavine.