Impact of fertilization by natural manure on the microbial quality of soil: Molecular approach.

The quality of soil is strongly bound by several interactions between chemical and biological components, including microbial composition, which are a key importance for soil performance. Cultural activities have a huge induction on soil health, through both modification of physicochemical proprieties and changing on soil microbial communities. This usually affects the safety of soil, and then the crop production and water. In the present work, the information on bacterial community composition was determined from a set of 6 soils collected from 2 farms in agricultural land of Marrakech (Morocco), one of which used poultry manure (PM) and the other cow manure (CM) as fertilizers. To profile this structure of the bacterial community Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA fragments has been used. These amendments resulted in the appearance of several novel bands and different relative intensities of bands between the control station and other sites studied. The stations most affected are those receiving a supply of manure rather high, which results in an organic and bacterial load in the soil. The results showed a bacterial diversity very important indicating a fecal contamination like Bacteroides, Pseudomonas, Staphylococcus,… etc. Bacteria pertain to the phylum Firmicutes and Bacteroidetes were noted to be the dominant ribotype in amended soil. Moreover, this work demonstrates also the existence of pathogens strains in soil amended by poultry manure (PM) belonging to the Clostridiales order and Pseudomonadales. The pathogenic bacteria detected posing a hazard of human contagion when they are used for soil practice.

High NaCl concentrations induce the nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers.

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

Genetics and biotechnology of the H(2)-uptake [NiFe] hydrogenase from Rhizobium leguminosarum bv. viciae, a legume endosymbiotic bacterium.

A limited number of strains belonging to several genera of Rhizobiaceae are capable of expressing a hydrogenase system that allows partial or full recycling of hydrogen evolved by nitrogenase, thus increasing the energy efficiency of the nitrogen fixation process. This review is focused on the genetics and biotechnology of the hydrogenase system from Rhizobium leguminosarum bv. viciae, a frequent inhabitant of European soils capable of establishing symbiotic association with peas, lentils, vetches and other legumes.

Regulation of nod factor sulphation genes in Rhizobium tropici CIAT899.

Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium. Broad host range of this species is related to its Nod factors wide spectrum. R. tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena. To study nodHPQ regulation, we carried out different interposon insertions in its upstream region. One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region. Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein. In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon. However, comparison between nodP::lacZ beta-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions. Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism. On the other hand, the transference of R. tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end.

Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex.

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5' and 3' regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

Sulfation of nod factors via nodHPQ is nodD independent in Rhizobium tropici CIAT899.

A cosmid from the Rhizobium tropici CIAT899 symbiotic plasmid, containing most of the nodulation genes described in this strain, has been isolated. Although this cosmid does not carry a nodD gene, it confers ability to heterologous Rhizobium spp. to nodulate R. tropici hosts (Phaseolus vulgaris, Macroptilium atropurpureum, and Leucaena leucocephala). The observed phenotype is due to constitutive expression of the nodABCSUIJ operon, which has lost its regulatory region and is expressed from a promoter present in the cloning vector. Thin-layer chromatography (TLC) analysis of the Nod factors produced by this construction shows that it is still capable of synthesizing sulfated compounds, suggesting that the nodHPQ genes are organized as an operon that is transcribed in a nodD-independent manner and is not regulated by flavonoids.

Characterization of Rhizobium tropici CIAT899 nodulation factors: the role of nodH and nodPQ genes in their sulfation.

We have purified and characterized the nodulation factors produced by Rhizobium tropici CIAT899. This strain produces a large variety of nodulation factors, these being a mixture of sulfated or nonsulfated penta- or tetra-chito-oligosaccharides to which any of six different fatty acyl moieties may be attached to nitrogen of the nonreducing terminal residue. In this mixture we have also found methylated or nonmethylated lipo-chitin oligosaccharides. Here we describe a novel lipo-chitin-oligosaccharide consisting of a linear backbone of 4 N-acetylglucosamine residues and one mannose that is the reducing-terminal residue and bearing a C18:1 fatty acyl moiety on the nonreducing terminal residue. In addition, we have identified, cloned, and sequenced R. tropici nodH and nodPQ genes, generated mutations in the nodH and nodQ genes, and tested the mutant strains for nodulation in Phaseolus and Leucaena plants. Our results indicate that the sulfate group present in wild-type Nod factors plays a major role in nodulation of Leucaena plants by strain CIAT899 of R. tropici.