Saprophytic Actinomycetes Promote Nodulation in <Emphasis Type="Italic">Medicago sativa</Emphasis>-<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> Symbiosis in the Presence of High N

Saprophytic rhizoactinomycetes isolated from the root nodule surface of the nitrogen-fixing actinorhizal plant Discaria trinervis, Streptomyces MM40, Actinoplanes ME3, and Micromonospora MM18, previously shown to stimulate nodulation in Frankia-Discaria trinervis symbiosis, were assayed as co-inoculants with Sinorhizobium meliloti 2011 on Medicago sativa. When plants were fertilized with a low level of N (0.07 mM), the inoculation of the actinomycetes alone did not show any effect on plant growth. Meanwhile, when actinomycetes were co-inoculated with S. meliloti, nodulation and plant growth were significantly stimulated compared to plants inoculated with only S. meliloti. The analysis of nodulation kinetics of simultaneously or delayed co-inoculations suggests that the effect of the actinomycetes operates in early infection and nodule development counteracting the autoregulation of nodulation by the plant. Because the actinomycete effect was found in the symbiotic nitrogen-fixing state of the plant, we investigated the effects of the actinomycetes, in single inoculation or co-inoculation with S. meliloti, on plants grown under a high level of N (7 mM) that was inhibitory for nodulation by S. meliloti. The inoculation of the actinomycetes alone did not show any effect on plant growth although high N was available. Unexpectedly, the co-inoculation of actinomycetes with S. meliloti on plants grown with high N (7 mM) significantly stimulates nodulation, clearly counteracting the inhibition of nodulation by high N. These results corroborate that the interaction of rhizoactinomycetes would interfere with the autoregulation of nodulation in alfalfa mediated by high N, opening new research lines of potential agronomical applications.     

Resting forms of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

The ability of the symbiotrophic rhizobium Sinorhizobium meliloti P221 to produce cells having all the properties of resting forms (RFs) during the development cycles of the culture or after addition of the threshold concentrations of anabiosis autoinducers was demonstrated. The numbers, properties, and ultra-structure of S. meliloti resting forms depended on the conditions of growth and poststationary-phase incubation. In the four-month poststationary-phase, cultures grown in media deficient in some nutrient elements and energy sources (nitrogen, phosphorus, or oxygen), numerous cells (24–76% of the number of CFUs in the stationary-phase cultures) exhibiting a high degree of heat resistance and reversibly inhibited metabolic activity (the absence of endogenous respiration) were detected. According to their ultrastructure, all the resting forms detected in starving cultures were divided into three groups: (1) cystlike resting cells (CRCs) with thick cell envelopes and compacted nucleoids, (2) CRCs containing numerous (up to three-quarters of their volumes) polyhydroxyalkanoate inclusions, and (3) RFs similar to Azotobacter cysts. The resting forms obtained in the culture grown at high concentrations (5 × 10⁻⁵ M) of C₁₂-AHB, a chemical analogue of microbial anabiosis autoregulators, were incapable of endogenous respiration and retained the colony-forming ability. The CFU number after plating of these resting forms was twice as high as in the control culture; the heat resistance of these cells (55°C, 10 min) was an order of magnitude higher. The bacterial cells obtained from the resting forms either had a mixed (Swa⁺Gri⁺) type of motility in semisolid agar, typical of the dominant phenotype of the parent cells, or switched to the Gri⁺ type. Emergence of different motility phenotypes depended on the conditions of RF formation. More severe stress conditions of RF formation induced the emergence of the Gri⁺ type of cell motility. The results obtained can be used for development of a new generation of bacterial preparations based on bacterial CRCs which are able to preserve their viability for a long time and are highly resistant to stress impacts.     

Functional difference between <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> NifA and <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> NifA

The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix^- phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.     

Transcriptome analysis of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>during symbiosis

Background  

Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners. In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis. We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. ssp. <Emphasis Type="Italic">acuminata</Emphasis> and its root nodulation by <Emphasis Type="Italic">Frankia</Emphasis>

A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources. The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2M of BAP but not without it. Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1M of BAP and 87mM sucrose. Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1M IBA. Alnus acuminata did not developed well on WPM. Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia. The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen. Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant.     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

Comprehensive metabolite profiling of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> using gas chromatography-mass spectrometry

A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula. A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition. S. meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step. Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS. The different compounds were identified by comparison with the NIST 98 database and available standards. From about 200 peaks in each chromatogram 65 compounds have been identified so far. A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools. A principal component analysis (PCA) was able to distinguish S. meliloti cells grown on different carbon sources based on their metabolite profile. A comparison of the metabolite composition of a S. meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant. Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine. This finding further emphasises the importance of integrating metabolic data into post-genomic research.     

Functional analysis of the two cyclophilin isoforms of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E. coli BL21 in the presence of abiotic stress conditions such as increased heat and salt concentration. Our results support and strengthen previous high-throughput studies implicating S. meliloti cyclophilins in various stress conditions.     

<Emphasis Type="Italic">tolC</Emphasis> mutant of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> strain CXM1-188 fails to establish effective symbiosis with alfalfa

The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5 mutagenesis in the effective strain CXM1-188. The mutant did not produce fluorescent halos in UV light on the LB medium containing Calcofluor white, which suggests that modification occurred in the production of exopolysaccharide EPS1. Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS media, and this was most likely connected with the absence of the second exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula white round Fix^? nodules or nodules of irregular shape. These nodules possessed the structure usually described for nodules of EPS1 mutants. According to the data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63 contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome. This gene encodes the protein sharing homology with the TolC protein, a component of a type I secretion system responsible for the export of protein toxins and proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to be exported by the type I secretion system, was tested in cultural supernatants of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was ascertained that secretory proteins ExsH and ExpE1 are absent in the culture medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved in the excretion of proteins ExsH and ExpE1.     

Occurrence of islands in genomes of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> native isolates

Genomes of 184 Sinorhizobium meliloti native isolates were studied to test the occurence of islands Sme21T, Sme19T, and Sme80S previously described in the model strain Rm1021. This analysis was conducted using PCR methodology involving specific primers. It was demonstrated that, in the examined geographically distinct populations of S. meliloti from the Northern Caucasus (NCG) and the Aral Sea region (PAG), the strains containing genomic islands were observed with similar frequency (0.55 and 0.57, respectively). Island Sme80S, denoted as an island of “environmental adaptivity,” was identified predominantly (frequency of 0.38) in genomes of strains which exhibited a lower level of salt tolerance and was isolated in PAG, a modern center of introgressive hybridization of alfalfa subjected to salinity. Island Sme21T designated as “ancestral” was observed in genomes of strains isolated in NCG, the primary center of host-plant biodiversity, 10-fold more often than in strains from PAG. An island Sme19T, which predominantly carries genes encoding transposases, was observed in genomes of strains in both populations with average frequency of 0.10. The analysis of linkage disequilibrium (LD) based on the assessment of probability for detection of different islands combinations in genomes revealed an independent inheritance of islands in salt-sensitive strains of various geographic origin. In contrast, the absence of this trend was noted in the majority of the examined combinations of salt-tolerant strains. It was concluded that the structure of chromosome in PAG strains which predominantly possessed a salt-sensitive phenotype was subjected to active recombinant processes, which could predetermine the intensity of microevolutionary processes in bacterial populations and facilitate an adaptation of bacteria in adverse environmental effect.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

Altered susceptibility to infection by <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Nectria haematococca</Emphasis> in alfalfa roots with altered cell cycle

Most infections of plant roots are initiated in the region of elongation; the mechanism for this tissue-specific localization pattern is unknown. In alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter, the cell cycle in roots is completed in 48 h instead of 24 h, and border cell number is decreased by more than 99%. These plants were found to exhibit increased root-tip infection by a fungal pathogen and reduced nodule formation by a bacterial symbiont. Thus, the frequency of infection in the region of elongation by Nectria haematocca was unaffected, but infection of the root tip was increased by more than 90%; early stages of Sinorhizobium meliloti infection and nodule morphology were normal, but the frequency of nodulation was fourfold lower than in wild-type roots.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.