Sinorhizobium Meliloti Strain Research Articles

Effects of different strains of Sinorhizobium meliloti on alfalfa (Medicago sativa L.) biomass yield

The effects of highly effective strains of Sinorhizobium meliloti on biomass and crude protein yields of alfalfa (Medicago sativa L.) were investigated in a three-year field experiment focusing on the advantage of alfalfa inoculation with N microbiological fertilizer over N mineral fertilization on chernozem soil. Strains 236 and 234 were found in this experiment to have significant effect on the yields of dry matter and proteins, by comparison to untreated control plants. In the third year of utilization, dry matter yield of alfalfa plants inoculated with strains 236 and 234 increased around 31% in the first cutting and 53% and 65% in the second one, compared to control plants. Strain 236 was found to increase the yield of crude proteins by 29.7% (second cutting) and 39.6% (third cutting) in the second year, and 27.5% (first cutting) and 50.3% (second cutting) in the third year of utilization, while strain 234 increased significantly the content of proteins in the third cutting of the second year (27%) and in the third year of utilization (51% in the first and 53% in the second cutting). The results indicate that effective strains of S. meliloti may be applied as N microbiological fertilizers on soils with optimal physical and chemical characteristics and autochthonous populations that may not have satisfactory effectiveness in nitrogen fixation.

Long-Term Field Release of Bioluminescent Sinorhizobium meliloti Strains to Assess the Influence of a recA Mutation on the Strains' Survival

A field release experiment was carried out to study the fate of the isogenic, firefly luciferase (luc) gene-tagged Sinorhizobium meliloti strains L1 (RecA-) and L33 (RecA+) in the environment. Both strains were released at concentrations of approximately 10(6) cfu g(-1) soil in replicate and randomized field plots, which had been sown with alfalfa (Medicago sativa). The survival of both strains during the following 7 years could be subdivided into three phases: a sharp decline for more than two orders of magnitude within the first 4 months (phase I), followed by fluctuations around an average number of 10(4) cfu g(-1) soil for nearly 4 years (phase II), and a further decline to approximately 60 cfu g(-1) (phase III). At most sampling dates, no significant differences in the survival of both strains were detected, indicating that the recA gene function was dispensable under these environmental conditions. During the field inoculation, both strains were dispersed accidentally by wind in small numbers to noninoculated field plots. Strain L33 established at a concentration of more than 10(3) cfu g(-1) soil with subsequent seasonal fluctuations. Although strain L1 must have been disseminated to a similar extent, it could never be recovered from noninoculated field plots, indicating that the recA mutation interfered with the strain's capability to establish there. At the beginning of the field experiment, an indigenous alfalfa-nodulating population was below the limit of detection. In the following years, however, an indigenous population arose, which finally outcompeted both strains for saprophytic growth and alfalfa nodulation. RecA- strain L1 was outcompeted for alfalfa nodulation slightly faster than its RecA+ counterpart L33. The diversity of the indigenous population was characterized by employing the Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction fingerprint method. Typing of 2731 root nodule isolates revealed a total of 38 fingerprint groups. More than 80% of the isolates could be grouped into six dominant fingerprint groups, indicating that a few dominant bacterial strain types had outcompeted the released strains.

Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021

K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The K(R)5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix(+)) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and gas chromatography (GC-MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.

A Proteomic Approach Towards the Analysis of Salt Tolerance in Rhizobium etli and Sinorhizobium meliloti Strains

Soluble proteins from the salt-tolerant Rhizobium etli strain EBRI 26 were separated by two-dimensional (2D) gel electrophoresis and visualised by Commassie staining. Six proteins are highly expressed after induction by 4% NaCl compared to the non-salt-stressed cells. These proteins have pI between 5 and 5.5 and masses of approximately 22, 25, 40, 65, 70, and 95 kDa. These proteins were analysed by Matrix-assisted laser adsorption ionization time of flight (MALDI-TOF) after digestion with trypsin. Despite having very good peptide mass fingerprint data, these proteins could not be identified, because the genome sequence of R. etli is not yet published. In a second approach, soluble proteins from salt-induced or non-salt-induced cultures from R. etli strain EBRI 26 were separately labelled with different fluorescent cyano-dyes prior to 2D difference in gel electrophoresis. Results revealed that 49 proteins are differentially expressed after the addition of sodium chloride. Fourteen proteins are overexpressed and 35 were downregulated. The genome of Sinorhizobium meliloti, a closely related species to R. etli, has been published. Similar experiments using Sinorhizobium meliloti strain 2011 identified four overexpressed and six downregulated proteins. Among the overexpressed protein is a carboxynospermidin decarboxylase, which plays an important role in the biosynthesis of spermidin (polyamine). The enzyme catalase is among the downregulated proteins. These proteins may play a role in salt tolerance.

Effect of elevated temperature and water availability on CO 2 exchange and nitrogen fixation of nodulated alfalfa plants

The aim of this study was to investigate the effects of predicted temperature increases and drought conditions of Mediterranean environments on N 2-fixing alfalfa plants. One-month-old plants inoculated with Sinorhizobium meliloti strain 102F78 were grown in growth chambers under different temperature (25/15 or 28/18 °C, day/night) and water availability (control or drought) regimes. Elevated temperature and drought reduced plant dry mass and leaf area, especially when both stresses were combined. The inhibitory effect of elevated temperature on plant growth was a consequence of decreased CO 2 and N 2 fixation rates. A photosynthetic decrease resulted from the inhibition of rubisco activity, probably associated with a lower activation state. An absence of differences in photosynthesis in relation to water availability suggests that drought decreased plant growth due to its negative effect on leaf area. Rising temperature and drought affected the nitrogen content negatively, although effects differed. Elevated temperature inhibited nodule activity drastically, whereas the inhibitory effect resulting from drought centred on nodule dry mass (DM) production. Plants exposed to a combination of elevated temperature and drought were the most negatively affected.

Plasmid‐associated genes in the model micro‐symbiont Sinorhizobium meliloti 1021 affect the growth and development of young rice seedlings

Sinorhizobium meliloti strain 1021 and its closely related strain Rm2011 inhibit rice seedling (Oryza sativa L. cv. Pelde) growth and development under certain rice-growing conditions. Experiments showed that inoculation of seedlings with approximately less than 10 cells of 1021 was sufficient to cause this inhibition. By using a series of plasmid-cured and plasmid-deleted derivatives of Rm2011, it was found that interactions between genes encoded on pSymA, and possibly pSymB, of Rm2011, affected rice growth and development by affecting both/either the plant and/or the bacteria. Further studies found that genes potentially related to indole-3-acetic acid (IAA) synthesis and nitrate metabolism, encoded on pSymA, were involved in rice growth inhibition in Sm1021- and Sm2011-treated rice seedlings. We conclude that the rice growth inhibition by S. meliloti Sm1021 is pSymA-associated and is induced by environmental nitrate.

Replication regions of Sinorhizobium meliloti plasmids

The replication ( rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA − strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75–79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O’Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232–237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.

Interaction between Medicago truncatula lines and Sinorhizobium meliloti strains for symbiotic efficiency and nodule antioxidant activities

The relationships between symbiotic performance and nodular antioxidant enzymes were studied for the associations between three Medicago truncatula lines and three Sinorhizobium meliloti strains. The results showed that the variability in symbiotic efficiency was dependent on the bacterial partner, host plant and their interaction. The contribution of each factor to the total amount of variance differed with the measured parameter. The aerial biomass production and nitrogen‐fixing capacity were affected similarly by the three factors, whereas root and nodule biomass and catalase (CAT, E.C. 1.11.1.6), guaiacol peroxidase (POX, E.C. 1.11.1.7) and ascorbate peroxidase (APX, E.C. 1.11.1.11) antioxidant activities were mainly influenced by the M. truncatula line. The nodule number was dependent on the bacterial strain, and superoxide dismutase (SOD, E.C. 1.15.1.1) was dependent mainly on the plant–rhizobium interaction. A highly significant correlation was found between nitrogen‐fixing activity, shoot biomass production, total nodule protein content and catalase activity. The other nodular antioxidant enzymes were differentially expressed between associations and showed no clear correlation with symbiotic efficiency.

Determination of nitrogen fixation effectiveness in selected Medicago truncatula isolates by measuring nitrogen isotope incorporation into pheophytin

Effectiveness is a term used to describe the input that a bacterial nitrogen-fixing symbiosis makes to plant nitrogen metabolism. In legumes, effectiveness is considered a polymorphic trait where specific interactions between the plant and symbiotic rhizobia contribute to the success of the interaction. Evaluation of effectiveness using model legumes like Medicago truncatula may open new avenues for genetic studies. In previous work, an isotope dilution mass spectrometry method, which uses the effect of nitrogen fixation on the nitrogen isotope composition of chlorophyll in plants grown on 15N fertilizer as a measure of effectiveness, was developed for estimating the contribution of symbiotic nitrogen fixation to plant nitrogen content. This 15N-dilution assay was used to evaluate the level of nitrogen fixation effectiveness in three Medicago truncatula lines that have been used as parents in generating recombinant inbred lines. Three Sinorhizobium meliloti strains, USDA 1600, 102F51 and MK506, differ in this measure of effectiveness on three lines of M. truncatula: Jemalong A17, DZA315.16 and F83005.5. Plant–rhizobia combinations grown in two different conditions showed comparable differences in effectiveness.

Increased yield and persistence of several annual medic species and Medicago sativa by inoculation with selected strains of Sinorhizobium meliloti and S. medicae

A broad range of genotypes of Medicago sativa, and annual medics including M. polymorpha, M. tornata and M. littoralis were inoculated with strains of Sinorhizobium meliloti or S. medicae of differing effectiveness for symbiotic N2 fixation then sown at 4 field locations. Dry matter production over 2 seasons was strongly related to plant density, which in turn was related to symbiotic effectiveness. Eighteen months after sowing at Esperance, Western Australia, lucerne inoculated with strain WSM922 showed 79% higher plant density and 43% more production than control strain CC169. At Broomehill, no significant differences existed in dry matter production between lucerne cultivars inoculated with strains WSM922, WSM826 and U45. Across all lucerne genotypes, inoculation with WSM922 outyielded those inoculated with CC169 by 99%. At Jerramungup, the difference in yield between these 2 inoculant strains was 44%. Results were consistent with those previously obtained under controlled conditions and emphasised the necessity to remain aware of the symbiotic requirements of newly produced cultivars. An analysis of nodule occupancy at 1 site using PCR-RAPDs revealed the dominance of a particular rhizobial strain (WSM922) in uninoculated plots which had become colonised over 3 seasons. The necessity for separate inoculant species of Sinorhizobium in Australia to satisfy the symbiotic requirements of the acid and alkaline groups of medics was reaffirmed.

Host range and saprophytic competence of Sinorhizobium meliloti — a comparison of strains for the inoculation of lucerne, strand and disc medics

Strains of Sinorhizobium meliloti were compared for their saprophytic competence (ability to survive and colonise) in mildly acidic (pHCa 4.8–5.4) soils, using a ‘cross-row’ technique at 3 field locations in the south-east of South Australia. Some strains of rhizobia had greater saprophytic competence than others. Strain WSM879 performed consistently well, nodulating 36% of lucerne seedlings (mean of 3 sites and 4 sampling regions) compared with former inoculant strain WSM826 which nodulated 27% of lucerne seedlings. At one site, strain WSM879 was compared with the former and current Australian inoculant strains (WSM826 and RRI128, respectively). Here, all 3 strains nodulated a similar percentage of lucerne seedlings. However, the addition of 5 t/ha of lime to the soil at this site increased the percentage of lucerne plants nodulated from 23 to 43%. This increase was due to a combination of better strain survival and colonisation and indicates there remains some potential to further improve these aspects of strain performance. The growth of 4 of the rhizobial strains from the field trials was measured on acidified agar media (between pH 4.0 and 7.5). There was virtually no colony growth (<10% of growth at pH 7.0) by strains WSM826, RRI128 and WSM879, at or below pH 6.0. Although strain MSUR52a was still able to grow (40% of potential) at pH 6.0 (in the absence of aluminium) this was not always reflected in better nodulation of lucerne seedlings by this strain in the field. Inclusion of aluminium in the media increased the sensitivity of the strains to acidity. The ability of 6 selected S. meliloti strains to form effective symbioses with 15 plant hosts (from Medicago sativa, Medicago littoralis and Medicago tornata) was compared. All S. meliloti strains formed effective symbioses with all plant hosts. Overall, strain RRI128 was the most effective strain with both the lucernes and the annual medics, resulting in shoot weights similar to those of plants supplied with mineral nitrogen.

Effect of salinity on root-nodule conductance to the oxygen diffusion in the Medicago truncatula– Sinorhizobium meliloti symbiosis

In order to determine the effect of salinity on the nodule conductance, oxygen uptake by the nodulated roots was measured by registering the concentration of O 2 as a function of time in a tight incubator of known volume containing the nodulated roots of Medicago truncatula. Four lines, namely TN8.20 and TN6.18, originated from local populations, F83005.5 originated from Var (France) and Jemalong 6, a cultivar from Australia, were hydroponically grown in 250 ml glass bottles under semi-controlled conditions in a glasshouse, after germination and inoculation with the strain Sinorhizobium meliloti 2011. The saline treatment was applied gradually to reach 75 mM after 2 weeks. Results show that oxygen uptake increased significantly with salinity in TN6.18 and F83005.5, but not in Jemalong nor in TN8.20. Without salt, Jemalong showed a significantly higher O 2 uptake of 240 μmol O 2 per h per plant than the mean of 130 μmol O 2 per h per plant for other lines. Salinity increased significantly the nodule conductance in all genotypes. This salt effect was significantly higher for TN6.18 than for TN8.20, and for Jemalong than for F83005.5. Without salt there was less genotypic variation in nodule conductance in the range of 5–8 μm s –1 for F83005.5 and TN8.20, respectively. Thus the sensitivity to salinity appears to be associated with an increase in nodule conductance that supports the increased respiration of N 2-fixing nodules under salinity.

Production of amino acids by Rhizobium, Mesorhizobium and Sinorhizobium strains in chemically defined media.

Five strains of Rhizobium spp, one strain of Mesorhizobium loti and two strains of Sinorhizobium meliloti were tested for their ability to grow in chemically-defined medium lacking growth factor. Qualitative and quantitative production of aspartic acid, serine, glutamic acid, glycine, histidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine was determined by the use of mannitol as sole carbon source. Strains of Rhizobium spp. and Sinorhizobium sp. produced all the amino acids analysed with the exception of cysteine and high biological levels of serine, glycine and alanine were detected after 2 days of culture in mineral medium. Strain U226 of M. loti only produced small amounts of amino acids and glutamic acid, histidine, arginine, cysteine, methionine, lysine and phenylalanine was not liberated into the media.

Heterologous complementation of the exopolysaccharide synthesis and carbon utilization phenotypes of Sinorhizobium meliloti Rm1021 polyhydroxyalkanoate synthesis mutants

A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described d-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.

Sinorhizobium meliloti strain 1021 produces a low-molecular-mass capsular polysaccharide that is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid harboring a phospholipid anchor.

Sinorhizobium meliloti strain 1021 possesses the particularity to synthesize biologically inefficient capsular polysaccharides (KPS). It has been assumed that this class of compounds is not produced in high-molecular-mass (HMM) forms, even if many genetic analyses show the existence of expression of genes involved in the biosynthesis of capsular polysaccharides. The expression of these genes that are involved in the export of a KPS throughout the membrane and in the attachment of a lipid moiety has never been related to a structurally characterized surface polysaccharide. It is now reported that S. meliloti strain 1021 produces low-molecular-mass polysaccharides (4-4.5 kDa) that are exclusively composed of beta-(2-->7)-linked 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo) residues. These compounds are considered precursor molecules of HMM KPS, whose biosynthesis is arrested in the case of S. meliloti strain 1021. For the first time, the phospholipid anchor of a rhizobial KPS has been found, and its structure could be partially identified-namely, a phosphoglycerol moiety bearing a hydroxy-octacosanoic acid. When compared to other rhizobial KPS (composed of dimeric hexose-Kdo-like sugar repeating units), the Kdo homopolymer described here may explain why a complementation of S. meliloti strain 1021 Exo B mutant with an effective rkpZ gene restoring an active higher KPS size does not completely lead to the fully effective nitrogen fixing phenotype.

Global transcriptional analysis of the phosphate starvation response in Sinorhizobium meliloti strains 1021 and 2011.

The global response to phosphate starvation was analysed at the transcriptional level in two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011. The Pho regulon is known to be induced by PhoB under conditions of phosphate limitation. Ninety-eight genes were found to be significantly induced (more than three-fold) in a phoB -dependent manner in phosphate-stressed cells, and phoB -independent repression of 86 genes was observed. Possible roles of these genes in the phosphate stress response are discussed. Twenty new putative PHO box sequences were identified in regions upstream of 17 of the transcriptional units that showed phoB -dependent, or partially phoB -dependent, regulation, indicating direct regulation of these genes by PhoB. Despite the overall similarity between the phosphate stress responses in Rm1021 and Rm2011, lower induction rates were found for a set of phoB -dependent genes in Rm1021. Moreover, Rm1021 exhibited moderate constitutive activation of 12 phosphate starvation-inducible, phoB -dependent genes when cells were grown in a complex medium. A 1-bp deletion was observed in the pstC ORF in Rm1021, which results in truncation of the protein product. This mutation is probably responsible for the expression of phosphate starvation-inducible genes in Rm1021 in the absence of phosphate stress.

Nitrogen fixation and growth of annual Medicago–Sinorhizobium associations at low temperature

Annual Medicago (medics) represents a great potential of forage production in areas suffering from shortage of livestock feed; however, low winter temperature delays the growth and reduces the nitrogen fixation of medics in Mediterranean dry areas with cool to cold winter. In this context, the identification of medics able to fix nitrogen and produce dry matter at low temperature is necessary to extend the grazing period. The experiment was carried out in controlled growth chambers at two low temperatures 7 and 9 °C, temperature known to limit growth and N 2 fixation of Medics in the field. Three accessions were used, two of Medicago aculeata, accessions 80 (A) and 5099 (B) and one of M. rigidula, accession 716 (R). Each accession was inoculated with Sinorhizobium strains appropriate for nodulation under low temperature ( Sinorhizobium meliloti strain M620 with accession A, strain M508 with B and strain BZI with R). The percentage of nitrogen in the shoots derived from atmosphere was quantified using 15 N dilution technique with barley as reference plant. Results demonstrated that responses of accessions depend on temperature, nitrogen and inoculated strain. Among the accessions tested, M. aculeata 5099 was more adapted to temperature and grew faster than the others at the lowest temperature. Strain M53 performed better with M. rigidula 716 than with M. aculeata 80 and strain M620 with M. aculeata 5099 than with M. aculeata 80 for yield and N nutrition of plants. M508 for M. aculeata 5099 and BZI for M. rigidula 716 leaded to more N fixed per plant. Nitrogen fixation in inoculated treatment of accession M. aculeata × M508 accounted for 88 and 78% of total plant N at 9 and 7 °C versus 49 and 51% for accession M. rigidula × M53. It was concluded that the selection of associations medics × Sinorhizobium is a promising way to improve yield and N 2 fixation at low temperature in areas where N fertilization cannot be possible.

Instability of Cryptic Plasmids in Strain Sinorhizobium meliloti P108 in the Course of Symbiosis with Alfalfa Medicago sativa

Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains SXM1, DM7-R, and P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation experiment (120 days) was studied. The isolated clones of strains SXM1 and DM7-R manifested stable inheritance of plasmids, whereas 12.7–14.0% of clones with changed plasmid profile were detected in a population of clones from strain P108. These segregants were designated as P108c. Segregants P108c exhibited significantly decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, compared to the original strain P108. It was established that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230 kb) of segregants P108c. It was concluded that genetic rearrangements are possible in rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the natural vegetation period of alfalfa.

Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021.

Sequencing of the Sinorhizobium meliloti strain 1021 genome led to the detection of 6204 open reading frames, 41 % of which have no hypothetical function. To help annotate this genome, a transcriptome analysis was carried out with a dedicated microarray consisting of 146 genes belonging to three different classes: (i) no hypothetical function; (ii) potentially involved in oxidative stress responses; (iii) known to participate in oxidative stress responses (e.g. catalase and superoxide dismutase genes). This transcriptome analysis, together with biological experiments and in silico investigations, identified new genes induced by exogenous H(2)O(2). The smc01944 gene was the most strongly induced: quantitative PCR showed that the amount of smc01944 mRNA increased 50-fold following the addition of 10 mM H(2)O(2), whereas the amount of katA mRNA (encoding a catalase) only increased 10-fold. Smc01944 is a non-haem chloroperoxidase (Cpo). The only member of this family to have been so far characterized is encoded by prxC of Pseudomonas fluorescens. Unexpectedly, the NH(2)-terminus of Smc01944 includes a signal peptide and Smc01944 is secreted into the supernatant. Interestingly, smc01944 is preceded by smc01945, encoding an OhrR-like regulator (MarR family). Thus, Smc01944 is the first exported Cpo encoded by a gene possibly regulated by an OhrR regulator. It was also shown that smc01944 is induced by t-butyl and cumene hydroperoxides but only slightly by menadione. The study of Smc01944 described in this work showed that the oxidative stress response of S. meliloti seems to differ from that of other bacteria characterized to date.

Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island.

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.