Strain HAMBI Research Articles

Neorhizobium tomejilense sp. nov., first non-symbiotic Neorhizobium species isolated from a dryland agricultural soil in southern Spain

We describe for the first time a non-symbiotic species of the recently described genus Neorhizobium, lacking nodulation or nitrogen fixation genes. The strains were isolated from a dryland agricultural soil in southern Spain where no record of legume cultivation is available, thus we propose the name Neorhizobium tomejilense sp. nov. (type strain T17_20T, LMG 30623T and CECT 9621T). N. tomejilense exhibit a clear distinct lineage from the other Neorhizobium species, Neorhizobium galegae, Neorhizobium alkalisoli and Neorhizobium huautlense, based on polyphasic evidence. Phylogenetic marker analysis of 16S rDNA, atpD, glnII, recA, rpoB and thrC genes and genomic identity data derived from the draft genomic sequences showed that N. tomejilense strains clearly separated from the other Neorhizobium species and that N. galegae represents the closest species, with Average Nucleotide Identities (ANIb) ranging from 90% (for type strain HAMBI 540T) to just under 95.0% (for two N. galegae sv. officinalis strains). Genomes from N. galegae and N. tomejilense, however, clearly differed in important traits, such as the number of rRNA operon copies or the number of tRNAs. Phenotypic characterisation of N. tomejilense also displayed differences with the other Neorhizobium species. Whole-cell matrix-assisted laser-desorption time-of-flight mass spectrometry (WC MALDI-TOF-MS) fingerprint analysis and the dendrogram derived from the fingerprint profiles, showed a clearly distinct group formed by the three N. tomejilense isolates (T17_20T, T20_22 and T11_12) from the other Neorhizobium especies.

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors

BackgroundThe species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.ResultsThe genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed.ConclusionsStrains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-500) contains supplementary material, which is available to authorized users.

Stable isotope labelling reveals that NaCl stress decreases the production ofEnsifer(Sinorhizobium)arborislipochitooligosaccharide signalling molecules

Ensifer (Sinorhizobium) arboris is a symbiont of salt-tolerant leguminous trees in the genera Acacia and Prosopis that are utilized in the prevention of soil erosion and desertification and in phytoremediation of salinized soil. Signalling between the plant and the rhizobia is essential for the formation of effective symbiosis that increases the success of reclaiming saline sites. We assessed the effect of salt stress on the growth and the production of lipochitooligosaccharide signalling molecules (LCOs) of S. arboris HAMBI 2361, an LCO-overproducing derivative of the S. arboris type strain HAMBI 1552. The strain tolerated NaCl up to 750 mM. To obtain both qualitative and quantitative information on the LCO production under salt stress, we devised a method where LCOs were differentially labelled by stable isotopes of nitrogen, (14)N and (15)N, and analysed by mass spectrometry. Under control conditions, the strain produced altogether 27 structural LCO variants. In 380 mM NaCl, 13 LCO variants were produced in detectable amounts, and six of these were reliably quantified, ranging from one-tenth to one-third of the non-stressed one.

Evaluation of the roles of two compatible solutes, glycine betaine and trehalose, for the Acacia senegal–Sinorhizobium symbiosis exposed to drought stress

Acacia senegal (Mimosoideae) is a leguminous, nitrogen-fixing tree that grows in arid areas of Africa and the Near East. In this work, we studied the effects of drought stress on the development of symbiosis between A. senegal seedlings and Sinorhizobium arboris. We also evaluated if two exogenous compatible solutes, glycine betaine and trehalose, are advantageous for the A. senegal–Sinorhizobium symbiosis and if these solutes are capable of protecting two Sinorhizobium strains from salt stress (NaCl) and osmotic stress induced by polyethylene glycol (PEG 6000). A. senegal seedlings exposed to severe drought stress developed more root hairs than plants grown under moderate stress. After inoculation with the GUS marked S. arboris strain HAMBI 2180 the hairs were deformed but infection threads occurred only occasionally. Non-typically deformed hairs were dwarfed and swollen. Severely stressed roots contained less nodules but more nodule initials than moderately stressed ones. Nodules formed showed a lowered glucuronidase activity and signs of premature senescence. The numbers of culturable rhizobia in soil mix were reduced from 107 to 106 CFU g−1. Thirty days after inoculation severely drought-stressed A. senegal seedlings were wilted. Regarding endogenous glycine betaine, A. senegal appeared to be a non-accumulator plant but was able to translocate foliar-applied glycine betaine into roots. Both glycine betaine (0.01 M) and trehalose (0.01, 0.05 and 0.09 M) protected cell cultures of the wild type S. arboris strain HAMBI 1552 and S. saheli strain HAMBI 1496 from osmotic stress (9 and 17% PEG). In the case of salt stress only trehalose had a favourable effect. Application of 0.0003 M glycine betaine or trehalose into A. senegal soil mix exposed to severe drought stress maintained the numbers of culturable rhizobia at the same level as in moderately stressed soils. The presence of glycine betaine in the soil mix also helped A. senegal seedlings survive under severe drought.

Persistence, population dynamics and competitiveness for nodulation of marker gene-tagged Rhizobium galegae strains in field lysimeters in the boreal climatic zone

A non-indigenous wild-type strain Rhizobium galegae HAMBI 540, which specifically nodulates perennial goat’s rue ( Galega orientalis), and its marker gene-tagged derivatives R. galegae HAMBI 2363( luc), R. galegae HAMBI 2368( gusA21) and R. galegae HAMBI 2364( gusA30) were used to evaluate the persistence, population dynamics and competitiveness for nodulation of rhizobia under field conditions in Finland. The lysimeters were filled with clean or diesel oil-polluted (3000 μg g −1) agricultural soil. During the first 2 years of the field release luc- and gusA21-tagged strains could be effectively detected by cultivation, reinforced with colony polymerase chain reaction. The population densities remained relatively stable from 10 4 to 10 5 cfu g −1 dry soil from spring until late autumn. Replicate limiting dilution polymerase chain reaction analysis gave comparable results with cultivation with strain HAMBI 2363 until 49 weeks after inoculation. GUS activity of strain HAMBI 2368 could be stably detected in nodules and soil. On the other hand, luc activity weakened clearly in cold conditions along with decreased metabolic activity of rhizobia. The competitive ability for nodulation of the gusA30-tagged strain decreased slowly with time compared to the wild-type strain. Moderate soil pollution did not have significant effects on target bacteria or plant growth. Limited vertical movement of target bacteria outside the rhizosphere was detected from percolated water.

The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl- N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl- N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.

Activation of the nodA promoter by the nodD genes of Rhizobium galegae induced by synthetic flavonoids or Galega orientalis root exudate

Rhizobial nodD genes produce transcriptional regulators that, together with appropriate inducer compounds, activate the other symbiotic nodulation ( nod) genes and initiate the nodule formation process. Two nodD homologues, nodD1 and nodD2, are present in the Rhizobium galegae strain HAMBI 1174. In this work we analysed their ability to induce the nodA promoter with synthetic inducers known to activate nod genes in other rhizobia. According to phylogenetic analysis, the inducer-specific carboxy-terminal part of the R. galegae nodD protein sequence groups together with those of Rhizobium leguminosarum and Sinorhizobium meliloti. However, the respective inducer compounds for their NodD proteins are not highly effective with R. galegae nodD products. The best inducer discovered with R. galegae nodD1 was the root exudate of the host plant of R. galegae, Galega orientalis. HPLC analyses revealed the presence of many divergent flavonoid compounds in the G. orientalis root exudate. The most effective HPLC fractions induced R. galegae nodD1 up to the level obtained by intact G. orientalis root exudate while apigenin and luteolin, which were also present in the root exudate, were only moderate inducers. A UV-Vis diode array spectrum of the most active peak indicated that the main inducer present in the G. orientalis root exudate is an unidentified chalcone-type compound. In the Galega– R. galegae interaction the first recognition between the NodD protein and the flavonoid inducer secreted from the roots of Galega is specific for these organisms, and thus partly responsible of the strict host specificity of this symbiosis.

Symbiotic properties of sinorhizobia isolated from Acacia and Prosopis nodules in Sudan and Senegal

The host specificity, infection process and effectiveness of nodules produced by several African sinorhizobial strains on different Acacia and Prosopis species (Leguminosae, Mimosoideae) were studied. Sinorhizobium arboris strain HAMBI 1552T, S. kostiense strains HAMBI 1489T and HAMBI 1493, S. saheli strain HAMBI 1496 and S. terangae bv. acaciae strain ORS 1058 induced nitrogen fixing nodules on seedlings of the following African or Latin American species (marked with *): A. angustissima*, A. mellifera, A. nilotica, A. oerfota (synonym A. nubica), A. senegal, A. seyal, A. sieberiana, A. tortilis subsp. raddiana, P. chilensis*, P. cineraria, P. juliflora and P. pallida*. All strains increased plant yield significantly compared with uninoculated seedlings watered with nitrogen-free medium, but none appeared to be superior. The sinorhizobial strains were unable to effectively nodulate Sesbania rostrata (Papilionoideae).

Effect of heat stress on cell activity and cell morphology of the tropical rhizobium, Sinorhizobium arboris

The effect of heat stress on the growth, physiological state, cell activity and cell morphology of the tropical Sinorhizobium arboris strain HAMBI 2190 was studied. The cells were chromosomally tagged with the firefly luciferase gene, luc. Since the bioluminescence phenotype is dependent on cellular energy reserves it was used as an indicator of the metabolic status of the cell population under various heat conditions. Variations in the numbers and lengths of growth phases between individual cultures indicated that the growth pattern at 40°C was disturbed compared to growth at 37 or 28°C. In addition, the cell morphology was changed radically. The number of culturable cells and the luciferase activity declined when the cultures were incubated at 40°C. By contrast, under all conditions studied, the cells could be stained with 5-(and 6-)sulfofluorescein diacetate, indicating esterase activity. This demonstrated that although the culturability and cellular energy reserves decreased considerably during heat stress, a majority of the of S. arboris cell population maintained basal enzyme activity.

Effect of heat stress on cell activity and cell morphology of the tropical rhizobium, Sinorhizobium arboris

The effect of heat stress on the growth, physiological state, cell activity and cell morphology of the tropical Sinorhizobium arboris strain HAMBI 2190 was studied. The cells were chromosomally tagged with the firefly luciferase gene, luc. Since the bioluminescence phenotype is dependent on cellular energy reserves it was used as an indicator of the metabolic status of the cell population under various heat conditions. Variations in the numbers and lengths of growth phases between individual cultures indicated that the growth pattern at 40°C was disturbed compared to growth at 37 or 28°C. In addition, the cell morphology was changed radically. The number of culturable cells and the luciferase activity declined when the cultures were incubated at 40°C. By contrast, under all conditions studied, the cells could be stained with 5-(and 6-)sulfofluorescein diacetate, indicating esterase activity. This demonstrated that although the culturability and cellular energy reserves decreased considerably during heat stress, a majority of the of S. arboris cell population maintained basal enzyme activity.

The effect of heat stress on the symbiotic interaction between Sinorhizobium sp. and Acacia senegal

Sinorhizobium sp. strain HAMBI 2180, marked with the β-glucuronidase (GUS) encoding gusA gene, was used for studying the effect of heat stress on the symbiotic interaction between Acacia senegal seedlings and Sinorhizobium sp. in growth cabinet conditions. The infection process, observed during 18 days after inoculation, was delayed at a root temperature of 40°C compared to a root temperature of ≤38°C, but nodulation was retarded already at 38°C. At 42°C no nodules were formed. At this root temperature the development of lateral roots and root hairs was one week delayed. Although the roots had normally deformed root hairs in addition to abnormally deformed ones, only disintegrated infection threads were present. However, the ability to form symbiosis was reversible. Nitrogen-fixing nodules developed on those parts of the roots which were formed after completion of the thermal stress at 42°C. A root temperature of 42°C did not affect the numbers of culturable rhizobia in the A. senegal rhizosphere.

Polyamine-induced changes in symbiotic parameters of the Galega orientalis-Rhizobium galegae nitrogen-fixing system

Plants of goat's rue (Galega orientalis) inoculated with Rhizobium galegae strain HAMBI 540 were grown in the presence of putrescine (Put), spermidine (Spd) or spermine (Spm), and several symbiotic characteristics were investigated to delineate the influence of polyamines (PA) on this nitrogen-fixing system. All three PA exerted a concentration-dependent effect on the nodule parameters tested. The increment of nodulation ability and nodule biomass accumulation was extreme (from 2.4- to 4.0-fold) when plants were subjected to 10 and 50 μM of various PA. However, at 100 μM a negative effect was observed. The acetylene-reduction activity of nodulated roots was increased also in response to treatment with the lower PA concentrations. The level of nitrogenase activity supported by succinate was significantly higher in bacteroids isolated from PA-treated nodules than in bacteroids from control nodules. The symbiotic parameters were also dependent on the type of PA used; the most effective being the diamine Put, while Spm showed a smaller physiological effect with respect to the others. Polyamines altered the ultrastructure of Galega nodule infected cells. After treatment with these substances, pronounced changes in the relative volume of the main components of infected cells and their compartments were observed. The significance of the structural observations and morphometric analyses, their relationship to differences in nitrogen fixation and possible modes of action are discussed.

The effect of heat stress on the symbiotic interaction between Sinorhizobium sp. and Acacia senegal

Sinorhizobium sp. strain HAMBI 2180, marked with the β-glucuronidase (GUS) encoding gusA gene, was used for studying the effect of heat stress on the symbiotic interaction between Acacia senegal seedlings and Sinorhizobium sp. in growth cabinet conditions. The infection process, observed during 18 days after inoculation, was delayed at a root temperature of 40°C compared to a root temperature of ≤38°C, but nodulation was retarded already at 38°C. At 42°C no nodules were formed. At this root temperature the development of lateral roots and root hairs was one week delayed. Although the roots had normally deformed root hairs in addition to abnormally deformed ones, only disintegrated infection threads were present. However, the ability to form symbiosis was reversible. Nitrogen-fixing nodules developed on those parts of the roots which were formed after completion of the thermal stress at 42°C. A root temperature of 42°C did not affect the numbers of culturable rhizobia in the A. senegal rhizosphere.

Characterization of two lipopolysaccharide types isolated from Rhizobium galegae.

Lipopolysaccharides (LPS) of Rhizobium galegae, a symbiotically nitrogen-fixing species of root-nodule bacteria, were isolated by the phenol-water method from strain HAMBI 1461, the LPS of which resembled enterobacterial smooth type LPS, and from strains HAMBI 1174 and HAMBI 1208, the LPSs of which resembled rough type LPS. The results of PAGE analysis of LPSs, Bio-Gel P2 gel filtration of polysaccharide fractions and the presence of deoxysugars and 4-O-methyl-deoxysugar both in the rough and smooth LPSs suggested that rough LPS contained a short O-antigenic polysaccharide for which we propose the name short O-chain LPS. Accordingly, the smooth LPS is called long O-chain LPS. Despite of the differences in the structure of LPS of R. galegae, all strains were equally effective in nodulating their hosts. The short O-chain LPS of R. galegae showed many features similar to those of phylogenetically related agrobacteria.

Assessment of competitiveness of rhizobia infecting Galega orientalis on the basis of plant yield, nodulation, and strain identification by antibiotic resistance and PCR.

Competition between effective and ineffective Rhizobium galegae strains nodulating Galega orientalis was examined on the basis of plant growth, nodulation, antibiotic resistance, and PCR results. In a preliminary experiment in Leonard's jars, ineffective R. galegae strains HAMBI 1207 and HAMBI 1209 competed in similar manners with the effective strain R. galegae HAMBI 1174. In a pot experiment, soil was inoculated with 0 to 10(5) HAMBI 1207 cells per g before G. orientalis was sown. Seeds of G. orientalis were surface inoculated with 2 x 10(4) and 2 x 10(5) cells of HAMBI 1174 per seed (which represent half and fivefold the commercially recommended amount of inoculant, respectively). Plant yield and nodulation by the effective strain were significantly reduced, with as few as 10(2) ineffective rhizobia per g of soil, and the inoculation response was not improved by the 10-fold greater dose of the inoculant. Bacteria occupying the nodules were identified by antibiotic resistance and PCR with primers specific for R. galegae HAMBI 1174, R. galegae, and genes coding for bacterial 16S rRNA (bacterial 16S rDNA). Sixty-two large nodules examined were occupied by the effective strain HAMBI 1174, as proven by antibiotic resistance and amplification of the strain-specific fragment. From 20 small nodules, only the species-specific fragment could be amplified, and isolated bacteria had the same antibiotic resistance and 16S PCR restriction pattern as strain HAMBI 1207. PCR with our strain-specific and species-specific primers provides a powerful tool for strain identification of R. galegae directly from nodules without genetic modification of the bacteria.

Stability of Markers Used for Identification of Two Rhizobium galegae Inoculant Strains after Five Years in the Field

The stability of identification markers was examined for two Rhizobium galegae inoculant strains after 5 years in the field. The two strains are genetically closely related, but differ in their lipopolysaccharides. Strain HAMBI 540 has lipopolysaccharide of the rough type, whereas that of strain HAMBI 1461 is of the smooth type. The properties that were examined for 10 field isolates of each inoculant type were symbiotic phenotype, phage type, intrinsic antibiotic resistance, maximum growth temperature, lipopolysaccharide and total soluble protein patterns, immunological properties, DNA restriction profiles, and DNA hybridization patterns, which were determined by using nifHDK and recA sequences as probes. Of these properties, all remained stable in soil, with the exception of some variation in intrinsic antibiotic resistance and the acquisition of an extra EcoRI restriction fragment by one of the isolates. Thus, both the rough and the smooth lipopolysaccharide phenotypes persisted equally well in soil.

Lipopolysaccharide and protein patterns ofRhizobiumsp. (Galega)

Strains of Rhizobium sp. (Galega) (R. galegae), R. meliloti, R. leguminosarum, and R. loti were compared for their lipopolysaccharide (LPS) and whole cell protein patterns. Antigenic properties of these LPS and proteins were tested by immunoblotting with rabbit antiserum raised against R. galegae strain HAMBI 540. The LPS and protein patterns of R. galegae strains differed from those of the other rhizobia tested. By immunoblotting, a species-specific R. galegae LPS antigen and two proteins specific for R. galegae were identified. Our results support the suggestion that R. galegae strains form a distinct taxonomic group within the genus Rhizobium.

DNA homology, phage-typing, and cross-nodulation studies of rhizobia infecting Galega species

The taxonomic status of Rhizobium nodulating Galega sp. is poorly defined. Earlier reports suggest that the rhizobia infecting G. officinalis should be included in the R. leguminosarum species or in the "fast-growing lupin – cowpea complex." Therefore organisms nodulating G. orientalis and G. officinalis were studied using cross nodulation, DNA homology, and phage typing. No cross-nodulation relationship between the Galega rhizobia and rhizobial strains from the major taxonomic groups was found. The mean relative DNA homology of 10 strains of Rhizobium nodulating Galega with reference DNA from strain HAMBI 540, which forms effective nodules on G. orientalis, was 77 ± 9%, compared with 19 ± 6% for 20 strains from other host plants. The Galega rhizobia formed a unique phage-typing group, not related to the major groups of fast growers, in agreement with the DNA homology and cross-nodulation results. These data indicate that the Galega rhizobia form a specific taxonomic group within the genus Rhizobium.