M. R. Espuny

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

NolR Regulates Diverse Symbiotic Signals of Sinorhizobium fredii HH103

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Str(r)) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysaccharide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.

Opening the “black box” of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899

BackgroundTranscription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes—five—and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants.MethodsThe three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.).ResultsPhenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions.ConclusionsWe report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene—nodD4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.

Polysaccharides and lipopolysaccharides and infectivity of Rhizobium trifolii

Abstract The extracellular polysaccharides obtained from infective and noninfective strains of Rhizobium trifolii were analysed to investigate if infective properties were correlated with polysaccharide composition. The results indicate that soluble extracellular polysaccharides were not a determinant factor in infectivity. However, electrophoresis of bacterial surface lipopolysaccharides showed a relation between the loss of infectivity and changes in electrophoretic mobility.

An exoB mutant of Rhizobium sp. is effective in indeterminate nodules of Hedysarum coronarium

A Rhizobium sp. (Hedysarum coronarium) calcofluor dark (Cal-) mutant, named Cal10, was obtained following Tn5mob-insertion mutagenesis. It is affected in the synthesis of exopolysaccharide and presents an altered lipopolysaccharide that is not recognized by a polyclonal antibody against the lipopolysaccharide of the parental strain. The residual exopolysaccharide obtained from the mutant lacks galactose and the high-molecular-mass acidic fraction. This mutant was complemented by plasmid pD56 that restores the production of exopolysaccharide, the alteration of lipopolysaccharide and the Cal phenotype. The data presented indicate that the gene in which the mutant is defective is homologous to the exoB gene of Rhizobium meliloti and fails to synthesize UDP-glucose 4′-epimerase. The Cal10 mutant was Fix+ on H. coronarium (sulla) although it develops an indeterminate type of nodule, indicating that exopolysaccharide is not essential for a successful nodulation in this symbiotic association.

ISRf1, a transposable insertion sequence from Sinorhizobium fredii

Sinorhizobium fredii strain HH103, a nitrogen-fixing bacterial symbiont of plants, contains an insertion sequence (IS) that can transpose into plasmid pMUS248 and activate a promoterless TcR gene that is normally not expressed. We have cloned and characterized this element, which we designate ISRf1. The IS is 1002 bp in length, contains a single 513-bp open reading frame (ORF), is flanked by imperfect 36-bp terminal inverted repeats, and creates 5-bp target duplications. Two copies of ISRf1 are present in the genome of HH103, but it is absent from 12 other Sinorhizobium and Rhizobium strains. The element transposes at a frequency of 2.7 x 10(-6) per generation per cell.

Alfalfa nodulation by Sinorhizobium fredii does not require sulfated Nod-factors

Rhizobium strain 042B(s) is able to nodulate both soybean and alfalfa cultivars. We have demonstrated, by mass spectrometry, that the nodulation (Nod) factors produced by this strain are characteristic of those produced by Sinorhizobium fredii, which typically nodulates soybean; they have 3-5 N-acetylglucosamine (GlcNAc) residues, a mono-unsaturated or saturated C16, C18 or C20 fatty-acyl chain, and a (methyl)fucosyl residue on C6 of the reducing-terminal GlcNAc. In order to study Rhizobium strain 042B(s) and its nodulation behaviour further, we introduced an insertion mutation in the noeL gene, which is responsible for the presence of the (methyl)fucose residue on the reducing terminal GlcNAc of the Nod-factors, yielding mutant strain SVQ523. A plasmid (pHM500) carrying nodH, nodP and nodQ, the genes involved in sulfation of Nod-factors on C6 of the reducing-terminal GlcNAc, was introduced into SVQ523, generating SVQ523.pHM500. As expected, strain SVQ523 produces unfucosylated Nod-factors, while SVQ523.pHM500 produces both unfucosylated and unfucosylated sulfated Nod-factors. Plant tests showed that soybean nodulation was reduced if the inoculant (SVQ523.pHM500) produced sulfated Nod-factors. In the Asiatic alfalfa cultivar Baoding, SVQ523 (absence of a substitution at C6) failed to nodulate, but both 042B(s) (fucosyl at C6) and SVQ523.pHM500 (sulfate at C6) formed nodules. In contrast, SVQ523 showed enhanced nodulation capacity with the western alfalfa cultivars ORCA and ARC. These results indicate that Nod-factor sulfation is not a requisite for S. fredii to nodulate alfalfa.

Analysis of Surface Infectivity Determinants in Rhizobium

Recent work has focused attention to the study of interactions between host lectins and rhizobial surface carbohydrates. We have examined the possible role of cell wall lipopolysaccharides (LPS) on the infective capability of Rhizobium.

Isolation and Characterization of an Insertion Element from Rhizobium fredii

Rhizobium fredii HH103 strain contains an insertion sequence (IS) that can transpose into plasmid pMUS248 and activate a promoterless TcR gene that is normally not expressed. We have cloned and characterised this element, which we designated ISRfl. This insertion element (Genome Sequence Database Accession No Y08939) is distinct from all previously characterised insertion elements of the Family Rhizobiaceae and shows the following characteristics: 1) It is 1,002 bp in length. 2) It is sandwiched between a pair of directly repeated AT-rich pentamers (TTACA) that form the apparent target duplication for the element. 3) The ends of the element itself contain 36 bp terminal inverted repeats with a 5 bp mismatch. 4) The terminal inverted repeats of ISRf1 flank a 930 bp central region that forms the core of the element. There is a single 513 bp ORF with a high probability of encoding a hydrophobic protein. We could not find any significant homology between this polypeptide and any sequence deposited in protein sequence databases. 5) Portions of the noncoding region of ISRf1 do, however, bear a significant structural resemblance to transposon Tn5403 from Klebsiella pneumoniae. The left and right terminal inverted repeats of ISRf1 harbour only four and six mismatches with respect to the corresponding 38 bp terminal inverted repeats of Tn5403 (Accession No. X75779). In addition, a block of bases that lies between positions 51 and 146 of the R. fredii sequence is 74% identical to bases 632 to 738 of the K. pneumoniae element. This homology corresponds almost precisely to a noncoding spacer region that separates tnpR and tnpA, the divergently oriented resolvase and transposase of Tn5403.

NodD2 in Multicopy Complements NodD1 Mutants of Rhizobium fredii for Nodulation of Soybean

All known R. fredii strains contain two copies of nodD, which are termed nodD1 and nodD2. Mutations in nodD1 abolish the ability to form nodules. Mutations in nodD2 delay nodulation. We have inserted, by marker exchange experiments, the omega cassette (Ω) into the NruI site of nodD1 of R. fredii USDA257 and HH103. Both mutants are unable to nodulate, to induce a nodAp-lacZ fusion in the presence of flavonoids and to produce Nod-factors. DNA hybridization experiments, using nodD1, nodD2 and the Ω. cassette as probes, showed that the nodD2 gene is not affected in the HH103 NodD1− mutant. Hence, nodD2 is not able to replace the nodD1 gene for the trascriptional activation of nodulation genes and nodulation ability. However, a multicopy vector containing the nodD2 gene from USDA257 (called pHBK330) restored nodulation ability to both NodD1− mutants. USDA257 NodD1− carrying pHBK330 produced low quantities of Nod-factors in the presence of flavonoids, or even in their absence. Apparently, the amounts of Nod-factors produced with and without flavonoids are very similar. This fact indicates that, in the presence of extra copies of nodD2, this production is constitutive and independent of flavonoids. Although nodD2 also restored nodulation ability of HH103 NodD1−, we could not demonstrate the production of Nod-factors in the presence of extra copies of nodD2. Moreover, isolates from soybean nodules produced by HH103 NodD1− (pHBK330) did not maintain pHBK330. Instead, they have plasmids than were larger than expected. Because these plasmids code for Tc-resistance (the marker of pHBK330), it is reasonable to assume that they are recombinant derivatives of pHBK330 and the genome of HH103.