Francisco Javier Ollero

Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.).

BackgroundRhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown.ResultsGenome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins.ConclusionsAvailability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.

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.

Rice and bean AHL-mimic quorum-sensing signals specifically interfere with the capacity to form biofilms by plant-associated bacteria.

Many bacteria regulate their gene expression in response to changes in their population density in a process called quorum sensing (QS), which involves communication between cells mediated by small diffusible signal molecules termed autoinducers. n-acyl-homoserine-lactones (AHLs) are the most common autoinducers in proteobacteria. QS-regulated genes are involved in complex interactions between bacteria of the same or different species and even with some eukaryotic organisms. Eukaryotes, including plants, can interfere with bacterial QS systems by synthesizing molecules that interfere with bacterial QS systems. In this work, the presence of AHL-mimic QS molecules in diverse Oryza sativa (rice) and Phaseolus vulgaris (bean) plant-samples were detected employing three biosensor strains. A more intensive analysis using biosensors carrying the lactonase enzyme showed that bean and rice seed-extract contain molecules that lack the typical lactone ring of AHLs. Interestingly, these molecules specifically alter the QS-regulated biofilm formation of two plant-associated bacteria, Sinorhizobium fredii SMH12 and Pantoea ananatis AMG501, suggesting that plants are able to enhance or to inhibit the bacterial QS systems depending on the bacterial strain. Further studies would contribute to a better understanding of plant-bacteria relationships at the molecular level.

Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103

In this work we show that the Sinorhizobium fredii HH103 ttsI gene is essential for the expression of the tts genes and secretion of nodulation outer proteins (Nops). Moreover, we demonstrate for the first time, to our knowledge, that the nod box preceding ttsI is necessary for Nops secretion. TtsI is responsible for the transcriptional activation of nopX, nopA, rhcJ and rhcQ. We confirm that the S. fredii HH103 ttsI gene is activated by NodD1 and repressed by NolR. In contrast, NodD2 is not involved in the regulation of ttsI expression. Despite the dependence of expression of both ttsI and nodA on NodD1 and flavonoids, clear differences in the capacity of some flavonoids to activate these genes were found. The expression of the ttsI and nodA genes was also sensitive to differences in the pH of the media. Secretion of Nops in the ttsI mutant could not be complemented with a DNA fragment containing the ttsI gene and its nod box, but it was restored when a plasmid harbouring the ttsI, rhcC2 and y4xK genes was transferred to the mutant strain. The symbiotic effect of Nops secretion was host-dependent but independent of the type of nodule formed by the host legume. Nops are beneficial in the symbiosis with Glycine max and Glycyrrhiza uralensis, and detrimental in the case of the tropical legume Erythrina variegata.

Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia ☆

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant-microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.

The Absence of Nops Secretion in Sinorhizobium fredii HH103 Increases GmPR1 Expression in Williams Soybean

Sinorhizobium fredii HH103 secretes through the type III secretion system at least eight nodulation outer proteins (Nops), including the effector NopP. These proteins are necessary for an effective nodulation of soybean. In this work, we show that expression of the nopP gene depended on flavonoids and on the transcriptional regulators NodD1 and TtsI. Inactivation of nopP led to an increase in the symbiotic capacity of S. fredii HH103 to nodulate Williams soybean. In addition, we studied whether Nops affect the expression of the pathogenesis-related genes GmPR1, GmPR2, and GmPR3 in soybean roots and shoots. In the presence of S. fredii HH103, expression of pathogenesis-related (PR) gene PR1 was induced in soybean roots 4 days after inoculation and it increased 8 days after inoculation. The absence of Nops provoked a higher induction of PR1 in both soybean roots and shoots, suggesting that Nops function early, diminishing plant defense responses during rhizobial infection. However, the inactivation of nopP led to a decrease in PR1 expression. Therefore, the absence of NopP or that of the complete set of Nops seems to have opposite effects on the symbiotic performance and on the elicitation of soybean defense responses.

Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinate- and indeterminate nodule-forming legumes and overproduce an altered EPS.

Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

CONSTRUCTION OF MULTIPURPOSE GENE CARTRIDGES BASED ON A NOVEL SYNTHETIC PROMOTER FOR HIGH-LEVEL GENE EXPRESSION IN GRAM-NEGATIVE BACTERIA

A series of gene cartridges containing a novel synthetic promoter (Psyn) was constructed. The Psyn sequence is based on the consensus of a number of naturally occurring promoters and displays strong activity in Escherichia coli and Rhizobium leguminosarum. In a direct comparison, Psyn proved to be about twice as strong as the tac promoter in E. coli, while the difference in Rhizobium was about tenfold. A small Psyn cartridge was constructed by adding a Shine-Dalgarno sequence, an ATG codon, and a removable lac operator, whose excision can convert the regulated cartridge into a constitutively expressed unit. A second cassette was obtained by the addition of a lacIq gene in order to provide autonomous regulation also in hosts lacking lacI functions, such as R. leguminosarum. A promoterless lacZ gene was inserted to monitor the activity. This gene can be either replaced with genes of interest, or used for gene fusions by means of conveniently positioned restriction sites. A third cassette was generated by adding a mercury-resistance determinant as a selectable marker, suitable for monitoring tagged bacteria released into environments. In such cases, where a non-antibiotic-resistant marker is preferable, the use of mercury chloride adds the advantage of inhibiting fungal growth when plating soil suspensions. The presence of the second marker, lacZ driven by the strong Psyn, facilitates the selection. Furthermore, the Psyn fragment can be used as a specific probe for the detection of released bacteria engineered with any of the above constructs.