NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis.

The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wild-type strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species.

Rhizobial strains isolated from nodules of Medicago marina in southwest Spain are abiotic-stress tolerant and symbiotically diverse.

The isolation and characterisation of nitrogen-fixing root nodule bacteria from Medicago marina, a tolerant legume species, were studied in two areas from southwest Spain. A total of 30 out of 82 isolates with distinct ERIC-PCR fingerprints were analysed on the basis of molecular (PCR-RFLP of the 16S-23S rDNA intergenic spacer region (IGS) with two endonucleases, analysis of the 16S rDNA and symbiotic nodC gene sequences, plasmid profiles and SDS-PAGE of LPS, including the partial sequence of the housekeeping gene glnII and the symbiotic gene nodA of some representatives), physiological (utilisation of sole carbon sources, tolerance to antibiotics, NaCl, heavy metals, temperature and pH) and symbiotic parameters (efficacy on M. marina, M. minima, M. murex, M. orbicularis, M. polymorpha, M. sativa and M. truncatula). All the bacteria isolated from M. marina nodules belonged to Ensifer meliloti, except for one strain that belonged to E. medicae. To determine the nodulation range of M. marina, 10 different Ensifer species were tested for their ability to nodulate on this plant. E. kummerowiae CCBAU 71714 and the E. medicae control strain M19.1 were the only Ensifer species tested that developed nitrogen-fixing nodules on this plant. Most of the M. marina-nodulating strains showed tolerance to stress factors and all of them shared the presence of a gene similar to cadA, a gene that encodes for a PIB-type ATPase, which is a transporter belonging to the large superfamily of ATP-driven pumps involved in the transport of metals across cell membranes.

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.

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.

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.

NopM and NopD are rhizobial nodulation outer proteins: identification using LC-MALDI and LC-ESI with a monolithic capillary column.

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLC-MS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD.

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.

Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S. fredii strains.

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.