Flavonoid-inducible modifications to rhamnan O antigens are necessary for Rhizobium sp. strain NGR234-legume symbioses.

Rhizobium sp. strain NGR234 produces a flavonoid-inducible rhamnose-rich lipopolysaccharide (LPS) that is important for the nodulation of legumes. Many of the genes encoding the rhamnan part of the molecule lie between 87 degrees and 110 degrees of pNGR234a, the symbiotic plasmid of NGR234. Computational methods suggest that 5 of the 12 open reading frames (ORFs) within this arc are involved in synthesis (and subsequent polymerization) of L-rhamnose. Two others probably play roles in the transport of carbohydrates. To evaluate the function of these ORFs, we mutated a number of them and tested the ability of the mutants to nodulate a variety of legumes. At the same time, changes in the production of surface polysaccharides (particularly the rhamnan O antigen) were examined. Deletion of rmlB to wbgA and mutation in fixF abolished rhamnan synthesis. Mutation of y4gM (a member of the ATP-binding cassette transporter family) did not abolish production of the rhamnose-rich LPS but, unexpectedly, the mutant displayed a symbiotic phenotype very similar to that of strains unable to produce the rhamnan O antigen (NGRDeltarmlB-wbgA and NGROmegafixF). At least two flavonoid-inducible regulatory pathways are involved in synthesis of the rhamnan O antigen. Mutation of either pathway reduces rhamnan production. Coordination of rhamnan synthesis with rhizobial release from infection threads is thus part of the symbiotic interaction.

High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234.

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

nodD2 of Rhizobium sp. NGR234 is involved in the repression of the nodABC operon.

Transcriptional regulators of the lysR family largely control the expression of bacterial symbiotic genes. Rhizobium sp. NGR234 contains at least four members of this family: two resemble nodD, while two others are more closely related to syrM. Part of the extremely broad host range of NGR234 can be attributed to nodD1, although the second gene shares a high degree of DNA sequence homology with nodD2 of R. fredii USDA191. A nodD2 mutant of NGR234 was constructed by insertional mutagenesis. This mutant (NGR omega nodD2) was deficient in nitrogen fixation on Vigna unguiculata and induced pseudonodules on Tephrosia vogelii. Several other host plants were tested, but no correlation could be drawn between the phenotype and nodule morphology. Moreover, nodD2 has a negative effect on the production of Nod factors: mutation of this gene results in a fivefold increase in Nod factor production. Surprisingly, while the structure of Nod factors from free-living cultures of NGR omega nodD2 remained unchanged, those from V. unguiculata nodules induced by the same strain are non-fucosylated and have a lower degree of oligomerization. In other words, developmental regulation of Nod factor production is also abolished in this mutant. Competitive RNA hybridizations, gene fusions and mobility shift assays confirmed that nodD2 downregulates expression of the nodABC operon.

Rhizobium sp. strain NGR234 NodZ protein is a fucosyltransferase.

Rhizobium sp. strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents. Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells. Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid. Crude extracts of this transconjugant possess fucosyltransferase activity. Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors. Pentameric chitin is the preferred substrate. These data suggest that fucosylation occurs before acylation of the Nod factors.

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host-specificity gene.

Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N-acetyl-D-glucosamine of LCOs is substituted at C6 with 2-O-methyl-L-fucose which can be acetylated or sulphated. We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeE, which is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum. Furthermore, mutation of noeE (NGRdelta noeE) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus. The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRdelta noeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2-O-methylfucose residue. Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase.

Molecular basis of symbiosis between Rhizobium and legumes.

Access to mineral nitrogen often limits plant growth, and so symbiotic relationships have evolved between plants and a variety of nitrogen-fixing organisms. These associations are responsible for reducing 120 million tonnes of atmospheric nitrogen to ammonia each year. In agriculture, independence from nitrogenous fertilizers expands crop production and minimizes pollution of water tables, lakes and rivers. Here we present the complete nucleotide sequence and gene complement of the plasmid from Rhizobium sp. NGR234 that endows the bacterium with the ability to associate symbiotically with leguminous plants. In conjunction with transcriptional analyses, these data demonstrate the presence of new symbiotic loci and signalling mechanisms. The sequence and organization of genes involved in replication and conjugal transfer are similar to those of Agrobacterium, suggesting a recent lateral transfer of genetic information.

Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 nod factors.

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a N-linked methyl group on the glucosaminyl terminus. Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.

Organization of host-inducible transcripts on the symbiotic plasmid of Rhizobium sp. NGR234.

In a systematic approach to identify genes involved in the early steps of the legume-Rhizobium symbiosis, we studied transcription patterns of symbiotic plasmid-borne loci. A competitive hybridization procedure was used to identify DNA restriction fragments carrying genes whose expression is enhanced by plant root exudates or by purified flavonoids. Fragments containing induced genes were then located on the physical map of the 500 kb pNGR234a. New inducible loci as well as previously described genes were identified and their time course of induction determined. After initial induction, transcription of loci such as nodABC and the host-specificity genes nodSU decreased to undetectable levels 24 h after incubation with purified flavonoids. In contrast, expression of other loci is detectable only after several hours of induction. Surprisingly, many genes remained transcribed in the nodD1- mutant suggesting the presence of other flavonoid-dependent activators in NGR234. The hsnl region, which is involved in host specificity, was shown to carry several inducible but independently regulated transcripts. Sequencing analysis revealed several open reading frames whose products, based on sequence similarities, may be involved in L-fucose metabolism and its adjunction to the Nod factors.

Subtraction hybridisation and shot-gun sequencing: a new approach to identify symbiotic loci.

Traditionally, new loci involved in the Rhizobium-legume symbiosis have been identified by transposon mutagenesis and/or complementation. Wide dispersal of the symbiotic loci in Rhizobium species NGR234, as well as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques. As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, subtractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of most nodulation genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmid library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonoids. To target genes not present in the closely related strain R. fredii USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation. Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins. Amongst these, a symbiotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.

Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp. strain NGR234 to nodulate Leucaena spp.

Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod-box promoter from NGR234 are sufficient to confer this extended host-range phenotype to L. leucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod-box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5'-end of nodS of USDA257. Consequently, the deduced NodS protein of USDA257 is shorter than its counterpart from NGR234, and the distance between the nod-box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod-box and extends into the upstream leader sequence. Transcriptional fusions with both nod-boxes confirmed that the promoter from NGR234 is flavonoid-inducible, and that the nod-box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS-containing Xhol fragment as hybridization probe. Flavonoid-induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRI fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives of Bradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild-type, leucaena-nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species.

Transposable elements for efficient manipulation of a wide range of gram-negative bacteria: promoter probes and vectors for foreign genes.

We describe here the construction and use of a series of modified transposons based on the insertion sequence IS1. Like their parent, omegon-Km [Fellay et al., Gene 76 (1989) 215-226], these elements permit efficient insertional mutagenesis of a variety of Gram-negative bacteria. The presence of a functional pBR322 origin of replication within the transposable element facilitates subsequent cloning of the mutated gene. The omegon-Km system was previously shown to function in Pseudomonas putida, Rhizobium leguminosarum and Paracoccus denitrificans. The results we present here demonstrate that its use can be extended to Xanthomonas campestris, a plant pathogen, and to the microaeroduric Zymomonas mobilis. Derivative transposons carrying unique restriction sites for ScaI, NdeI, XbaI and XhoI have been constructed, allowing the cloning and introduction of foreign genes. We have also constructed two derivatives which can be used to generate operon fusions upon insertion and are thus useful for isolating and characterising indigenous promoters. One carries a promoterless chloramphenicol acetyl-transferase (CAT)-encoding gene (cat) and the second, the entire promoterless Escherichia coli lac operon. We demonstrate the utility of the cat promoter probe in X. campestris to target conditional promoters inducible by high salt or subject to repression by glucose.

Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria.

To combine the features of the omega interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the omega interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (alph A) which is expressed well in many Gram-negative bacteria. The IS1 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain in which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.

Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria.

We have constructed a series of derivatives of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg2+ resistance gene (ApR, CmR, TcR, KmR or HgR) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these omega derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing an omega interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected. Thus, the selection and/or characterization of omega insertional mutations can be carried out in these bacterial species.