2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant

When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps⁺ strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.     

Varying the abundance of O antigen in Rhizobium etli and its effect on symbiosis with Phaseolus vulgaris

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region alpha, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from lps region alpha elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris. The latter strains were mutated in lps region alpha and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamine.     

Tyrosinase from Rhizobium etli is involved in nodulation efficiency and symbiosis-associated stress resistance

Tyrosinase (EC 1.14.18.1) is a monophenol oxidase responsible for the synthesis of the black pigment known as melanin. The tyrosinase gene (melA) is plasmid-encoded in many rhizobial species. In Rhizobium etli CFN42, the genetic location of melA in the symbiotic plasmid (p42d) and its RpoN-NifA regulation suggest an involvement in symbiosis. In this work, we analyzed the symbiotic phenotype of a streptomycin-resistant derivative of CFN42 (CE3), a melA mutant (SP2) and a complemented strain (SP66), demonstrating that melA inactivation reduced nodule formation rate and diminished total nodule number by 27% when compared to the CE3 strain. The nitrogen fixation capacity of the mutant strain was not affected. Also, in vitro assays were performed where the resistance of CE3, SP2 and SP66 strains to H(2)O(2) was evaluated; the melA mutant strain was consistently less resistant to peroxide. In another series of experiments, Escherichia coli W3110 strain expressing R. etli melA displayed enhanced resistance to p-hydroxybenzoic, vanillinic and syringic acids, which are phenolic compounds frequently found in the soil. Our results are the first to demonstrate a specific role for tyrosinase in R. etli: this enzyme is required during early symbiosis, apparently providing resistance against reactive oxygen species and phenolic compounds generated as part of the plant protective responses.     

Regulatory role of Rhizobium etli CNPAF512 fnrN during symbiosis

The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN(2) region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of R. etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation. Inoculation of Phaseolus vulgaris with an R. etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the R. etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of fixN and fixG under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.     

The type and yield of lipopolysaccharide from symbiotically deficient rhizobium lipopolysaccharide mutants vary depending on the extraction method

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.     

The Rhizobium etli argC gene is essential for Arginine biosynthesis and nodulation of Phaseolus vulgaris

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.     

The Rhizobium etli trpB gene is essential for an effective symbiotic interaction with Phaseolus vulgaris.

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.     

The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli

A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a DeltarelA DeltaspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Omega element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-beta-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.     

Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.     

Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.     

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.     

Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.     

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation.

By its inability to grow on sulfate as the sole sulfur source, a mutant strain (CTNUX8) of Rhizobium etli carrying Tn5 was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a cysG (siroheme synthetase)-homologous gene. By RNase protection assays, it was established that the cysG-like gene had a basal level of expression in thiosulfate- or cysteine-grown cells, which was induced when sulfate or methionine was used. Unlike its wild-type parent (strain CE3), the mutant strain, CTNUX8, was also unable to grow on nitrate as the sole nitrogen source and was unable to induce a high level of nitrite reductase. Despite its pleiotropic phenotype, strain CTNUX8 was able to induce pink, effective (N2-fixing) nodules on the roots of Phaseolus vulgaris plants. However, mixed inoculation experiments showed that strain CTNUX8 is significantly different from the wild type in its ability to nodulate. Our data support the notion that sulfate (or sulfite) is the sulfur source of R. etli in the rhizosphere, while cysteine, methionine, or glutathione is supplied by the root cells to bacteria growing inside the plant.     

A site-specific recombinase (RinQ) is required to exert incompatibility towards the symbiotic plasmid of Rhizobium etli

The replication/partition region of the symbiotic plasmid p42d of Rhizobium etli CE3 is characterized by the presence of the repABC operon. A recombinant plasmid containing this region is able to replicate in a R. etli derivative cured from p42d, with the same stability and copy number shown by the parental plasmid. However, when this construct is introduced into the wild-type strain, instead of exerting incompatibility against the p42d, it forms a stable cointegrate with it. In this paper, we show that a site-specific resolvase, and its action sites are essential factors to displace the symbiotic p42d. We propose a model for this novel incompatibility mechanism.     

Genetic locus and structural characterization of the biochemical defect in the O-antigenic polysaccharide of the symbiotically deficient Rhizobium etli mutant, CE166. Replacement of N-acetylquinovosamine with its hexosyl-4-ulose precursor

The O-antigen polysaccharide (OPS) of Rhizobium etli CE3 lipopolysaccharide (LPS) is linked to the core oligosaccharide via an N-acetylquinovosaminosyl (QuiNAc) residue. A mutant of CE3, CE166, produces LPS with reduced amounts of OPS, and a suppressed mutant, CE166 alpha, produces LPS with nearly normal OPS levels. Both mutants are deficient in QuiNAc production. Characterization of OPS from CE166 and CE166 alpha showed that QuiNAc was replaced by its 4-keto derivative, 2-acetamido-2,6-dideoxyhexosyl-4-ulose. The identity of this residue was determined by NMR and mass spectrometry, and by gas chromatography-mass spectrometry analysis of its 2-acetamido-4-deutero-2,6-dideoxyhexosyl derivatives produced by reduction of the 4-keto group using borodeuteride. Mass spectrometric and methylation analyses showed that the 2-acetamido-2,6-dideoxyhexosyl-4-ulosyl residue was 3-linked and attached to the core-region external Kdo III residue of the LPS, the same position as that of QuiNAc in the CE3 LPS. DNA sequencing revealed that the transposon insertion in strain CE166 was located in an open reading frame whose predicted translation product, LpsQ, falls within a large family of predicted open reading frames, which includes biochemically characterized members that are sugar epimerases and/or reductases. A hypothesis to be tested in future work is that lpsQ encodes UDP-2-acetamido-2,6-dideoxyhexosyl-4-ulose reductase, the second step in the synthesis of UDP-QuiNAc from UDP-GlcNAc.     

Synthesis of the flavonoid-induced lipopolysaccharide of Rhizobium Sp. strain NGR234 requires rhamnosyl transferases encoded by genes rgpF and wbgA

In the presence of flavonoids, Rhizobium sp. strain NGR234 synthesizes a new lipopolysaccharide (LPS), characterized by a rhamnan O-antigen. The presence of this rhamnose-rich LPS is important for the establishment of competent symbiotic interactions between NGR234 and many species of leguminous plants. Two putative rhamnosyl transferases are encoded in a cluster of genes previously shown to be necessary for the synthesis of the rhamnose-rich LPS. These two genes, wbgA and rgpF, were mutated. The resulting mutant strains synthesized truncated rough LPS species rather than the wild-type rhamnose-rich LPS when grown with flavonoids. Based on the compositions of these purified mutant LPS species, we inferred that RgpF is responsible for adding the first one to three rhamnose residues to the flavonoid-induced LPS, whereas WbgA is necessary for the synthesis of the rest of the rhamnan O-antigen. The NGR234 homologue of lpsB, which, in other bacteria, encodes a glycosyl transferase acting early in synthesis of the core portion of LPS, was identified and also mutated. LpsB was required for all the LPS species produced by NGR234, in the presence or absence of flavonoids. Mutants (i.e., of lpsB and rgpF) that lacked any portion of the rhamnan O-antigen of the induced LPS were severely affected in their symbiotic interaction with Vigna unguiculata, whereas the NGR?wbgA mutant, although having very few rhamnose residues in its LPS, was able to elicit functional nodules.     

Identification of an ATP-binding cassette transporter for export of the O-antigen across the inner membrane in Rhizobium etli based on the genetic, functional, and structural analysis of an lps mutant deficient in O-antigen

For O-antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenyl pyrophosphate-bound O-antigen oligosaccharide subunits or polysaccharide occurs before ligation to the core region of the LPS molecule. In this study, we identified by mutagenesis an ATP-binding cassette transporter in Rhizobium etli CE3 that is likely responsible for the translocation of the O-antigen across the inner plasma membrane. Mutant FAJ1200 LPS lacks largely the O-antigen, as shown by SDS-polyacrylamide gel electrophoresis and confirmed by immunoblot analysis. Furthermore, LPS isolated from FAJ1200 is totally devoid of any O-chain glycosyl residues and contains only those glycosyl residues that can be expected for the inner core region. The membrane component and the cytoplasmic ATP-binding component of the ATP-binding cassette transporter are encoded by wzm and wzt, respectively. The Tn5 transposon in mutant FAJ1200 is inserted in the wzm gene. This mutation resulted in an Inf- phenotype in bean plants.