Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover

Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.     

A Hydrophobic Mutant of Rhizobium etli Altered in Nodulation Competitiveness and Growth in the Rhizosphere

We isolated and characterized CE3003, a Tn5-induced mutant with altered colony morphology derived from Rhizobium etli CE3. CE3003 produced domed colonies and was highly hydrophobic as indicated by its ability to partition into hexadecane, whereas its parent produced flat colonies and was hydrophilic. On bean plants, CE3003 induced nodules and reduced acetylene. CE3003 and CE3 grew at similar rates when they were grown separately or together in culture medium or inoculated singly onto bean seeds. However, when they were mixed at a 1:1 ratio and applied to seeds, CE3003 achieved significantly lower populations than CE3 in the rhizosphere. Five days after coinoculation of CE3 and CE3003, the population of the mutant was less than 10% of the population of CE3 in the bean rhizosphere. To determine the nodulation competitiveness of the mutant, it was coinoculated with CE3 at various ratios at planting, and the ratio of the nodules occupied by each strain was determined 21 days later. A 17,000-fold excess of CE3003 in mixed inocula was required to obtain equal nodule occupancy by the two strains. A genomic library of strain CE3 was mobilized into CE3003, and we identified a cosmid, pRA3003, that restored the parental colony morphology and hydrophilicity to the mutant. Restoration of the parental colony morphology was accompanied by recovery of the ability to grow competitively in the rhizosphere and to compete for nodulation of beans. The data show an association between cell surface hydrophobicity, nodulation competitiveness, and competitive growth in the rhizosphere in mutant CE3003.     

Genetic Basis for Rhizobium etli CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis.O antigens typically constitute the distal portions of lipopolysaccharides (LPS) and help determine the diverse surface characteristics of Gram-negative bacteria. These repeat unit carbohydrate polymers vary tremendously in structure and, as a family, they exhibit all known sugars and sugar modifications, linked in myriad ways forming homopolymers and heteropolymers. Control of polymer length also varies, allowing highly uniform to completely random lengths. Great diversity of O-antigen structures even within a species is well known. Moreover, O antigens of a single strain can vary according to growth and environmental conditions. One such condition is the presence of a multicellular host (5, 18, 36, 40, 42, 44).Rhizobium etli CE3 fixes nitrogen inside root nodules it incites on the common bean Phaseolus vulgaris. The O antigen of its LPS (Fig. ​(Fig.1)1) is essential for bacterial infection during development of this symbiosis (41). In addition, at least two alterations occur in the O antigen when R. etli CE3 is grown in the presence of either the host plant or plant exudates. The content of the multiply O-methylated terminal fucose is decreased (19, 44), whereas the 2-O methylation of internal fucoses (2OMeFuc) increases twofold (Fig. ​(Fig.1)1) (15, 44). In addition to the multiply O-methylated terminal fucose and 2OMeFuc, methylation occurs always on 6-deoxytalose and likely on glucuronic acid to yield 3-O-methyl-6-deoxytalose (3OMe6dTal) and methyl-esterified glucuronyl (MeGlcA) residues (Fig. ​(Fig.1)1) (22); however, the incidence of these methylations is not known to vary with growth condition. The genetics responsible for the variable O methylations and the additions of the residues they modify have not been elucidated.Open in a separate windowFIG. 1.R. etli CE3 O-antigen structure (22). The portion of the LPS conceptually defined as O antigen begins with N-acetyl-quinovosamine (QuiNAc) at the reducing end followed by a mannose (Man) residue and a fucose (Fuc) residue. Attached to this fucose is the repeating unit consisting of one fucose residue, one 3-O-methyl-6-deoxytalose residue (3OMe6dTal), and one glucuronyl methyl ester residue (MeGlcA). The sugars of the repeating unit are added sequentially exactly five times (in most molecules). An O-acetyl group is present in each of the repeating units, but its location is unknown at this time. Growth in TY culture results in one 2-O-methylfucose (2OMeFuc) per O antigen on average (22). The O-antigen backbone is capped with a 2,3-di-O-methylfucose (referred to as the terminal residue in this report) on which additional O methylation at the 4-position is variable as indicated by parentheses. Growth of the bacteria in the presence of the host plant or plant exudates induces the increase of 2-O methylation of internal fucose (2OMeFuc) residues and decreased relative amount of the terminal residue (44).Most mutations affecting the known R. etli CE3 O-antigen structure map to a 28-kb genetic cluster on the chromosome (Fig. ​(Fig.2)2) (previously referred to as lps region α [8, 19, 37, 40, 45]). Genes and mutations within this cluster previously have been given the designations lps (9) and lpe (19). Recently, the new designation wre has been sanctioned by the Bacterial Polysaccharide Gene Database for this genetic cluster and other genes specifically devoted to the R. etli CE3 O antigen, in keeping with the system of nomenclature for bacterial polysaccharide genes (47).Open in a separate windowFIG. 2.R. etli CE3 O-antigen genetic cluster. (A) The R. etli CE3 chromosomal O antigen genetic cluster spans nucleotides 784527 to 812262 of the genome sequence (28) and consists of 25 putative ORFs. ORFs relevant to the present study are enlarged, and the relative locations of mutations are indicated. White triangles indicate mutations created by insertion of antibiotic cassettes, and black triangles indicate mutations created by Tn5 mutagenesis. The strain numbers carrying these mutations are indicated above the triangles. (B) The solid bars represent the extents of R. etli CE3 DNA cloned for complementation analysis. The scale and positions match those of the lower map in panel A.Duelli et al. (19) identified a 3-kb genetic locus that is required for the presence of the 2,3-di-O-methylfucose or 2,3,4-tri-O-methylfucose at the terminus of the O antigen. Now known to be near one end of the O-antigen genetic cluster (Fig. ​(Fig.2),2), the DNA sequence reported by Duelli et al. encompasses nucleotides 807701 to 810147 of the subsequently determined genome sequence (28). Sequence and annotation of the 3-kb locus have since been revised. In place of the four open reading frames (ORFs) suggested previously (19), the current annotation predicts two ORFs: wreA and wreC (Fig. ​(Fig.2).2). The wreA ORF is predicted to encode a methyltransferase (19), but the predicted WreC polypeptide sequence matches no known methyltransferase or glycosyltransferase or any other polypeptide sequence in the database (Fig. ​(Fig.3).3). When it became clear that this locus was part of the larger O-antigen genetic cluster, the nucleotide sequence suggested that three genes contiguous to wreA also might encode functions needed for synthesis and addition of the terminal fucose. The results to be shown bore out predictions of this hypothesis.Open in a separate windowFIG. 3.Conserved domain predictions. Spanning nucleotides 804817 to 810147 of the genome sequence (28), ORFs RHE_CH00766, RHE_CH00767, RHE_CH00768, RHE_CH00769, and RHE_CH00770 were named wreB, wreD, wreF, wreA, and wreC, respectively. Previously, wreF, wreA, and wreC were referred to as nlpe2, lpeA, and nlpe1, respectively (19). ORF RHE_CH00755, spanning nucleotides 791286 to 794093, was named wreM. Predicted positions of conserved domains are indicated by amino acid positions. Abbreviations: GT, conserved glycosyltransferase domain; MT, conserved methyltransferase domain. Gray boxes indicate the predicted transmembrane domains.The gene responsible for the other conditionally variable O-antigen methylation, the 2-O methylation of internal fucose residues (2OMeFuc), had not been identified in prior published work. However, among mutants isolated by random Tn5 mutagenesis, a few had been shown to lack 2OMeFuc entirely (44). We show here that the transposon insertions were located in the bifunctional gene wreM. Furthermore, results of directed insertion mutagenesis confirm two separate enzymatic domains encoded by this gene, with the α domain being required for the 2-O methylation activity and mutation of the other domain resulting in a truncated O antigen. Mutants from the directed mutagenesis that appeared to have no LPS defects other than the lack of 2OMeFuc served as tools to assess the importance of just this structural feature in the symbiosis with P. vulgaris.     

Proteome analysis of aerobic and fermentative metabolism in Rhizobium etli CE3

Rhizobium etli undergoes a transition from an aerobic to a fermentative metabolism during successive subcultures in minimal medium. This metabolic transition does not occur in cells subcultured in rich medium, or in minimal medium containing either biotin or thiamine. In this report, we characterize the aerobic and fermentative metabolism of R. etli using proteome analysis. According to their synthesis patterns in response to aerobic (rich medium, minimal medium with biotin or minimal medium with thiamine) or fermentative (minimal medium without supplements) growth conditions, proteins were assigned to five different classes: (i) proteins produced only in aerobic conditions (e.g., catalase-peroxidase KatG and the E2 component of pyruvate dehydrogenase); (ii) protein produced under both conditions but strongly induced in aerobic metabolism (e.g., malate dehydrogenase and the succinyl-CoA synthetase beta subunit); (iii) proteins that were induced equally under all conditions tested (e.g., AniA, DnaK, and GroEL); (iv) proteins downregulated during aerobic metabolism, and (v) proteins specific to only one of the conditions analyzed. Northern blotting studies of katG expression confirmed the proteome data for this protein. The negative regulation of carbon metabolism proteins observed in fermentative metabolism is consistent with the drastic physiological changes which occur during this process.     

Quantitative Study of Nodulation Competitiveness in Rhizobium Strains

We compared the nodulation competitiveness of three strains of Rhizobium leguminosarum by counting the number of nodules formed on faba bean plants after the application at sowing time of different concentrations of the strains to soils already containing Rhizobium strains of the same species. A relationship of type y = axⁿ was found to exist between the ratio of the nodules formed by the applied inoculum strain to the nodules formed by the soil strains and the ratio of Rhizobium cells in the inoculum to the cells in the soil. This relationship was also confirmed in another competition experiment in which two R. meliloti strains of identical competitiveness were mixed in various proportions. The relationship can also be applied to the majority of results reported in the literature. Should it prove to be more widely applicable, it could be used to estimate the relative competitiveness of Rhizobium strains and thus predict the performance of an inoculum in a given soil.     

The extracellular proteome of Rhizobium etli CE3 in exponential and stationary growth phase

Background

The extracellular proteome or secretome of symbiotic bacteria like Rhizobium etli is presumed to be a key element of their infection strategy and survival. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. To find out the possible role of secreted proteins we analyzed the extracellular proteome of R. etli CE3 in the exponential and stationary growth phases in minimal medium, supplemented with succinate-ammonium.

Results

The extracellular proteins were obtained by phenol extraction and identified by LC-ESI MS/MS. We identified 192 and 191 proteins for the exponential and stationary phases respectively. Using the software Signal P, we predicted signal peptides for 12.95% and 35.60% of the proteins identified in the exponential and stationary phases, respectively, which could therefore be secreted by the Sec pathway. For the exponential growth phase, we found in abundance proteins like the ribosomal proteins, toxins and proteins belonging to the group "defence mechanisms". For the stationary growth phase, we found that the most abundant proteins were those with unknown function, and in many of these we identified characteristic domains of proteases and peptidases.

Conclusions

Our study provided the first dataset of the secretome of R. etli and its modifications, which may lead to novel insights into the adaptive response of different stages of growth. In addition, we found a high number of proteins with unknown function; these proteins could be analyzed in future research to elucidate their role in the extracellular proteome of R. etli.     

Rhizobium etli CE3 bacteroid lipopolysaccharides are structurally similar but not identical to those produced by cultured CE3 bacteria

Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin.     

Thiamine limitation determines the transition from aerobic to fermentative-like metabolism in Rhizobium etli CE3

Both thiamine and biotin when added to minimal medium subcultures reversed the fermentative-like metabolism exhibited by Rhizobium etli CE3. Thiamine auxotrophs lacking thiCOGE genes were used to investigate the role of thiamine in this medium. A thiC1169∷ miniTn 5lacZ1 thiamine auxotroph subjected to the above subcultures resulted in growth arrest, reduced pyruvate-dehydrogenase activity, and a smaller amount of poly-β-hydroxybutyrate compared with the CE3 strain. Moreover, thiC and thiEb genes were overexpressed as result of thiamine limitation. The absence of classical thi genes suggests that thiamine is synthesized with low efficiency by an alternative pathway. Low levels of thiamine cause the CE3 strain to exhibit a fermentative-like metabolism.     

Increased Bean (Phaseolus vulgaris L.) Nodulation Competitiveness of Genetically Modified Rhizobium Strains

Rhizobium leguminosarum bv. phaseoli strain collections harbor heterogeneous groups of bacteria in which two main types of strains may be distinguished, differing both in the symbiotic plasmid and in the chromosome. We have analyzed under laboratory conditions the competitive abilities of the different types of Rhizobium strains capable of nodulating Phaseolus vulgaris L. bean. R. leguminosarum bv. phaseoli type I strains (characterized by nif gene reiterations and a narrow host range) are more competitive than type II strains (that have a broad host range), and both types are more competitive than the promiscuous rhizobia isolated from other tropical legumes able to nodulate beans. Type I strains become even more competitive by the transfer of a non-Sym, 225-kilobase plasmid from type II strain CFN299. This plasmid has been previously shown to enhance the nodulation and nitrogen fixation capabilities of Agrobacterium tumefaciens transconjugants carrying the Sym plasmid of strain CFN299. Other type I R. leguminosarum bv. phaseoli transconjugants carrying two symbiotic plasmids (type I and type II) have been constructed. These strains have a diminished competitive ability. The increase of competitiveness obtained in some transconjugants seems to be a transient property.     

Genetic Determinants of Swarming in Rhizobium etli

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.     

Multiresistance genes of Rhizobium etli CFN42

Multidrug efflux pumps of bacteria are involved in the resistance to various antibiotics and toxic compounds. In Rhizobium etli, a mutualistic symbiont of Phaseolus vulgaris (bean), genes resembling multidrug efflux pump genes were identified and designated rmrA and rmrB. rmrA was obtained after the screening of transposon-generated fusions that are inducible by bean-root released flavonoids. The predicted gene products of rmrAB shared significant homology to membrane fusion and major facilitator proteins, respectively. Mutants of rmrA formed on average 40% less nodules in bean, while mutants of rmrA and rmrB had enhanced sensitivity to phytoalexins, flavonoids, and salicylic acid, compared with the wild-type strain. Multidrug resistance genes emrAB from Escherichia coli complemented an rmrA mutant from R. etli for resistance to high concentrations of naringenin.     

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.     

Rhizobitoxine Production by Bradyrhizobium elkanii Enhances Nodulation and Competitiveness on Macroptilium atropurpureum

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.