Conservation of a pseudomonad-like hydrocarbon degradative ferredoxin oxygenase complex involved in rhizopine catabolism in Sinorhizobium meliloti and Rhizobium leguminosarum bv. viciae

In Sinorhizobium meliloti the mocCABR genes have previously been shown to be required for rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolism. We show that the mocDE(F) gene cluster is also needed. MocDE(F), which is involved in the catabolism of 3-O-MSI to its demethylated form scyllo-inosamine (SI) has homology to components that would comprise a ferredoxin-oxygenase system. The mocCABRDE(F) suite of genes is required for 3-O-MSI catabolism in both S. meliloti and R. leguminosarum bv. viciae. However, SI catabolism in S. meliloti requires mocCABR, whereas only mocCA are required for its catabolism in R. leguminosarum suggesting the two species require different chromosomal genes which act in concert with moc genes for the catabolism of rhizopine.     

Chemical synthesis of scyllo-inosamine and catabolism studies in Sinorhizobium meliloti

Rhizopines such as scyllo-inosamine (SIA) and L-3-O-methyl-scyllo-inosamine (3-O-MSI) play an intricate role as nutritional mediators during the establishment of the symbiotic relationship between legumes and rhizobia. The mechanism of action is not well understood. One challenge is the availability of rhizopines, which occur in only minute amounts in plant nodules. We herewith report an efficient synthesis of scyllo-inosamine and its biochemical activity in specific bacteria. SIA was prepared in 7 steps and 32% overall yield from readily available myo-inositol. The chemically synthesized SIA was tested to determine whether it can serve as sole carbon and nitrogen source for Sinorhizobium meliloti wild-type strain L5-30 and for strains carrying mutations in the rhizopine degradation (moc) genes. The analysis of the phenotype of the mutant strains revealed that the moc genes previously shown to be essential for the breakdown of the rhizopines isolated from root nodules are also essential for the utilization of the chemically synthesized SIA.     

Early production of rhizopine in nodules induced by Sinorhizobium meliloti strain L5-30

The rhizopine L-3-O-methyl-scyllo-inosamine (3-O-MSI) is metabolized by approximately 10% of the strains of Rhizobium leguminosarum by. viciae and Sinorhizobium meliloti. Rhizopine strains enjoy a substantial competitive advantage in nodulation, which is manifest before 14 days post-inoculation, implying that rhizopine is produced before this time. We were able to detect this compound in the roots of alfalfa (Medicago sativum L. cv. Hunter River) four days after germination (six days post-infection) with S. meliloti strain L5-30 by gas chromatography-mass spectrometry (GC-MS). At four days, nodules were not visible, and the concentration of rhizopine was extremely low, estimated at 67 pg/gfw (picograms/gram fresh weight). The amount increased gradually but remained low until 16 days, when there was a 50-fold increase from day four, by which time nodules were well established. This pattern of synthesis is consistent with previous studies indicating that rhizopine synthesis is regulated by nifA/ntrA regulatory genes, which are maximally expressed in bacteroids at the onset of nitrogen fixation. However, the low level of rhizopine synthesis must be responsible for the early effects on competition for nodulation. Production of rhizopine at this time most likely results from micro-aerobic induction of mos genes in free-living bacteria, either in the infection threads or in the rhizosphere.     

The Rhizobium meliloti rhizopine mos locus is a mosaic structure facilitating its symbiotic regulation.

The Rhizobium meliloti L5-30 mos locus, encoding biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine, is shown to be a mosaic structure. The mos locus consists of four open reading frames (ORFs) (ORF1 and mosABC) arranged in an operon structure. Within this locus, several domains of homology with other prokaryotic symbiotic genes (nifH, fixA, fixU, and nifT) are present, suggesting that this locus may represent a hot spot for rearrangement of symbiotic genes. Unusually, these domains are present in the coding as well as noncoding regions of the mos locus. Proteins corresponding to those encoded by mosABC, but not ORF1, have been detected in nodule extracts by using antibodies. As ORF1 shows extensive homology with the 5' region of the nifH gene (P.J. Murphy, N. Heycke, S.P. Trenz, P. Ratet, F.J. de Bruijn, and J. Schell, Proc. Natl. Acad. Sci. USA 85:9133-9137, 1988) and a frameshift mutation indicates that expression of this ORF is not required for mos activity, we propose that the mos locus has acquired a duplicated copy of nifH, including the promoter region, in order to become symbiotically regulated. Surprisingly, since the functions are likely different, MosA has an amino acid sequence similar to that of the DapA protein of Escherichia coli. The central domain of MosB has extensive homology with a range of diverse proteins involved with carbohydrate metabolism in either antibiotic or outer-cell-wall biosynthesis. This region is also common to the regulatory proteins DegT and DnrJ, suggesting a regulatory role for MosB. The structure of MosC is consistent with its being a membrane transport protein.     

MosA, a protein implicated in rhizopine biosynthesis in Sinorhizobium meliloti L5-30, is a dihydrodipicolinate synthase

MosA is a gene product encoded on a pSym megaplasmid of Sinorhizobium meliloti L5-30. The gene is part of an operon reported to be essential for the synthesis of the rhizopine 3-O-methyl-scyllo-inosamine. MosA has been assigned the function of an O-methyltransferase. However, the reported sequence of this protein is very much like that of dihydrodipicolinate synthase (DHDPS), except for a 40 amino acid residue C-terminal domain. This similarity contradicts accepted ideas regarding structure-function relationships of enzymes. We have cloned and overexpressed the recombinant gene in Escherichia coli, and discovered that the reported sequence contains an error resulting in a frame-shift. The correct sequence contains a new stop codon, truncating the C-terminal 41 amino acid residues of the reported sequence. The expressed protein, bearing an N-terminal polyhistidine tag, catalyzes the condensation of pyruvate and aspartate beta-semialdehyde efficiently, suggesting that this activity is not a side-reaction, but an activity for which this enzyme has evolved. Electro-spray mass spectrometry experiments and inhibition by L-lysine are consistent with the enzyme being a DHDPS. E.coli AT997, a mutant host normally requiring exogenous diaminopimelate for growth, could be complemented by transformation with a plasmid bearing the gene encoding MosA. A role for this enzyme in rhizopine synthesis cannot be ruled out, but is called into question.     

Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30

Rhizopine (l-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5–30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5–30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.     

Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30

Rhizopine (l-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5–30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5–30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.     

An Experimental Test of the Rhizopine Concept in Rhizobium meliloti

In some Rhizobium-legume symbioses, compounds known as rhizopines are synthesized by bacteroids and subsequently catabolized by free-living cells of the producing strain. It has been suggested than rhizopines act as proprietary growth substrates and enhance the competitive ability of the producing strain in its interactions with the diverse microbial community found within the rhizosphere. Wild-type, rhizopine-producing Rhizobium meliloti L5-30 and mutant L5-30 strains deficient for either rhizopine synthesis or catabolism were inoculated onto lucerne host plants in competition experiments. These experiments demonstrated that no apparent advantage resulted from the ability to synthesize a rhizopine, whereas the ability to catabolize rhizopine provided a clear advantage when an organism was in competition with a strain without this ability. The results suggest that when an organism is in competition with a catabolism-deficient mutant, the ability to catabolize rhizopine results in enhanced rates of nodulation. The results of the experiments were not consistent with the hypothesis that the sole role of rhizopines is to act as proprietary growth substrates for the free-living population of the producing strain.     

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti--Medicago laciniata symbiosis

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti. e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa - S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.     

The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide biosynthesis.

exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix- nodules on alfalfa. However, mutants of R. meliloti Rm41 carrying the same exo lesions induce normal Fix+ nodules. We show that such induction is due to a gene from strain Rm41, which we call lpsZ+, that is missing in strain SU47. lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide. In both SU47 and Rm41, either lpsZ+ or exo+ is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.     

The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure.

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.     

Cloning and characterization of a novel beta-galactosidase-coding gene from Rhizobium meliloti

The Rhizobium meliloti (Rm) lacZ gene provides a convenient model to investigate patterns of gene regulation in these agronomically important bacteria. A gene encoding beta-galactosidase (beta Gal) activity was cloned from R. meliloti by complementing a lactose-negative Escherichia coli mutant. A series of Sau3A subclones was generated in pBR322, and the coding region for the beta Gal-coding gene was localized to a 2.4-kb core fragment. In E. coli 'maxicells', these lacZ subclones produced a 79-kDa polypeptide, irrespective of the fragment size demonstrating that the translation initiation signal(s) are located on the 2.4-kb fragment. Transposon Tn5 mutagenesis and BAL 31 deletion analysis showed that the expression of the Rm lacZ gene in E. coli was dependent on the tetracycline-resistance promoter of pBR322. The cloned sequence was required for beta Gal synthesis in Rhizobium since mutants generated by reverse genetics lack this enzyme and were specifically defective in lactose catabolism.     

Role of trehalose transport and utilization in Sinorhizobium meliloti--alfalfa interactions

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rml021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.     

Evolutionary dynamics of rhizopine within spatially structured rhizobium populations

Symbiosis between legumes and nitrogen-fixing bacteria is thought to bring mutual benefit to each participant. However, it is not known how rhizobia benefit from nodulating legume hosts because they fix nitrogen only after becoming bacteroids, which are terminally differentiated cells that cannot reproduce. Because undifferentiated rhizobia in and around the nodule can reproduce, evolution of symbiotic nitrogen fixation may depend upon kin selection. In some hosts, these kin may persist in the nodule as viable, undifferentiated bacteria. In other hosts, no viable rhizobia survive to reproduce after nodule senescence. Bacteroids in these hosts may benefit their free-living kin by enhancing production of plant root exudates. However, unrelated non-mutualists may also benefit from increased plant exudates. Rhizopines, compounds produced by bacteroids in nodules and catabolized only by related free-living rhizobia, may provide a mechanism by which bacteroids can preferentially benefit kin. Despite this apparent advantage, rhizopine genotypes are relatively rare. We constructed a mathematical model to examine how mixing within rhizobium populations influences the evolution of rhizopine genotypes. Our model predicts that the success of rhizopine genotypes is strongly dependent upon the spatial genetic structure of the bacterial population; rhizopine is more likely to dominate well-mixed populations. Further, for a given level of mixing, we find that rhizopine evolves under a positive frequency-dependent process in which stochastic accumulation of rhizopine alleles is necessary for rhizopine establishment. This process leads to increased spatial structure in rhizobium populations, and suggests that rhizopine may expand the conditions under which nitrogen fixation can evolve via kin selection.     

Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis.

We have developed a system for using TnphoA (TnphoA is Tn5 IS50L::phoA), which generates fusions to alkaline phosphatase (C. Manoil and J. Beckwith, Proc. Natl. Acad. Sci. USA 82:8129-8133, 1985), in Rhizobium meliloti. Active fusions expressing alkaline phosphatase can arise only when this transposon inserts in genes encoding secreted or membrane-spanning proteins. By confining our screening to 1,250 TnphoA-generated mutants of R. meliloti that expressed alkaline phosphatase, we efficiently identified 25 symbiotically defective mutants, all of which formed ineffective (Fix-) nodules on alfalfa. Thirteen of the mutants were unable to synthesize an acidic exopolysaccharide (exo::TnphoA) that is required for nodule invasion. Twelve of the mutations created blocked at later stages of nodule development (fix::TnphoA) and were assigned to nine symbiotic loci. One of these appeared to be a previously undescribed locus located on the pRmeSU47a megaplasmid and to encode a membrane protein. Two others were located on the pRmeSU47b megaplasmid: one was a new locus which was induced by luteolin and encoded a membrane protein, and the other was dctA, the structural gene for dicarboxylic acid transport. The remaining six loci were located on the R. meliloti chromosome. One of these was inducible by luteolin and encoded a membrane protein which determined lipopolysaccharide structure. Three additional chromosomal loci also appeared to encode membrane proteins necessary for symbiosis. The remaining two chromosomal loci encoded periplasmic proteins required for symbiosis.     

Plasmid-associated genes in the model micro-symbiont Sinorhizobium meliloti 1021 affect the growth and development of young rice seedlings

Sinorhizobium meliloti strain 1021 and its closely related strain Rm2011 inhibit rice seedling (Oryza sativa L. cv. Pelde) growth and development under certain rice-growing conditions. Experiments showed that inoculation of seedlings with approximately less than 10 cells of 1021 was sufficient to cause this inhibition. By using a series of plasmid-cured and plasmid-deleted derivatives of Rm2011, it was found that interactions between genes encoded on pSymA, and possibly pSymB, of Rm2011, affected rice growth and development by affecting both/either the plant and/or the bacteria. Further studies found that genes potentially related to indole-3-acetic acid (IAA) synthesis and nitrate metabolism, encoded on pSymA, were involved in rice growth inhibition in Sm1021- and Sm2011-treated rice seedlings. We conclude that the rice growth inhibition by S. meliloti Sm1021 is pSymA-associated and is induced by environmental nitrate.     

Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones

Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa. Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S. meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures. This is the first report of AHLs having acyl chains longer than 14 carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs. Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis. We also show that sinI and sinR mutants are still capable of producing several short-chain AHLs, one of which was identified as octanoyl HL. We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S. meliloti."     

Evidence that the exoH gene of Sinorhizobium meliloti does not appear to influence symbiotic effectiveness with Medicago truncatula 'Jemalong A17'

The purpose of this study was to identify strains of Sinorhizobium meliloti that formed either an effective or completely ineffective symbiosis with Medicago truncatula L. 'Jemalong A17' and, subsequently, to determine whether differences existed between their exoH genes. Sinorhizobium meliloti TII7 and A5 formed an effective and ineffective symbiosis with M. truncatula 'Jemalong A17', respectively. Using a multilocus sequence typing method, both strains were shown to have chromosomes identical with S. meliloti Rm1021 and RCR2011. The 2260-bp segments of DNA stretching from the 3' end of exoI through open reading frames of hypothetical proteins SM_b20952 and SM_b20953 through exoH into the 5' end of exoK in strains TII7 and Rm1021 differed by a single nucleotide at base 127 of the hypothetical protein SM_b20953. However, the derived amino acid sequences of the exoH genes of effective TII7, ineffective A5, and strain Rm1021 were shown to be identical with each other. Therefore, it would seem unlikely that the gene product of exoH is directly involved with the low efficiency of a symbiosis of strain Rm1021 with M. truncatula 'Jemalong A17'. Complementation or complete genome sequence analyses involving strains TII7 and A5 might be useful approaches to investigate the molecular bases for the differential symbiotic response with M. truncatula 'Jemalong A17'.