Mutants Of Rhizobium Leguminosarum Bv Research Articles

Malonate catabolism does not drive N2 fixation in legume nodules.

Malonyl-coenzyme A (CoA) decarboxylase, malonyl-CoA synthetase, and malonate transporter mutants of Rhizobium leguminosarum bv. viciae and trifolii fixed N2 at wild-type rates on pea and clover, respectively. Thus, malonate does not drive N2 fixation in legume nodules.

An acpXL mutant of Rhizobium leguminosarum bv. phaseoli lacks 27-hydroxyoctacosanoic acid in its lipid A and is developmentally delayed during symbiotic infection of the determinate nodulating host plant Phaseolus vulgaris.

Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus, Brucella melitensis, Bartonella henselae, and Legionella pneumophila, which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phaseoli strain 8002, which forms symbiosis with Phaseolus vulgaris, a determinate nodulating legume. Structural analysis using gas chromatography and mass spectrometry revealed that the mutant lipid A lacked the VLCFA. Compared to the parent strain, the mutant was more sensitive to the detergents deoxycholate and dodecyl sulfate and the antimicrobial peptide polymyxin B, suggesting a compromise to membrane stability. In addition, the mutant was more sensitive to higher salt concentrations. Passage through the plant restored salt tolerance. Electron microscopic examination showed that the mutant was developmentally delayed during symbiotic infection of the host plant Phaseolus vulgaris and produced abnormal symbiosome structures.

Characterization of a {gamma}-aminobutyric acid transport system of Rhizobium leguminosarum bv. viciae 3841.

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Rhizobium population dynamics in the pea rhizosphere of rhizobial inoculant strain applied in different formulations.

The effect of inoculant formulation on the population dynamics of rhizobia in the pea rhizosphere was investigated using a streptomycin-resistant mutant of Rhizobium leguminosarum bv. viceae NITRAGIN128C56G (128C56G strR). The isolate was formulated into liquid, peat powder, and granular peat carriers, and was tested on pea at field sites near Saskatoon, Saskatchewan, and Beaverlodge, Alberta, in 1996 and 1997. The liquid and peat powder formulations were applied to seed while the granular inoculant was applied to soil. In three out of four site years, population dynamics were similar among formulations: an initial decline or lag period lasting 2-5 days followed by an increase to approximately 10(5) colony-forming units (CFU)/seedling by 14-28 days after planting (DAP) and, where sampled, a continuing increase from 10(7) to 10(8) CFU/plant at 63 DAP. In these same site years, nodule number (not determined at Beaverlodge in 1997) and nodule occupancy at 60 days were not significantly different among formulations. In contrast, soil populations of 128C56G strR from the liquid formulation declined to near zero by 28 DAP at Beaverlodge in 1996, when soil moisture was excessive in spring because of high rainfall. Populations increased in this treatment after this time, but remained significantly lower than the populations of the other two formulations throughout the sampling period. Pea seed yields were not significantly different among treatments in either year at Beaverlodge, but were significantly higher with granular inoculant than the noninoculated control in Saskatoon. Within inoculated treatments at Saskatoon, there were no significant differences in grain yield.

Occurrence of lipopolysaccharide alterations among Tn5 mutants of Rhizobium leguminosarum bv. trifolii strain 24.1 with altered colony morphology.

Transposon mutants of Rhizobium leguminosarum bv. trifolii 24.1 showing less glossy or smaller colonies were screened for properties usually associated with lipopolysaccharide (LPS) defects in R. leguminosarum, i.e. motility, growth rate, tendency to agglutination in liquid media and symbiotic efficiency. Neither any of the above mutants nor the earlier isolated 24.12 strain, defective in LPS, showed all these properties changed simultaneously. According to PAGE/sodium deoxycholate analysis the mutant 24.12 was the only one producing defective lipopolysaccharide. GC-MS analysis revealed in this mutant qualitative changes in composition of its LPS in comparison with LPS isolated from the parent strain. Other Tn5 mutants produced LPSs similar in composition, however the proportion between LPS I and LPS II differed from that in the parent strain.

Role of Exopolysaccharides of Rhizobium leguminosarum bv. viciae as Host Plant-Specific Molecules Required for Infection Thread Formation During Nodulation of Vicia sativa

Mutants of Rhizobium leguminosarum bv. viciae bacteria that are affected in the biosynthesis of exopolysaccharides (EPS) are unable to effectively nodulate their host plants. By studying defined mutants, we show that R. legumi-nosarum bv. viciae strains require EPS for formation of infection threads in Vicia sativa (vetch) as well as for efficient induction of tight root hair curling. Results of coinoculation experiments with the EPS-deficient pssD111 mutant of R. leguminosarum bv. viciae in combination with heterologous EPS-producing strains indicated that vetch has certain structural requirements for rhizobial EPS to function in symbiosis. We hypothesize that EPS accelerates root hair curling and infection to such an extent that rhizobial root penetration precedes a plant defense response.

Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR.

A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by Tn5 in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71.3% identity over 892 bp). At the protein level there was 70% identity and 93.3% similarity over 267 amino acids with the ExoR protein of R. meliloti Rm1021. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM710. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar.

Defective Infection and Nodulation of Clovers by Exopolysaccharide Mutants of Rhizobium leguminosarum bv. trifolii

The biological and symbiotic properties of two types of exopolysaccharide deficient (Exo- phenotype) mutants of Rhizobium leguminosarum were investigated. One mutant, a pss1 derivative (ANU437) of R. l. bv. trifolii, produces very low levels of acidic exopolysaccharide (EPS) and of the acidic oligosaccharide (AOS) repeating unit lacking its O-acetyl substituent. The second strain, originally a R. I. bv. viciae derivative, but containing a clover pSym plasmid (Exo-1 mutant ANU54(pBRIAN)), did not produce any EPS polymer or detectable oligosaccharide repeating unit. The Exo- mutants formed no capsules and their growth in laboratory media was comparable to their parent strains but was inhibited by the addition of the phytoalexins medicarpin and kievitone, but not by the phytoalexin pisatin. Both Exo- mutants were slow to induce clover root hair curling (Hac+), and formed growing infection threads (Inf-) only on white clovers. However, the extent of these early symbiotic defects depended on the plant growth conditions used. Both mutants induced small non-nitrogen-fixing (Fix-) nodules on white and subterranean clovers. Within the white clover nodules induced by mutant ANU437, bacteria were released into plant cells. On white clovers at elevated light and temperature conditions, the Exo-1 mutant ANU54(pBRIAN) usually failed to initiate infection threads and thus formed empty nodules which do not contain any bacteria. In addition, this mutant formed only empty nodules without infection threads on subterranean clovers under all growth conditions tested. The outermost layers of plant cells in these empty nodules had thickened cell walls and deposited materials. These experiments show that EPS synthesis is essential for the induction of rapid root hair curling, proper infection thread formation and the ability to sustain the growth of induced nodules on various clovers.

Nodulation response of exopolysaccharide deficient mutants of Rhizobium leguminosarum bv. trifolii to addition of acidic exopolysaccharide

Several transposon Tn5-induced mutants of Rhizobium leguminosarum bv. trifolii 24.1 synthesized no detectable acidic exopolysaccharide (EPS) and were unable to induce nitrogen-fixing nodules on clover plants. Clover (Trifolium protense) co-inoculated with R. leguminosarum bv. trifolii 2407 Nod - mutant and the mutants deficient in exopolysaccharide production (Exo -) formed mixed infected nodules that did not fix nitrogen. The ability some of these Exo - mutants to induce nitrogen-fixing nodules on clover was restored in the presence of added homologous EPS from R. leguminosarum bv. trifolii 24.1 wild-type strain.

Disturbed gene expression and bacteroid development in Trifolium pratense root nodules induced by a Tn5 mutant of Rhizobium leguminosarum bv. trifolii defective in exopolysaccharide synthesis

A newly isolated mutant of the symbiotic bacterium Rhizobium leguminosarum bv. trifolii, Rt79, is deficient in exopolysaccharide synthesis. It induced nodules defective in nitrogen fixation on clover (Trifolium pratense L.). The expression of plant (ENOD2, leghemoglobin) and bacterial (ropA, nifH) genes was studied by in-situ hybridization. The absence of nifH transcripts was noticed. Mutant-induced nodules showed changes in nodule development, such as precocious degeneration of bacteroids and deviating bacteroid ultrastructure, from the moment that bacteria were released from infection threads, and reduction of the number of infected plant cells. Abnormal bacteroids still invaded plant cells but in the interzone between the pre-fixation zone and the nitrogen fixing zone the maturation process required for nitrogen fixation was eventually blocked. These observations as well as the gene-expression data point to a failure in bacteroid development from the onset of the infection process rather than to a sudden block in the infection process.

Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae MNF300 and R. leguminosarum bv. trifolii WU95 unable to accumulate 4-hydroxybenzoate lack 4-hydroxybenzoate hydroxylase. The capacity of these mutants to take up and grow on 4-hydroxybenzoate was restored by a 2.0 kb EcoRI-PstI DNA fragment. This contained only one ORF which had over 60% DNA sequence similarity with the structural gene for 4-hydroxybenzoate hydroxylase (pobA) from Pseudomonas spp. and Acinetobacter. Reported effects of metabolic inhibitors and substrate analogues on the apparent uptake of 4-hydroxybenzoate have now been shown to be due to their direct effect on 4-hydroxybenzoate hydroxylase. We propose that uptake of 4-hydroxybenzoate is via a metabolic 'drag' mechanism dependent on the activity of the pobA gene product.

The Rhizobium leguminosarum biovar viciae nodO gene can enable a nodE mutant of Rhizobium leguminosarum biovar trifolii to nodulate vetch

Analysis of the nodulation characteristics of transposon-induced mutants of Rhizobium leguminosarum bv. viciae revealed that nodO and the closely-linked rhi genes contribute to nodulation of peas (Pisum sativum) and the vetch Vicia hirsuta. Although mutation of nodO alone had no significant effect on nodulation of either legume, a double mutant lacking both nodO and nodE nodulated both legumes very poorly. Similarly, a double mutant lacking nodE and either rhiA or rhiB nodulated peas less efficiently than a nodE mutant. Thus, although mutations affecting only the rhi genes normally have no observed effect on nodulation, these genes do appear to contribute to pea nodulation. When transferred to a wild-type strain of Rhizobium leguminosarum bv. trifolii, neither nodO nor the rhi gene region conferred pea or vetch nodulating ability. However, in a nodE mutant of R. I. bv. trifolii, nodO did confer a significant level of vetch nodulating ability, indicating that the secreted NodO protein can play a role in determining legume recognition by R. I. bv. viciae.

Specific detection of rhizobia in root nodules and soil using the polymerase chain reaction

The polymerase chain reaction (PCR) amplification of specific DNA sequences, allows specific and sensitive detection of bacteria at the genus, species or strain level depending on the design of the oligonucleotide primers. In this study we utilized 20 mer primers that flanked a 300 bp region of the npt II gene of the transposon Tn5 thus allowing for the amplification of this region. Insertion of the Tn 5 element into rhizobia allowed for detection of these cells using PCR amplification. Using the npt II-specific primers and Tn5 insertion mutants of Rhizobium leguminosarum bv. phaseoli we were able to detect these specific rhizobia strains in root nodules of bean plants and in inoculated soils. Utilization of genus-specific gene sequences would allow for estimates of cells of that genus in environmental samples. Conversely, use of gene sequences common to rhizobia, e.g. nif and nod sequences, would give estimates of the population of rhizobia. This paper serves to illustrate the use of PCR, for detecting gene sequences in an environmental sample such as a root nodule.

Identification of a nodD-like gene in Frankia by direct complementation of a Rhizobium nodD-mutant.

Clones from a Frankia At4 gene bank were pooled into groups and mass conjugated into a nodD mutant of Rhizobium leguminosarum bv. viciae by triparental matings. When peas were inoculated with the pooled transconjugants, nodulation was observed. A plasmid, pAt2GX containing Frankia DNA, was isolated from bacteria recovered from these nodules. This plasmid was shown to complement a nodD mutant of R. leguminosarum bv. viciae. Thus pAt2GX contains a Frankia gene that is functionally equivalent to nodD of R. leguminosarum bv. viciae.

In‐situ localization of chalcone synthase mRNA in pea root nodule development

SummaryIn this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it is hypothesized that the Rhizobium Nod factor induces cell division in the root cortex by stimulating the production of flavonoids that function as auxin transport inhibitors. In nodules CHS mRNA is predominantly present in a region at the apex of the nodule consisting of meristematic and cortical cells. These cells are not infected by Rhizobium. Therefore it is postulated that CHS plays a role in nodule development rather than in a defence response. In roots CHS mRNA is located at a similar position as in nodules, suggesting that CHS has the same function in both root and nodule development.When nodules are formed by mutants of Rhizobium leguminosarum bv. viciae that are unable to secrete β(1‐2) glucan and to synthesize the O‐antigen containing LPS I, CHS genes are also expressed in regions of the nodule that are infected by Rhizobium. It is postulated that the impaired development of nodules formed by these mutants is due to an induction of a plant defence response.

Autoregulatory response of Phaseolus vulgaris L. to symbiotic mutants of Rhizobium leguminosarum bv. phaseoli

In Rhizobium-legume symbiosis, the plant host controls and optimizes the nodulation process by autoregulation. Tn5 mutants of Rhizobium leguminosarum bv. phaseoli TAL 182 which are impaired at various stages of symbiotic development, were used to examine autoregulation in the common bean (Phaseolus vulgaris L.). Class I mutants were nonnodulating, class II mutants induced small, distinct swellings on the roots, and a class III mutant formed pink, bacterium-containing, but ineffective nodules. A purine mutant (Ade-) was nonnodulating, while a pyrimidine mutant (Ura-) formed small swellings on the roots. Amino acid mutants (Leu-, Phe-, and Cys-) formed mostly empty white nodules. Each of the mutants was used as a primary inoculant on one side of a split-root system to assess its ability to suppress secondary nodulation by the wild type on the other side. All mutants with defects in nodulation ability, regardless of the particular stage of blockage, failed to induce a suppression response from the host. Only the nodulation-competent, bacterium-containing, but ineffective class III mutant induced a suppression response similar to that induced by the wild type. Suppression was correlated with the ability of the microsymbiont to proliferate inside the nodules but not with the ability to initiate nodule formation or the ability to fix nitrogen. Thus, the presence of bacteria inside the nodules may be required for the induction of nodulation suppression in the common bean.

Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C4-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.

The occurrence of unusual laminated structures rich in ?-1,4-glucans in plastids of Phaseolus vulgaris root-nodule cells infected by an ineffective C4-dicarboxylic-acid mutant of Rhizobium leguminosarum bv. phaseoli

Root nodules induced in Phaseolus vulgaris L. by the wild-type (WT) and a C4-dicarboxylic-acid mutant strain of Rhizobium leguminosarum biovar. phaseoli were compared on the basis of ultrastructure and cytochemistry of cellulose subunits. The mutant bacteroids failed to colonize infected host cells in a normal manner, and presented a premature degenerative appearance. Starch granules, rough endoplasmic reticulum and mitochondria were found to accumulate in the ineffective nodules. The most striking difference between effective and ineffective nodules was the presence of unusual spherical, laminated structures in plastids of mutant-infected host cells only. Cytochemical observations showed that these structures contain β-1,4-glucans. The presence of β-1,4-glucans within such structures may be caused by the activity of a cellulase which is produced by either the bacteroids or the host cell and is locally hydrolyzing the host cell-wall, thus releasing cellulose subunits into the cytoplasm. Another possibility is denovo synthesis of β-1,4-glucans in the host cell.

Isolation and characterization of mutants of Rhizobium leguminosarum bv. viciae 248 with altered lipopolysaccharides: possible role of surface charge or hydrophobicity in bacterial release from the infection thread

Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv. viciae on effective symbiosis and on a number of cell surface characteristics were studied. Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248. Ten class I LPS mutants completely lacked the O-antigen-containing LPS species. The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS. The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS. Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen. Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain. The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility. A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.