Chemotaxis by Rhizobium meliloti

Summary Chemotaxis by Rhizobium meliloti strain Ve 26 has been studied and conditions required for chemotaxis have been defined, using the Adler capillary assay technique. Several sugars and amino-acids were shown to be attractants with varying effectiveness for this organism: sugars are weak attractants (except gluconate) and amino-acids are good attractants (except unpolar amino-acids).     

Studies of Rhizobium meliloti isolated from salt-affected soils

Rhizobium meliloti strains have been isolated from salt-affected, non-cultivated soils of Greece. The effectiveness of the isolates was investigated in test-tubes using nitrogen-free salts agar or soil. In the latter case a new simple and rapid technique was used during which plants of Medicago saliva were grown in large test-tubes containing salt-affected soil under controlled sterile conditions. Field trial measurements in salt-affected soils indicate a good response to inoculation.     

Metabolism of some polyols by Rhizobium meliloti

The utilization of d-mannitol, d-arabitol, and d-sorbitol by Rhizobium meliloti was studied in extracts from mannitol-grown cells. Two different polyol dehydrogenases were induced by any of these polyols: (i) a nicotinamide adenine dinucleotide (NAD)-arabitol dehydrogenase and (ii) a NAD-sorbitol dehydrogenase, whereas polyol phosphate dehydrogenases were absent. d-Arabitol dehydrogenase was observed to act on both d-arabitol and d-mannitol, but d-sorbitol dehydrogenase acted specifically on d-sorbitol. d-Arabitol was oxidized to d-xylulose, d-mannitol and d-sorbitol were oxidized to d-fructose. An adenosine triphosphate-linked hexokinase which acts on d-fructose and absence of hexose isomerase were also detected in this organism.     

Structural modifications in Rhizobium meliloti Nod factors influence their stability against hydrolysis by root chitinases

Acylated chitooligosaccharide signals (Nod factors) trigger the development of root nodules on leguminous plants and play an important role in determining host specificity in the Rhizobium-plant symbiosis. Here, the ability of plant chitinases to hydrolyze different Nod factors and the potential significance of the structural modifications of Nod factors in stabilizing them against enzymatic inactivation were investigated. Incubation of the sulfated Nod factors of Rhizobium meliloti, NodRm-IV(S) and NodRm-V(S), as well as their desulfated derivatives NodRm-IV and NodRm-V, with purified chitinases from the roots of the host plant Medicago and the nonhost plant Vicia resulted in the release of the acylated lipotrisaccharide NodRm-III from NodRm-V, NodRm-IV and NodRm-V(S), whereas NodRm-IV(S) was completely resistant to digestion by both chitinases. Kinetic analysis showed that the structural parameters determining host specificity, the length of the oligosaccharide chain, the acylation at the nonreducing end and the sulfatation at the reducing end of the lipooligosaccharide, influence the stability of the molecule against degradation by chitinases. When the Nod factors were incubated in the presence of intact roots of Medicago, as well as of Vicia, the acylated lipotrisaccharide was similarly released in vivo from all Nod factors except NodRm-IV(S). In addition, a dimer-forming activity was observed in intact roots which also cleaved NodRm-IV(S). This activity was much greater in Medicago than in Vicia and increased upon incubation. The initial overall degradation rate of the Nod factors on Medicago was inversely correlated with their biological activities on Medicago roots. These results open the possibility that the activity of Nod factors on Medicago may partly be determined by the action of chitinases.     

Large-Scale Production of Rhizobium meliloti on Whey

Whey, a by-product of the cheese industry, can sustain the growth of fast-growing rhizobia. To avoid any latency of growth, rhizobial inoculum must be prepared under inducing conditions. In unsupplemented whey, the number of cells of Rhizobium meliloti Balsac reached 5 × 10⁹ CFU/ml in 48 h of incubation. This is comparable to the yield obtained with yeast-mannitol broth, the standard medium for the growth of rhizobia. In raw whey supplemented with yeast extract (1.0 g/liter) and phosphate (0.5 g/liter), the number of cells reached 10¹⁰ CFU/ml in 48 h of incubation. This is a twofold increase compared with the population normally obtained in industrial production. Whey represents a relatively inexpensive and efficient substrate medium for the large-scale production of fast-growing rhizobia.     

Structural studies of a novel exopolysaccharide produced by a mutant of Rhizobium meliloti strain Rm1021

The structure of a novel expolysaccharide obtained from a mutant of Rhizobium meliloti strain Rm1021 was elucidated by a combination of enzymic, chemical, and spectroscopic methods. The polysaccharide is composed of a disaccharide repeating-unit, beta-D-Glcp-(1----3)-alpha-D-Galp-(1----3), having a 6-O-acetyl group attached to most D-glucose residues and a 4,6-O-(1-carboxyethylidene) group attached to every D-galactose residue.     

Metabolism of Tryptophan and Tryptophan Analogs by Rhizobium meliloti

The alfalfa symbiont Rhizobium meliloti Rm1021 produces indole-3-acetic acid in a regulated manner when supplied with exogenous tryptophan. Mutants with altered response to tryptophan analogs still produce indole-3-acetic acid, but are Fix⁻ because bacteria do not fully differentiate into the nitrogen-fixing bacteriod form. These mutations are in apparently essential genes tightly linked to a dominant streptomycin resistance locus.     

Infectivity of Rhizobium meliloti as affected by extracellular polysaccharides

O livares , J., B edmar , E.J. & M artinez -M olina , E. 1984. Infectivity of Rhizobium meliloti as affected by extracellular polysaccharides. Journal of Applied Bacteriology 56 , 389–393.
The addition of crude extracellular polysaccharide (EPS) preparations from highly infective Rhizobium meliloti increased the infectivity of a poorly infective strain. The addition of crude EPS preparations from another species did not promote nodu-lation, suggesting the specificity of such an effect. Acid treatment of the crude EPS preparations resulted in total inhibition of nodulation.     

The Oligosaccharides Produced from Sucrose by Dematium pullulans

A tetrasaccharide produced from sucrose by Dematium pullulans was obtained in a crystalline state, and its properties and physical constants were studied. By elementary analysis, molecular weight, tetrazolium test, the Raybin test, aldose value, paper chromatographic examination of partial acid hydrolyzate and enzymic hydrolyzate, periodate oxidation, and molar optical rotation, the crystalline compound was identified as β-fructofuranosyl-1-kestose trihydrate.     

Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices.

The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles. Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells. Observations of R. meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s. Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation. Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops. These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops. We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.     

Analysis of Rhizobium meliloti Sym Mutants Obtained by Heat Treatment

Deletions in the pSym megaplasmid of Rhizobium meliloti were produced at a high frequency, and their lengths varied according to incubation temperature. Morphological differentiation into large and small colonies occurred after heat treatment. Small colonies elicited pseudonodules on alfalfa roots.     

Morphogenetic Rescue of Rhizobium meliloti Nodulation Mutants by trans-Zeatin Secretion

The development of nitrogen-fixing nodules is induced on the roots of legume host plants by Rhizobium bacteria. We employed a novel strategy to probe the underlying mechanism of nodule morphogenesis in alfalfa roots using pTZS, a broad host range plasmid carrying a constitutive trans-zeatin secretion (tzs) gene from Agrobacterium tumefaciens T37. This plasmid suppressed the Nod- phenotype of Rhizobium nodulation mutants such that mutants harboring pTZS stimulated the formation of nodulelike structures. Alfalfa roots formed more or fewer of these nodules according to both the nitrogen content of the environment and the position along the root at which the pTZS+ bacteria were applied, which parallels the physiological and developmental regulation of true Rhizobium nodule formation. This plasmid also conferred on Escherichia coli cells the ability to induce root cortical cell mitoses. Both the pattern of induced cell divisions and the spatially restricted expression of an alfalfa nodule-specific marker gene (MsENOD2) in pTZS-induced nodules support the conclusion that localized cytokinin production produces a phenocopy of nodule morphogenesis.     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti.

A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.     

Studies on cytokinin production by Rhizobium

Cytokinin was released into the medium by cultures of both Rhizobium japonicum and R. leguminosarum. Calculations show that the amount of cytokinin released during the logarithmic phase of growth by R. japonicum would be sufficient to initiate the cortical cell divisions necessary to form a root nodule. The substance released by R. japonicum was identified as a zeatin-like compound on the basis of paper chromatography in four solvent systems. Two solvents clearly separated the rhizobial product from N⁶-Δ²-isopentenyladenine and its ribonucleoside. The predominant intracellular cytokinin found in both enzymatic hydrolysates of sRNA and alkaline hydrolysates of total RNA also was similar to zeatin.     

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed.     

The effect of strA mutation on symbiotic nitrogen fixation by Rhizobium meliloti.

Studies on 3H-dihydrostreptomycin accumulation and binding to ribosomes showed that ineffective strain CMts17 carries strB type mutation changing its membrane permeability to the drug. Introduction of high level streptomycin resistance of strA type into strain CMts17 was correlated with acquisition of effectiveness and membrane permeability to the drug. This suggests that changes in membrane permeability, responsible for ineffectiveness of strain CMts17, can be reversed by strA mutation.