Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti.

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.

Crecimiento y formación de polisacáridos de cepas de Sinorhizobium meliloti en un fermentador tipo air-lift: influencia en la velocidad de nodulación en plantas de alfalfa / Crowth of Sinorhizobium meliloti strains and exopolysaccharide formation in an air-lift fermentor: influence on the nodulation rate in alfalfa plants

En este trabajo, se estudió la influencia de los polisacáridos formados por cepas de Sinorhizobium meliloti sobre la velocidad de nodulación en plantas de alfalfa. Las experiencias fueron realizadas en agitador rotatorio y en un fermentador con circulación de líquido por inyección de aire, empleando diferentes cepas de S. meliloti, El crecimiento bacteriano fue determinado por recuento de células viables y la concentración del exopolisacárido por precipitación con etanol. Se alcanzaron concentraciones máximas del orden de 1.10 (10) células viables/ml y de 11 g/l de polisacárido. Los estudios de crecimiento de plantas de alfalfa en cámara climatizada mostraron que se producía una reducción en el tiempo de aparición de nódulos de 10 a 4 días, cuando las mismas eran inoculadas con suspensiones diluidas 1/10, manteniendo las cepas sus propiedades simbióticas.

Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti.

Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants. One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion. However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective. The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs. These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis. Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti.

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.

Ultrastructural studies on nodules induced by pyrimidine auxotrophs of Sinorhizobium meliloti.

Twenty three pyrimidine auxotrophs of Sinorhizobium meliloti Rmd201 were generated by random mutagenesis with transposon Tn5. On the basis of biochemical characters these auxotrophic mutants were classified into car, pyrC and pyrE/pyrF categories. All auxotrophs induced white nodules which were ineffective in nitrogen fixation. Light and electron microscopic studies revealed that the nodules induced by pyrC mutants were more developed than the nodules of car mutants. Similarly the nodules induced by pyrE/pyrF mutants had more advanced structural features than the nodules of pyrC mutants. The nodule development in case of pyrE/pyrF mutants was not to the extent observed in the parental strain. These results indicated that some of the intermediates and/or enzymes of pyrimidine biosynthetic pathway of S. meliloti play a key role in bacteroidal transformation and nodule development.

Contaminación bacteriana y presencia de Sinorhizobium meliloti en aguas de los canales de riego del río Neuquén / Bacterial contamination and presence of Sinorhizobium meliloti in irrigation canal water from the NeuquÚn River

A survey of the changes in populations of heterotrophic bacteria, coliform microorganisms and S. meliloti was conducted in samples taken from the water irrigation channels of the NeuquÚn River (Argentina). Fifty-six water samples were collected during the spring-summer seasons of 1997-1999 years. Both the heterotrophic plate count bacterial and the number of coliforms oscillated between 110-5050 CFU/ml and 8-1400 CFU/100 ml, respectively, during the period this study was carried out. Fecal coliforms were detected in 91.1 of the water samples investigated. Moreover, the results showed that S. meliloti capable of nodulating alfalfa (Medicago sativa L.) Cuf 101 were present in 68 of the water samples and in effectiveness studies, no isolate out of 25 evaluated could be classified as superior N fixers. That is, they did not produce plants equal in weight to nitrate-grown plants (KNO3 0.05). All the S. meliloti strains were resistant to novobiocin and bacitracin, while 72 of the microsymbionts demonstrated resistance to between seven and ten antibiotics. Results presented in this study showed that irrigation waters of the NeuquÚn river could act as dispersal agents of both ineffective S. meliloti strains and thermotolerant coliform bacteria.

Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti.

Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied. Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant. The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation. Histology of the nodules induced by trpE and aro mutants exhibited striking similarities. Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone. Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency. A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation. The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted. These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S. meliloti. The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.