Characterization of extrachromosomal replicons present in the extended host range Rhizobium sp. LPU83.

In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, plasmid DNA can constitute a high percentage of the genome. We have characterized acid-tolerant isolates of rhizobia-here represented by the strain Rhizobium sp. LPU83-that have an extended nodulation-host range including alfalfa, the common bean, and Leucena leucocephala. In this study we analyzed the plasmids of R. sp. LPU83 in order to characterize their role in the evolution of Medicago symbionts and their involvement in symbiotic behavior. The pLPU83a plasmid was found to be transmissible with no associated phenotypic traits. The symbiotic plasmid pLPU83b could be transferred at very low frequencies under laboratory conditions only when pLPU83a was present; could restore nodulation to a strain cured of its symbiotic plasmid, S. meliloti A818; but could not restore the full nitrogen fixation associated with alfalfa.

Cultural conditions required for the induction of an adaptive acid-tolerance response (ATR) in Sinorhizobium meliloti and the question as to whether or not the ATR helps rhizobia improve their symbiosis with alfalfa at low pH.

Sinorhizobium meliloti associates with Medicago and Melilotus species to develop nitrogen-fixing symbioses. The agricultural relevance of these associations, the worldwide distribution of acid soils, and the remarkable acid sensitivity of the microsymbiont have all stimulated research on the responses of the symbionts to acid environments. We show here that an adaptive acid-tolerance response (ATR) can be induced in S. meliloti, as shown previously for Sinorhizobium medicae, when the bacteria are grown in batch cultures at the slightly acid pH of 6.1. In marked contrast, no increased tolerance to hydrogen ions is obtained if rhizobia are grown in a chemostat under continuous cultivation at the same pH. The adaptive ATR appears as a complex process triggered by an increased hydrogen-ion concentration, but operative only if other--as yet unknown--concomitant factors that depend on the culture conditions are present (although not provided under continuous cultivation). Although the stability of the ATR and its influence on acid tolerance has been characterized in rhizobia, no data have been available on the effect of the adapted state on symbiosis. Coinoculation experiments showed that acid-adapted indicator rhizobia (ATR+) were present in >90% of the nodules when nodulation was performed at pH 5.6, representing a >30% increase in occupancy compared with a control test. We show that the ATR represents a clear advantage in competing for nodulation at low pH. It is not yet clear whether such an effect results from an improved performance in the acid environment during preinfection, an enhanced ability to initiate infections, or both conditions. The practical use of ATR+ rhizobia will depend on validation experiments with soil microcosms and on field testing, as well as on the possibility of preserving the physiology of ATR+ bacteria in inoculant formulations.