Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp NGR234

In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properl y addressed. At present, there is no 'model system' to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhiz obium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculate d with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau aft er 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of pl ant development. Microscopic inspection of the extracts revealed that, typi cally, nodules contained c. 15 x 10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquot s of the extracts were plated on selective media, a substantial number of ' colony-forming units' was observed in all cases, indicating that a consider able fraction of the bacteroids had the potential to redifferentiate into g rowing bacteria. In nodules from the early developmental stages, the fracti on of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slig htly when the plants started to flower. In order to see how nodule senescen ce affected the survival and redifferentiation potential of bacteroids, som e plants were placed in the dark for 1 week. This led to typical symptoms o f senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisi ngly, the number of total and viable bacteroids g(-1) nodule FW remained vi rtually constant, and the fraction of total bacteroids yielding CFUs did no t decrease but significantly increased up to 75% of the bacteroids judged t o be viable after extraction. This result indicates that during nodule sene scence bacteroids might be induced to redifferentiate into the state of fre e-living, growing bacteria.