Distinct mechanisms for entering VNC state by different rhizobial strains

The two model systems investigated (Rhizobium ‘hedysari’ HCNT1 and Rhizobium meliloti 41) have been previously assayed for their ability to shift from a normal, aerobic, growth-supporting metabolism to an oxygen limiting, low expensive, base activities. Such metabolic conversion leads bacteria to stop reproduction although allowing them to survive. The biochemical pathways responsible for this low level metabolism are quite different for the two rhizobial strains. In the first case (R. ‘hedysari’ HCNT1) the internal energy budget has to be rapidly consumed to guarantee the entry to the VNC state. This can be accomplished by inducing nitrite reductase under oxygen limiting conditions so forcing the cells to reduce nitrite, presumably at the expense of endogenous ATP. The mutant strain previously obtained by inactivation of the gene encoding nitrite reductase (nirK) does not burn up ATP when exposed to the same oxygen limiting conditions in the presence of nitrite. R. meliloti 41 takes longer time to spend endogenous energy, probably due to the high accumulation of stock material (e.g. PHB and/or glycogen), thus remaining longer at higher energy levels. In this strain the carbon/energy flux can be regulated under low oxygen tension and the role of the previously described ORF1 in this regulation system has been investigated. All this has been verified by treating the cells in a way that drastically reduces the energy conservation (microaerobic to anaerobic incubation) followed by ATP determination and microscopic analysis after staining with DTAF (5-(4,6-dichlorotriazine-2-yl)aminofluoresceine), and CTC (5-cyano-2,3,ditolyl-tetrazolium-chloride). While in the case of HCNT1 the number of culturable cells is corresponding to the number of metabolic active cells, in the case of R. meliloti strain 41 the number of colony forming units resulted to be much lower. This indicates that the mechanisms adopted by the two strains under investigation follow different paths and that the so-called VNC state can be reached within a relatively large interval of internal energy charge, depending upon the bacterial strain. Soil and root colonisation, as well as plant infection test experiments have been performed in microcosm with wild type and mutant strains in condition promoting VNC induction. To evaluate the suitability of luc marker for strains under investigation a strain of R. meliloti luc has been also employed.