A possibile role of nirK in Rhizobium sullae HCNT1.

In response to a number of environmental stresses many bacterial species, including Vibrio vulnificus, Sinorhizobium meliloti, Micrococcus luteus, Escherichia coli and Helicobacter pylori, enter the viable-but-not-culturable (VBNC) status (McDougald et al., 1998). In this metabolic state they lose their ability to grow on media that usually sustain them and undergo such physiological and morphological changes as increased resistance to several physic and chemical factors, and changes in protein and lipid content. The recent increasing use of specific fluorescent dyes such as Syto 9, CTC (5-cyano-2,3-di-4-tolyl-tetrazolium chloride), AO (Acridine Orange), propidium iodide, made possible a proper identification of viability and the metabolic state of microbes (Basaglia et al. 1997). Sinorhizobium meliloti 41, a rhizobium nodulating Medicago sativa, enters VBNC status in liquid microcosms when O2 is depleted from the atmosphere of the incubation mixture (Toffanin et al., 2000; Casella et al. 2001). Plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and stably inherited in Sinorhizobium meliloti 41 (pRP4-luc) as a reporter gene. The strain obtained, S. meliloti 41 pRP4-luc and its parental strain, served as a model system for VBNC experiments both in vitro and in soil samples. Rhizobium sullae, formerly Rhizobium ’hedysari’, is a nitrogen fixing bacterium that induces symbiotic nodule formation on the legume Hedysarum coronarium (Squartini et al., 2002). Strain HCNT1, expressing a copper-containing nitrite reductase encoded by nirK, which is closely related to nitrite reductases in true denitrifiers, enters the same VBNC status when oxygen is limiting, but only when nitrite is present and converted to NO. Since HCNT1 cannot grow as a denitrifier and inactivation of nirK only resulted in the loss of NO production (Toffanin et al, 1996), the hypothesis that expression of nirK may induce the VBNC status has been investigated. Therefore, a comparison between the two systems, S. meliloti and R. sullae, is presented in order to verify the possible connection of nirK with the VBNC status. McDougald D. et al. 1998. FEMS Microbiology Ecology 25: 1-9 Basaglia M. et al. 1997. In: The biotechnology and ecological interactions of microbial inoculants". Granada, Spain. pp. 19-20 Toffanin A. et al. 2000. Biol. Fertil. Soils. 31 (6): 484-488 Casella S. et al. 2001. ISME-9, Amsterdam (The Netherlands), p. 191 Squartini A. et al. 2002. Int. J. Syst. Evol. Microbiol. 52: 1267-1276 Toffanin A. et al. 1996. Appl. Environ. Microbiol. 62 (11): 4019-4025