Abstract
Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the sigma(S)-dependent Escherichia coli promoter P(fic) and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of beta-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing beta-galactosidase from P(fic). Changes in cell size and expression of beta-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of beta-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil beta-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted.