Construction and characterization of a proU-gfp transcriptional fusion that measures water availability in a microbial habitat

Abstract

We constructed and characterized a transcriptional fusion that measures the availability of water to a bacterial cell. This fusion between the proU promoter from Escherichia coli and the reporter gene gfp was introduced into strains of E. coli, Pantoea agglomerans, and Pseudomonas syringae. The proU-gfp fusion in these bacterial biosensor strains responded in a quantitative manner to water deprivation caused by the presence of NaCl, Na(2)SO(4), KCl, or polyethylene glycol (molecular weight, 8000). The fusion was induced to a detectable level by NaCl concentrations of as low as 10 mM in all three bacterial species. Water deprivation induced proU-gfp expression in both planktonic and surface-associated cells; however, it induced a higher level of expression in the surface-associated cells. Following the introduction of P. agglomerans biosensor cells onto bean leaves, the cells detected a significant decrease in water availability within only 5 min. After 30 min, the populations were exposed, on average, to a water potential equivalent to that imposed by approximately 55 mM NaCl. These results demonstrate the effectiveness of a proU-gfp-based biosensor for evaluating water availability on leaves. Furthermore, the inducibility of proU-gfp in multiple bacterial species illustrates the potential for tailoring proU-gfp-based biosensors to specific habitats.

FIG. 1.

Map of pPProGreen (15.2 kb). The promoter proU was directionally cloned from the proU expression vector pOSEX4 (19) into the broad-host-range pPROBE-KT′ vector (38). The rrnB transcriptional terminators, with one copy (T1) downstream and four copies (T4) upstream of the fusion, surrounded the P_proU-gfp fusion. The proU promoter contains an upstream activating region (UAR) and a silencer (S) located in the proV gene. The transcriptional (+1) and translational (+64) start sites are shown. All of the restriction enzyme sites shown, including HindIII (H), SalI (S), BamHI (B), and EcoRI (E), were unique in pPProGreen.

FIG. 2.

Induction of P_proU-gfp in cells that were grown in 1/2-21C broth (A, B, and C) or on 1/2-21C plates (D, E, and F) containing various concentrations of NaCl. (A and D) E. coli DH5α(pPProGreen) (○) and DH5α(pVLacGreen) (□); (B and E) P. agglomerans BRT98(pPProGreen) (○) and BRT98(pVLacGreen) (□); (C and F) P. syringae B728a(pPProGreen) (○) and B728a(pPNptGreen) (□). The insets show induction over a narrower range of NaCl concentrations. The fluorescence of the cells was measured with a fluorometer. The induction ratio was the mean fluorescence of the cells relative to the mean fluorescence of cells grown with 0 mM NaCl. Values represent the mean ± standard error of the mean (n = 3).

FIG. 3.

Induction of P_proU-gfp in cells that were grown on 1/2-21C plates containing various levels of PEG 8000. P. agglomerans BRT98(pPProGreen) (□) and P. syringae B728a(pPProGreen) (▵) were grown for 24 h, and the fluorescence intensity of cell suspensions was measured with a fluorometer. The induction ratio was the mean fluorescence of the cells relative to the mean fluorescence of cells grown with 0% PEG 8000. Values represent the mean ± standard error of the mean (n = 3).

FIG. 4.

Spatial distribution of cells exhibiting fluorescence due to P_proU-gfp induction on bean leaves (P. vulgaris). (A) P. agglomerans BRT98(pPProGreen) on a bean leaf after 24 h of incubation under moist conditions (no visible bacterial fluorescence); (B) P. agglomerans BRT98(pPProGreen) on a bean leaf 4 h after transfer of the plants from moist conditions to ambient conditions (i.e., after 4 h of drying); (C) P. syringae B728a(pPProGreen) on a bean leaf after 4 h of drying; (D) P. agglomerans BRT98(pPProGreen) on a bean leaf after 8 h of drying; (E) P. syringae B728a(pPProGreen) on a bean leaf after 8 h of drying. Leaves were examined by epifluorescence microscopy. The bacterial cells exhibited green or bluish-green fluorescence, the plant epidermal cells exhibited red autofluorescence, the leaf trichomes exhibited bluish-green (B to D) or blue (E) autofluorescence, and the stomata exhibited blue autofluorescence (E). Magnification, ×100.

FIG. 5.

Induction of P_proU-gfp in P. agglomerans BRT98(pPProGreen) cells recovered from bean leaves at various times following inoculation. (A) Experiment 1, in which the cells were monitored for 30 min (n = 3); (B) experiment 2, in which the cells were monitored for 240 min (n = 2). The induction ratio was the mean fluorescence of the cells relative to the mean fluorescence of the cells at 0 min. Values represent the mean ± standard error of the mean for n samples. Data points indicated by the same letter do not differ by Fisher's least-significant difference test (P < 0.05).