Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa

Abstract

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated P_Rha) and found that P_Rha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated P_BAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the P_Rha and P_BAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in P_Rha and P_BAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible P_araBAD (P_BAD) and rhamnose-inducible P_rhaBAD (P_Rha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic P_Tac promoter is not subject to Vfr regulatory control.

Keywords: Pseudomonas aeruginosa; Vfr; arabinose; cyclic AMP; rhamnose.

FIG 1

P_Rha promoter activity is Vfr dependent. (A and B) P. aeruginosa strains PA103 (A) and PAK (B) carrying a chromosomally integrated P_Rha-lacZ transcriptional reporter were cultured in LB with the indicated concentrations of rhamnose. When the culture A₆₀₀ reached 1.0, the cells were harvested and assayed for β-galactosidase activity, reported in Miller units. *, P < 0.05 (ANOVA). (C) The indicated PA103 and PAK strains carrying the P_Rha-lacZ reporter, transformed with a vector control (pJN105) or a Vfr expression vector (pVfr), were cultured in LB with 0.005% rhamnose. Cells were assayed for β-galactosidase activity as described above. The reported data are the averages from at least three independent experiments. *, P < 0.05 (ANOVA).

FIG 2

P_BAD promoter activity is Vfr-dependent. (A) PA103 strains carrying a chromosomally integrated P_BAD-lacZ transcriptional reporter were cultured in LB with the indicated concentrations of arabinose. When the culture A₆₀₀ reached 1.0, the cells were harvested and assayed for β-galactosidase activity, reported in Miller units. (B) PA103 or a Δvfr mutant carrying the P_BAD-lacZ reporter, transformed with a vector control (pJN105) or a Vfr expression vector (pVfr), was cultured in LB. The culture was back-diluted to a A₆₀₀ of 0.8 in LB with 0.2% arabinose and incubated for an additional 45 min. The cells were then harvested and assayed for β-galactosidase activity. The reported data are the averages from at least three independent experiments. *, P < 0.05 (ANOVA).

FIG 3

Plasmid-based P_BAD promoter activity is Vfr dependent. PA103 exsA and exsA vfr strains carrying a chromosomally integrated P_exsD-lacZ transcriptional reporter were transformed with a plasmid encoding exsA under the transcriptional control of the P_BAD promoter. Cells were cultured in LB with the indicated concentrations of arabinose. When the culture A₆₀₀ reached 1.0, the cells were harvested and assayed for β-galactosidase activity, reported in Miller units (A), and ExsA protein levels by immunoblotting using ExsA antiserum (B). The reported β-galactosidase activity data are the averages from at least three independent experiments. *, P < 0.05 (t test).

FIG 4

Vfr directly binds to P_Rha and P_BAD promoter probes. (A) Wt PA103 and the Δvfr mutant carrying a chromosomally integrated P_Rha-lacZ transcriptional reporter lacking (− rhaT) or including (+ rhaT) the rhamnose transporter gene rhaT were cultured in LB with the indicated concentrations of rhamnose and assayed for β-galactosidase activity, reported in Miller units. The reported data are the averages from at least three independent experiments. (B and C) Radiolabeled P_Rha (B) and P_BAD (C) promoter probes were incubated alone (lane 1) or with the indicated concentrations of Vfr in the presence of the nonspecific competitor poly(dI·dC) and a second radiolabeled probe derived from algD that served as a negative control. Lane C is a control with the algD probe incubated with 200 nM Vfr. Reaction products were subjected to nondenaturing gel electrophoresis followed by phosphorimaging. The asterisk in panel B indicates the second promoter probe complex observed at the higher concentrations of Vfr tested. The phosphorimages are representative data from two independent experiments.

FIG 5

Influence of Hfq on Vfr-dependent control of the P_Rha promoter. (A) Hfq inhibits Vfr synthesis. (B and C) PA103 wt and Δhfq strains carrying a chromosomally integrated P_Rha-lacZ (B) or P_BAD-lacZ (C) transcriptional reporter were cultured in LB with the indicated concentrations of rhamnose or arabinose, respectively. When the culture A₆₀₀ reached 1.0, the cells were harvested and assayed for β-galactosidase activity, reported in Miller units. (D) PA103 carrying either empty vector control (pJN105) or an Hfq expression vector (pHfq) were cultured in LB with 0.2% arabinose and 0.005% rhamnose. Cells were assayed for β-galactosidase activity as described above. The reported data are the averages from at least three independent experiments. *, P < 0.05 (t test).

FIG 6

β-Galactosidase expressed from pUC18-mini-Tn7T-Gm-LacZ10 is unstable. PA103 cells carrying the P_Rha-lacZ reporter derived from pUC18-mini-Tn7T-Gm-LacZ10 (mini-Tn7), a P_Rha-lacZ derivative with a corrected lacZ gene (corrected mini-Tn7), and a strain with a mini-CTX-lacZ based reporter (mini-CTX) were harvested in a series of tubes containing Z buffer. Samples were assayed for β-galactosidase activity at the indicated times. The reported values represent the amount of β-galactosidase activity remaining at each time point relative to time zero. The reported data are the averages from least three independent experiments. *, P < 0.05 (ANOVA).

FIG 7

P_Tac promoter activity is Vfr independent. PA103 and a Δvfr mutant carrying a chromosomally integrated P_Tac-lacZ transcriptional reporter were cultured in LB with the indicated concentrations of IPTG. When the culture A₆₀₀ reached 1.0, the cells were harvested and assayed for β-galactosidase activity, reported in Miller units. The reported data are the averages from at least three independent experiments. There was no statistical difference (ANOVA) in P_Tac-lacZ transcriptional reporter activity between PA103 and the Δvfr mutant.