A GFP-lacZ bicistronic reporter system for promoter analysis in environmental gram-negative bacteria

Abstract

Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods.

Figure 1. Bicistronic reporter system based on GFP and lacZ.

A The reporter vector pRV1 containing truncated versions of the lacZ gene and Sm/Sp resistance marker could only be maintained in E. coli strains with supF tRNA. GFP is located upstream of the lacZ gene and is preceded by the EcoRI/BamHI cloning site for the target promoters. The oriT sequence allows the mobilization of pRV1 to new hosts by conjugation. B The mini-Tn10-based vector pLOF-hom.fg. was used for target strain modification. This vector contains the homologous fragment, which was introduced into the strains of interest to create single copy insertions in the chromosome.

Figure 2. Isolation of a non-fluorescent P. putida strain.

(i) P. putida KT2442, a rifampicin resistance variant of P. putida KT2440, was mutagenized with mini-Tn5 transposon pUT-ResKm. (ii) Single colonies were selected in minimal media and screened for the lack of fluorescence. Two colonies were selected and named P. putida UV1 and UV2. (iii) The Km resistance marker was removed from the strains by expressing ParA resolvase, which recognizes the two res sites flanking the Km marker. After marker elimination, the strains retained the non-fluorescence phenotype. (iv) The homologous fragment placed in the mini-Tn10 transposon was mobilized to the marker-less P. putida MEG1 strain (a deviant of P. putida UV1), generating the P. putida MEG3 strain. This strain was used as a host for the bicistronic reporter system with the target promoters.

Figure 3. Catabolic pathway for benzoate degradation in P. putida.

Benzoate activates the BenR regulator, which controls the expression of the ben operon. The enzymes encoded by the ben operon metabolize the conversion of benzoate to cis,cis-muconate (cis,cis-muc), which is the signal for CatR activation. CatR controls the expression of the cat operon for the metabolization of cis,cis-muconate, which is further converted to β-ketoadipate. Finally, β-ketoadipate is converted to TCA intermediates through the action of the pca pathway (gray arrows).

Figure 4. Design of bicistronic GFP-lacZ reporter strains.

(i) pRV1 containing the cloned promoters (labeled as P) was introduced into the P. putida MEG3 strain by conjugation. Colonies were selected in minimal media using the Sm antibiotic. (ii) The homology regions of the suicide vector pRV1 were recombined with the two segments placed in the chromosome, generating a functioning Sm/Sp resistance marker and lacZ gene. (iii) Finally, the correct insertion of the segments generated a strain with a stable reporter system that was sensitive to the Km antibiotic.

Figure 5. Dual reporter expression in P. putida MEG3 strains.

GFP expression is shown on the left, while lacZ expression is shown on the right. The strain with no promoter cloned (labeled as MEG3) was used as a control. The strains having Pb and Pc fused to the dual reporter system presented GFP and LacZ signals when cultured in the presence of 1 mM benzoate.

Figure 6. Assay of promoter activities in response to benzoate.

Overnight cultures were diluted in fresh minimal media supplemented with succinate as the sole carbon source. At the mid-exponential phase, the cells were exposed to 1 mM benzoate, and the GFP and the lacZ expression was assayed. A The GFP expression was measured in P. putida MEG3-Pb and MEG3-Pc after 4 hours of induction using flow cytometry. B β-galactosidase activity of P. putida MEG3-Pb and MEG3-Pc after 4 hours of induction. C Time course quantification of GFP expression in P. putida MEG3-Pb and MEG3-Pc in response to 1 mM benzoate. The vertical bars represent the standard deviation of the experiments performed in duplicate. D The quantification of GFP levels in the P. putida MEG3-Pb population in response to 1 mM benzoate. The distribution of cell fluorescence is shown along the induction curve. At each time point, 15,000 events were analyzed. E The quantification of GFP levels in the P. putida MEG3-Pc population in response to 1 mM benzoate. The experiments were performed as in D.