Genetic Selection Scheme for Isolation of Signal Transduction Pathway Mutants

MATERIALS AND METHODS

Strains and plasmids. Strains and plasmids used in this work are described in Table 1. Wild-type S. elongatus (previously known as Synechococcus sp. strain PCC 7942 and “Anacystis nidulans” R2, Pasteur Collection no. 7942) was grown in modified BG-11 medium (4) at 30°C and 150 μE m⁻² s⁻¹ irradiance in liquid culture or on solid plates containing 1.5% agar. Transformation of S. elongatus with plasmid constructs was carried out by the method detailed in references 8 and 9.

Escherichia coli DH5α (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) was used as the host for all plasmids and was propagated on Luria-Bertani broth (3) and Terrific Broth (13) media in the presence of antibiotics at standard concentrations (3).

Construction of the psbDII-codA clone.The pRTmcs-codA plasmid, which served as the starting vector for the construction of the psbDII-codA plasmid, was constructed as follows. The plasmid used for constructing pRTmcs-codA was pRT103, which was derived from pUC18/19 (15). In this plasmid, the cauliflower mosaic virus 35s promoter (cAMV-35s) and a plant polyadenylation site are located upstream and downstream, respectively, of the pUC18/19 multiple cloning site (15). The codA gene was amplified from E. coli by using PCR and cloned into the NcoI site of pRT103 to generate pRTmcs-codA (D. Morishige, personal communication).

A 256-bp fragment containing the −96 to + 160 region of the psbDII gene (1) was amplified by using PCR with Synechococcus species genomic DNA as the template. PCRs (25 μl each) contained the following: 100 ng of DNA, 1.5 mM MgCl₂, approximately 0.04 to 0.07 μM (each) primer, 50 mM KCl, 10 mM Tris-HCl [pH 9.0], 0.1% Triton X-100, and 0.25 units of Taq DNA polymerase (Promega Corporation, Madison, Wis.). Primer sequences for the amplification of the psbDII 256-bp fragment are as follows: forward primer, 5′-GGAATTCGAAGCCTGCGAGACAC-3′, and reverse primer, 5′-GACGTAAACCATCC-3′. Thirty cycles of amplification were performed as follows. Denaturation was at 92^°C for 30 s, annealing of the primer to template DNA was performed at 45 to 50^°C for 5 min, and elongation of the primer was at 72^°C for 1 min. Reactions were carried out in a PCRExpress thermal cycler (Hybaid, Middlesex, United Kingdom). The amplified psbDII promoter fragment carried an EcoRI site on the 5′ end (see the sequence of the forward primer in bold). The fragment was digested with EcoRI, purified, and then cloned into the EcoRI-SmaI digested plasmid pRTmcs-codA (a gift from D.T. Morishige, Department of Biochemistry, Texas A&M University). This cloning procedure generated an in-frame translational fusion between the psbDII promoter and the codA gene to generate pRTmcs-codA#5, where the promoter, ribosome binding site, and translational start site of the gene fusion comes from the psbDII gene. The identity of the pRTmcs-codA#5 clone was verified by DNA sequencing.

The entire fusion construct (psbDII-codA) was then excised from pRTmcs-codA#5 by digestion with XbaI and XhoI and ligated into XbaI- and XhoI-digested pAM1573 (2). pAM1573 is a neutral site II chromosomal integration vector developed in the laboratory of Susan Golden of Texas A&M University. This vector contains an E. coli origin of replication, a chloramphenicol cassette for the selection of recombinant E. coli and Synechococcus species transformants, and a multiple cloning site. Two fragments of the Synechococcus species chromosome termed neutral site II flank the antibiotic cassette and multiple cloning site. DNA constructs cloned between the two neutral site fragments will recombine into the Synechococcus species chromosome by a homologous recombination event. pAM1573 does not replicate in Synechococcus species, since it does not contain a Synechococcus species origin of replication and does not contain any Synechococcus species promoters (2). Cloning of the excised XhoI-XbaI fragment from pRT-mcs-codA into pAM1573 generated pAM1573 psbDII-codA. The pAM1573 psbDII-codA plasmid was transformed into Synechococcus strain AMC378, which carries a psbDII-lacZ fusion in chromosomal neutral site I (1), to give rise to strain DU201. A diagrammatic representation of the cloning of pAM1573 psbDII-codA is given in Fig. 1. Successful integration of the psbDII-codA construct into neutral site II of AMC378 was verified by PCR. This strain (DU201) was later used for chemical mutagenesis.

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FIG. 1.

Diagrammatic representation of the cloning strategy used to generate S. elongatus strain DU201.

To test whether the E.coli codA gene was actively expressed under the control of the psbDII promoter and whether the enzyme cytosine deaminase was active in Synechococcus DU201 cells, wild-type Synechococcus species and the DU201 strain were grown to the same optical density in 100-ml cultures, serially diluted, and plated on BG-11 agar medium containing 0.5 mg of 5′-fluorocytosine/ml. No viable colonies were formed from strain DU201 at any dilution, whereas wild-type Synechococcus species showed no decrease in cell viability when grown in the presence of 5′-fluorocytosine. This finding indicated that the codA gene was expressed under control of the psbDII promoter in strain DU201 and converted 5′-fluorocytosine to 5-fluorouracil, which resulted in the death of these cells.

RESULTS

We present here a scheme for the selection of mutants in a signal transduction pathway based on the toxicity of the chemical 5′-fluorocytosine. To test this selection strategy, we chose the psbDII high-light-regulated signal transduction pathway. The psbDII gene and its cis-acting regions have previously been well characterized (1, 4). Moreover, transcriptional and translational fusions of this gene with the reporter gene lacZ have previously been used to characterize promoter activity of this gene (1, 4). We constructed a chimeric fusion between the psbDII −86 to + 160 region and the open reading frame of the E. coli codA gene. In this hybrid gene, the promoter, ribosomal binding site, translational start site, and first 50 bases of the coding region were from the psbDII gene. This region of the psbDII gene had previously been used to generate translational lacZ fusions and had been demonstrated to show high-light induction comparable to the levels of induction shown by psbDII mRNA (4, 5). A Synechococcus strain, DU201, which contained the hybrid psbDII-codA gene in chromosomal neutral site II and the psbDII-lacZ fusion in neutral site I, was generated. Chemical mutagenesis using MMNG was chosen to generate random point mutations in the chromosome. At the time this selection method was developed, a foolproof transposon-based mutagenesis scheme had not been demonstrated for Synechococcus. Moreover, since we anticipated that mutations in the psbDII pathway could affect the viability of the mutant strain(s), we chose a mutagenesis method that would inflict the least possible alteration on gene sequences. Insertion of a transposon might prove deleterious, but a point mutation in the same locus might prove to have a less severe effect.

A preliminary screening step to assay the effect of the mutations generated on psbDII promoter activity utilized the psbDII-lacZ construct in chromosomal neutral site I (1). In this step, β-galactosidase assays were used to monitor the promoter activity of the psbDII gene. The β-galactosidase levels would reflect the effect of a mutation that altered the psbDII promoter activity level in any of the isolates compared to that of the nonmutagenized control strain. This screening step separated those strains with mutations in the codA gene from those with mutations in the psbDII high-light pathway. Strains with mutations in the codA gene should show 5′-fluorocytosine resistance but have normal levels of psbDII activity. Strains that carried a mutation in the psbDII high-light pathway should show 5-fluorocytosine resistance and should also have altered psbDII promoter activity, as measured by β-galactosidase levels, compared to that of the nonmutagenized DU201 control strain. Table 2 gives the distribution of different phenotypes for psbDII promoter activity retrieved after mutagenesis and selection with 5′-fluorocytosine-based selection criteria. The control strain used for comparison was nonmutagenized DU201.

Of 72 5′-fluorocytosine-resistant independent isolates, 7 isolates showed wild-type levels of psbDII activity, 5 strains had increased psbDII activity, and 24 strains had decreased psbDII activity, as monitored by β-galactosidase assays (Table 2). Thirty-six mutants that were isolated have yet to be characterized for psbDII activity with this assay. Strains that had impaired growth or had different pigmentation profiles than that of the wild type were chosen for further study, and β-galactosidase assays in triplicate were carried out for these representative strains. The results of these assays are shown in Fig. 2.

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FIG. 2.

Expression of psbDII-lacZ fusions in isolated mutant strains of S. elongatus. Specific activity (nanomoles of O-nitrophenyl-β-d-galactopyranoside per minute per milligram of protein) was determined for each psbDII-lacZ reporter strain. β-Galactosidase levels were corrected by subtracting background levels from a promoterless lacZ strain, AMC181. The graphed values represent the means of the results of triplicate experiments, with the standard deviations indicated. The histograms show β-galactosidase levels for strain DU201 (control 1, control 2, and control 3) with mutant strains U, Z, JU4, J, and K 0 h before (hatched bars) and 2 h after (black bars) the shift to high light (800 μE m⁻² s⁻¹). The graphed values for the data for control 1, control 2, and mutants U, Z, and JU4 are the means of the results of triplicate independent experiments, with the standard deviations indicated. The data for control 3 and mutant strains J and K were from one experiment. Due to the poor growth of strains J and K in high light, it was difficult to repeat the experiments.

These results show that mutant U has elevated psbDII promoter activity, as assessed by β-galactosidase assays (Fig. 2). This mutant very likely contains a defect in the protein factor that binds to the negative element mapped to the psbDII UTR (1). Mutants Z and JU4, however, show decreased psbDII promoter activity (Fig. 2), implying that the defect is in the gene encoding the factor that binds to the upregulatory element in the psbDII UTR (1). All three mutants do maintain high-light induction of the psbDII gene, indicating that binding site 1 in the psbDII UTR is still functional in a wild-type manner. Mutants K and J, however, lost high-light induction of the psbDII-lacZ reporter gene (Fig. 2), indicating that the mutations in these two strains affect the protein factor(s) that binds to the light-regulating element in the psbDII UTR (1).

Three mutants, mutants J, K, and Z, also showed impaired growth rates. These mutants were initially isolated without shaking in liquid culture. As seen in Fig. 3, all three mutants had lower growth rates when grown under high-light intensity in liquid culture than that of control strain DU201. In particular, mutant Z could not be grown under high-light conditions, as this strain rapidly bleached and died under these conditions. Thus, no β-galactosidase values were obtained for this strain.

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FIG. 3.

Growth curve for control strain DU201 and selected mutant strains under high-light conditions. The following symbols represent the indicated strains: ▪, strain DU201; •, strain K; ▴, strain J; and ♦, strain Z. OD₇₅₀, optical density at 750 nm.

DISCUSSION

A genetic selection scheme was designed for the isolation of mutants in the psbDII high-light-regulating pathway that controls activity of the psbDII promoter. The psbDII high-light-regulating pathway in S. elongatus was chosen to test this scheme, since this gene had previously been well characterized in this organism and insertional mutagenesis of psbDII was shown to not be lethal to this organism (5). The advantage of the selection strategy proposed here is that the viable colonies that arise are all mutant in nature. The wild-type cells are killed by selection on 5′-fluorocytosine, allowing easy isolation of mutant colonies.

In this study, mutations were created by treatment of strain DU201 with the chemical agent MMNG (8). However, this scheme lends itself to use with a variety of mutagenic strategies and agents. Mutations can as easily be created by transposon or interposon mutagenesis. A scheme for these types of mutagenesis in Synechococcus species has recently been designed and would be appropriate for use with this selection strategy (2). In particular, this strategy is useful where a visible or easily identifiable mutant phenotype is not available for purposes of selection. This screen uses resistance to 5′-fluorocytosine as the phenotypic selection criterion for mutant identification.

It was anticipated that this selection scheme would give rise to cells with decreased psbDII promoter activity in high light. However, we isolated five independent mutant strains that showed elevated psbDII activity in low and high light, which implies that these strains also produce elevated levels of cytosine deaminase. If this were indeed the case, these mutants would not have been isolated as viable colonies by this selection method. It is very likely that these mutants possess a secondary mutation in the codA gene that obliterates cytosine deaminase function, thereby conferring a 5′-fluorocytosine-resistant phenotype on these cells that allowed them to be isolated in this screen. Identification of strains with secondary mutations in the codA gene could be carried out by replacing the lacZ gene in neutral site I with a second copy of the psbDII-codA construct, which would cause these strains to be sensitive to 5′-fluorocytosine. With replica plating on medium without the selective agent, the strains could be kept viable, while at the same time the second site mutation in the codA gene could be confirmed. This strategy would then also allow for the isolation of mutations in the signal transduction pathway that confer elevated levels of expression of the target gene.

The mutants isolated by this approach that have presently been characterized had impaired function of the target gene, psbDII. Mutants J, K, Z, JU4, and U all had psbDII promoter activities that differed significantly from those of the wild type. Previous studies with the psbDII promoter have shown that a mutation in binding site 1 negates high-light induction of psbDII, while mutations in binding sites 2 and 3 affect positive and negative regulatory elements, respectively (1). Thus, mutants J and K should have mutations in the protein factor that binds to binding site 1, while mutants Z and JU4 should have abnormal protein factors that bind to binding site 2 (1). It is anticipated that mutant U is aberrant for the protein factor binding to binding site 3 or to the direct repeat region within the psbDII open reading frame (1).

In addition, mutants J and K, which showed no high-light induction of the psbDII promoter, also showed aberrant growth rates in high light. It has been demonstrated that loss of psbDII function, while not lethal in Synechococcus species, causes the organism to be less competitive than the wild type in mixed-culture experiments (5). Thus, these mutants show phenotypes similar to that of the previously reported psbDII-null mutant. It has been demonstrated by gel shift analysis that the upstream regions of the psbDII, psbAII, and psbAIII genes bind the same trans-acting factors (4). It is possible that mutations in one or more of these protein factors could have a pleiotropic effect, affecting not only expression of the psbDII gene but also expression of other high-light-inducible genes like psbAII and psbAIII in Synechococcus species. This effect could account for the low growth rate and defects in pigmentation seen in isolates J and K.

Rescue of these mutations with genomic DNA allows us to precisely pinpoint the site of the mutations and to identify those genes that are part of the psbDII high-light pathway. Rescue of the mutations, however, confers wild-type regulation on the psbDII promoter, which in turn leads to expression of the codA gene and death of the rescued mutant on 5′-fluorocytosine. To overcome this problem, rescued mutants should be replica-plated on medium with and without 5′-fluorocytosine after transformation with genomic DNA to obtain viable rescued cells. Indeed, the sensitivity of the rescued strains to 5′-fluorocytosine can be used as an indication that a successful rescue of the mutant phenotype has taken place with the consequent reconstitution of the wild-type signal transduction pathway.

The psbDII promoter used in this selection method is active under both conditions tested, i.e., low- and high-light intensities. Since our goal was to isolate mutants that were affected by both the low- and high-light expression of this gene, this characteristic of the promoter was crucial for our outcome. The choice of promoter to be used in this selection scheme can greatly influence the outcome of the mutants isolated. A promoter that is tightly regulated in a temporal or cell-specific manner can also be used with this selection scheme, and the expression window of the promoter can be utilized to select for mutants that are aberrant in either temporal or cell-specific regulation of the signal transduction pathway under analysis. Indeed, the utility of this selection method can be extended by using an inducible promoter-operator system like lacI-lacZ to control the expression of the codA gene in a time- or cell-specific manner for the isolation of mutants defective in either temporal or cell-specific expression of the signaling pathway under study.

The ability to generate and screen random mutations is a crucial step in isolating the components and understanding the functioning of a signal transduction pathway (6, 11). We have described a genetic selection scheme that lends itself to the isolation of mutants defective in a cellular signal transduction pathway where the resulting viable colonies are mutant, thus allowing for easy selection of mutant phenotypes. Identification of the mutated loci could be done by transformation of the mutant with a genomic library and scoring for rescue of the mutant phenotype (8). If mutagenesis were by a transposon, then isolation of the transposon would indicate the mutated locus of the gene (2). This selection scheme is easy to carry out once the codA construct is generated and can be used in systems where an intrinsic codA gene is absent or where the native codA gene has been deleted. The genetic selection scheme described here extends the tools available for the isolation and characterization of mutations in a given signal transduction pathway.