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Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes serious infections in burned patients and immunocompromised hosts (4, 22). Damage caused by P. aeruginosa is due to the production of several extracellular and cell-associated virulence factors (7, 23). Exotoxin A, which is the most toxic of the extracellular virulence factors, catalyzes the transfer of an ADP-ribosyl moiety onto elongation factor 2 in eukaryotic cells (6). This results in inhibition of protein synthesis and cell death (6, 21). It is known that exotoxin A production by P. aeruginosa is controlled by both positive and negative regulatory genes (17). We have previously described two P. aeruginosa genes, ptxR and ptxS, that regulate exotoxin A synthesis (3, 5). The ptxR gene codes for a protein that belongs to the LysR family of transcriptional activators and enhances exotoxin A synthesis at the transcriptional level (5). The ptxS gene codes for a protein that belongs to the GalR-LacI family of transcriptional repressors (3). Available evidence suggests that PtxS interferes with the enhancement of exotoxin A synthesis by ptxR (3).

Despite previous analyses, the mechanism(s) through which the expression of ptxS and ptxR is regulated is not known. We have recently provided evidence which suggests that ptxS negatively autoregulates its own synthesis in P. aeruginosa (15). The level of -galactosidase activity produced by a ptxS-lacZ fusion plasmid in the ptxS isogenic mutant PAO1::ptxS was four- to fivefold higher than that produced by its parent strain, PAO1 (15). DNA gel shift experiments showed that PtxS specifically binds to a 52-bp fragment within the ptxS upstream region (15). In addition, DNase protection experiments localized PtxS binding to a 20-bp fragment (15). This 20-bp fragment contains a 14-bp sequence with complete dyad symmetry (a palindromic sequence). In this study, we used site-directed mutagenesis to determine if the 14-bp sequence constitutes a putative PtxS operator site. We have also tried to determine the role of specific nucleotides within the 14-bp sequence in PtxS binding and ptxS expression.

Methods. For site-directed mutagenesis, the oligonucleotides that were used to construct a 14-bp deletion within the ptxS upstream region were 5'-CCTTCTGGTATTTT-3'-deletion of 14 bp-5'-AACTCCTGGCATCC-3' and its complement (accession number AF012100 [3]). The 3' end of the 14-bp palindrome is located 71 bp 5' of the ptxS translation initiation codon. Different oligonucleotides that were used in the synthesis of specific nucleotide mutations corresponded to the region 5'-GGTATTTTCTGAAACCGGTTTCAACTCCTGGC-3' of the ptxS upstream region. Each oligonucleotide was modified to incorporate the required mutations in specific nucleotides. Plasmid pBS9 was used as a template for the site-directed mutagenesis. Plasmid pBS9 is a pKS vector in which the 722-bp BamHI/KpnI ptxR-ptxS intergenic fragment was cloned (15). Site-directed mutagenesis experiments were accomplished using a Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) according to the manufacturer's recommendations. The deletion of the 14-bp sequence and the incorporation of specific mutations were confirmed by nucleotide sequence analysis. DNA gel shift experiments were performed as previously described (2, 15) using specific primers to amplify the 38- and 52-bp fragments from the pBS9 derivatives by PCR (20). The 52-bp fragment was used as a probe in previous DNA gel shift experiments (15). The 3' end of the 52-bp fragment is located 58 bp 5' of the ptxS translation start site (3). The 38-bp fragment is the 52-bp fragment from which the 14-bp sequence was deleted. The 38- and 52-bp fragments were end-labeled with [-³²P]ATP (Amersham, Arlington Heights, Ill.) using T4 polynucleotide kinase (2). The source of the PtxS protein for DNA binding experiments was the lysate of the Escherichia coli strain K38/pJAC17, in which ptxS was overexpressed as previously described (15).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   (A) Schematic diagram of the ptxS upstream region. The locations of the potential promoter region, the 52-bp fragment, the 14-bp palindromic sequence, and the essential restriction sites are indicated. The 5' end of the ScaI site is located 9 bp 3' of the ptxS translation start codon, while the 3' end of the KpnI site is located 7 bp 5' of the ptxS translation start codon. (B) Localization of the ptxS promoter activity. DNA fragments that carry the indicated deletions from the 5' end of the ptxS upstream region were synthesized by PCR and cloned into the lacZ fusion vector pSW205 (14). Recombinant plasmids were introduced into the P. aeruginosa strains PAO1 and PAO::ptxS, and the level of -galactosidase activity was determined as previously described (10). Values are the averages of results of three independent experiments ± the standard errors of the means.

Identification of the region that carries the ptxS promoter. The 3' end of the 52-bp fragment is located 58 bp 5' of the ptxS translation start codon (Fig. 1A). Prior to the mutagenesis experiments, we tried to determine if the 52-bp fragment to which PtxS binds carries a ptxS promoter. Based on our computer analysis of the ptxS upstream region, we had identified a possible ptxS promoter region that overlaps with the PtxS binding site and carries most of the conserved nucleotides within the ⁷⁰ promoters (15). Despite several attempts, we were not successful in determining the ptxS transcriptional start site. Therefore, we tried to localize the ptxS promoter using the previously described lacZ translational fusion vector pSW205 (14). DNA fragments that carry different nested deletions from the 5' end of the 742-bp BamHI/ScaI fragment (which contains the entire ptxS upstream region) were synthesized by PCR (Fig. 1A). Plasmids pBS33, pBS29, pBS31, pBS35, and pBS36 carry 718, 696, 575, 472, and 125 bp of the ptxS upstream region, respectively (Fig. 1B). In comparison with plasmid pBS8 (which carries the intact 742-bp BamHI/ScaI fragment), plasmids pBS33 and pBS29 produced significantly lower levels of -galactosidase activity (Fig. 1B). Whether this difference in the levels of -galactosidase activity is due to the presence of either more than one ptxS promoter or binding sites for a positive regulatory factor is not known at this time. However, plasmids pBS31, pBS35, and pBS36 produced no detectable -galactosidase activity (Fig. 1B). These results indicate that, despite its observed homology to the ⁷⁰ promoters (15), at least the 500-bp region immediately 5' of the ptxS translation start site does not carry a ptxS promoter. Therefore, it is unlikely that the ptxS promoter overlaps with the PtxS binding site. We have also excluded the possibility that the detected levels of -galactosidase activity were influenced by PtxS. Higher levels of -galactosidase activity were obtained when the above-described recombinant plasmids were introduced into the ptxS isogenic mutant PAO::ptxS (due to the derepression of ptxS expression) (Fig. 1B).

Analysis of PtxS binding to the 14-bp palindromic sequence. Computer analysis revealed that the 20-bp ptxS upstream fragment to which PtxS binds contains a 14-bp sequence with complete dyad symmetry (palindrome) that may constitute a potential PtxS operator site (Fig. 2). This palindromic sequence is located 71 bp 5' of the ptxS initiation codon (Fig. 1A). The 14-bp sequence contains the three most conserved nucleotides (5'-AA-C-3') within the operator sites of the GalR-LacI repressors (18). Therefore, in this study, we tried to determine if PtxS binds to this palindromic sequence and the roles of the specific nucleotides within the palindromic sequence in PtxS binding. The deletion of the palindrome, as well as specific mutations, was accomplished using plasmid pBS9, which carries the 722-bp BamHI/KpnI fragment of the ptxS upstream region (15) (Fig. 1A). As shown in Fig. 3, PtxS did not bind to the 38-bp deletion fragment rather than the 52-bp probe, which supports the possibility that the 14-bp sequence is the PtxS binding site. The first conserved nucleotide that we mutated was the adenine at position 5 within the right half of the palindrome (a transition of A to C; plasmid pBS9A5) (Fig. 2). This mutation reduced PtxS binding (Fig. 3). Quantitative analysis revealed that in comparison to PtxS binding to the intact 52-bp fragment, PtxS binding to the mutated 52-bp fragment was significantly reduced (five- to sixfold, data not shown). In addition to mutating this base, we changed the other adenine at the same position within the left half of the symmetrical sequence (a transition of A to C; plasmid pBS9A5/5') (Fig. 2). As shown in Fig. 3, changing the adenine in both halves of the dyad symmetrical sequence eliminated PtxS binding to the 52-bp fragment. We then determined the effects of mutating the two other conserved nucleotides at positions 4 (a transition of A to C; plasmid pBS9A4) and 7 (a transition of C to A; plasmid pBS9C7) within the right half of the palindrome (Fig. 2). While the mutation of A at position 4 caused a reduction in PtxS binding, the mutation of C at position 7 eliminated PtxS binding (Fig. 3). Furthermore, we examined the effect of a mutation in a nucleotide within the palindromic sequence other than the conserved AA-C nucleotides. We synthesized a 52-bp fragment in which the G at position 2 within the right half of the palindromic sequence was replaced with T (plasmid pBS9G2) (Fig. 2). However, this mutation had no effect on PtxS binding to the 52-bp fragment (Fig. 3).

View larger version (20K): [in this window] [in a new window]   FIG. 2.   DNA sequence of the potential PtxS operator (dyad symmetry) site within the ptxS upstream region. Boxed regions indicate the right (bottom, 3' sense sequence) and left (top, 5' antisense sequence) halves of the dyad symmetry. The nucleotides are numbered (1 to 7) for the right half and (1' to 7') for the left half. Filled circles indicate the positions of the nucleotides that were mutated in this study. The types of the mutations are indicated. Plasmids that carry either deletion of the dyad symmetrical sequence or specific nucleotide mutations were divided into two groups. In group I, the deletion and individual mutations were generated in plasmid pBS9 (which carries the 722-bp fragment) by site-directed mutagenesis. For expression experiments, the 722-bp BamHI/KpnI fragment was deleted from pBS8 and replaced by either the 708-bp BamHI/KpnI fragment from pBS9 or the 722-bp BamHI/KpnI fragments from pBS9 derivatives (group II). Plasmid pBS8 is the same as the previously described plasmid pBS8-4 (15).

View larger version (87K): [in this window] [in a new window]   FIG. 3.   DNA gel shift experiments to examine the effects of different mutations on the binding of the 52-bp fragment to the partially purified PtxS. The lysate of the E. coli strain K38/pJAC17 was used as a source of the PtxS protein as previously described (15). We used the 38-bp deletion fragment and the 52-bp fragments that carried different mutations that were synthesized from pBS9 or its derivatives by PCR in the DNA gel shift assay as described in the text. Each DNA binding mixture contained 105 cpm of the labeled 52-bp fragment and approximately 2 µg of the cell lysate. With the exception of the reaction mixture with pBS9, all DNA binding reaction mixtures contained the 52-bp fragments that were obtained from their respective plasmids. The binding reaction mixture with pBS9 contained the 38-bp deletion fragment. In the control lane, the 50-bp fragment was incubated with the lysate of the E. coli strain K38/pT7-5 only. , the DNA binding reaction mixtures containing the probe only; +, the DNA binding reaction mixtures in which the probe was incubated with cell lysate. The faint band in pBS9 was not detected in several repeated experiments (data not shown). Similar faint bands that migrated at the same lower distance were also detected in pBS9A5/5' and pBS9C7 (in some but not all experiments), even though these reaction mixtures contained the 52-bp fragment and not the 38-bp deletion fragment.

The effects of different mutations on ptxS expression. The expression of ptxS that is carried on fragments that contain different mutations in PAO1 was determined using the previously described lacZ translational fusion vector pSW205 (14). As shown in Fig. 4, levels of -galactosidase activity by plasmids carrying either a deletion of the 14-bp sequence or specific mutations within the 14-bp sequence were significantly higher than that produced by pBS8, which contains the putative PtxS operator. The highest level of ptxS expression (increase in -galactosidase activity) was detected with plasmid pBS8, which carries a deletion of the 14-bp sequence (48-fold increase) (Fig. 4). Plasmids pBS8A5, pBS8A5/5', and pBS8C7, in which the A, A and A', and C at positions 5, 5 and 5', and 7, respectively, were mutated, produced comparable increases in ptxS expression (an approximately 38-fold increase in -galactosidase activity) (Fig. 4). The increase in the level of ptxS expression by plasmid pBS8G2, which carries the transition of G to T at position 2, was relatively smaller than that produced by other plasmids (23-fold) (Fig. 4). The smallest increase in ptxS expression was detected with plasmid pBS8A4 (a transition of A to C at position 4) (7.5-fold) (Fig. 4). These increases in the level of ptxS expression are higher than the four- to fivefold increase that we usually detect when ptxS is expressed in the ptxS isogenic mutant PAO::ptxS (15). To determine if this enhancement in ptxS expression varies in the absence of PtxS, the fusion plasmids were introduced in the PAO::ptxS strain. As shown in Fig. 4, with the exception of the pBS8A4 plasmid, all other fusion plasmids produced higher levels of -galactosidase activity than that produced by PAO::ptxS/pBS8. The levels of -galactosidase activity produced by pBS8A5/5', pBS8A4, pBS8G2, and pBS8C7 in both PAO1 and PAO::ptxS were comparable (Fig. 4). However, the levels of -galactosidase activity produced by pBS8 and pBS8A5 in PAO::ptxS were higher than those produced in PAO1 (Fig. 4).

View larger version (52K): [in this window] [in a new window]   FIG. 4.   Effects of the deletion and different base pair mutations on ptxS expression. Fusion plasmids containing different mutations were introduced into the P. aeruginosa strains PAO1 and PAO::ptxS by electroporation. Cells were grown in TSB-DC medium, and the level of -galactosidase activity was determined as previously described (10). The description of each plasmid is given in Fig. 2. Values are the averages of results of three independent experiments ± the standard errors of the means. For the sake of conformity, the previously described plasmid pBS8-4 is referred to here as pBS8.