Development of tools for the genetic manipulation of Pseudomonas aeruginosa

Journal of Microbiological Methods 58 (2004) 203 – 212 www.elsevier.com/locate/jmicmeth

Development of tools for the genetic manipulation of Pseudomonas aeruginosa Sang-Jin Suh a,*, Laura A. Silo-Suh a, Dennis E. Ohman b b

a Department of Biological Sciences, Auburn University, 101 Life Sciences Building, Auburn, AL 36849, USA Department of Microbiology and Immunology, Medical College of Virginia, Campus of Virginia Commonwealth University, and Veterans Affairs Medical Center, Richmond, VA 23298, USA

Received 29 October 2003; received in revised form 29 January 2004; accepted 26 March 2004 Available online 4 May 2004

Abstract To facilitate study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, several genetic tools were developed. These tools include a series of cassettes carrying (a) the minimal sequence for the origin of transfer (oriT) of RP4 plasmid for introducing plasmid into P. aeruginosa via conjugation, (b) a minimal sequence for P. aeruginosa replicon (stabilizing fragment or SF) for maintenance of plasmids in P. aeruginosa, and (c) the transcriptionally non-polar tetracycline resistance gene (TcR) for insertional mutagenesis. Additional genetic constructs include (d) two conjugative and suicide lacZ reporter fusion plasmids for studying gene expression at the transcriptional or translational level, (e) a gentamicin resistant promoter-probing mini-Tn5 lacZ, and (f) a tightly regulated T7 promoter/repressor system to control gene expression in P. aeruginosa. D 2004 Elsevier B.V. All rights reserved. Keywords: Pseudomonas replicon; oriT cassette; TcR cassette; Regulated promoter

1. Introduction Pseudomonas aeruginosa is a gram-negative bacterium that is found in various environmental niches including water, soil, hospital environments, in association with plants, and in human infections. As an opportunistic pathogen, this bacterium primarily infects patients who are immunocompromised, suffer from severe burns, or have the genetic disorder cystic fibrosis (CF) (Cross et al., 1983). The ubiquitous

* Corresponding author. Tel.: +1-334-844-1666; fax: +1-334844-1654. E-mail address: [email protected] (S.-J. Suh). 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.03.018

environmental distribution of P. aeruginosa indicates that this bacterium has the ability to survive under various environmentally stressful conditions. The complete genome sequence of P. aeruginosa revealed that this bacterium has a large genome of 6.3 million nucleotides and a large number of genes devoted to nutrient acquisition and catabolism in addition to an unusually large number of transcriptional regulators (Stover et al., 2000). Presence of a large number of transcriptional regulators is indicative of the ability of P. aeruginosa to sense, respond, and adapt successfully to various environmental signals. Thus, together with nutritional versatility, the ability to withstand environmental insults potentially contributes to the ubiquitous distribution of P. aeruginosa.

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The advent of molecular technology such as the DNA array has facilitated genomic studies to elucidate potential mechanisms of P. aeruginosa gene regulation and pathogenesis. However, it is still necessary to manipulate the bacterium at the genetic level in order to elucidate functions of the genes that have been identified via genomic studies. This often requires isolation of mutants as well as verification of DNA array studies via reporter fusion expression studies. Elegant genetic tools have been developed to facilitate genetic manipulation of P. aeruginosa, especially for studying gene expression using reporter fusions (Schweizer, 1992, 1993b; West et al., 1994; Hoang et al., 1998, 2000; Becher and Schweizer, 2000; Schweizer and Chuanchuen, 2001). However, many genetic tools are still lacking for the manipulation of P. aeruginosa. One of the most useful molecular constructs for an investigator is cassettes that can be easily isolated and cloned into a gene or a plasmid of interest. Thus, we developed three genetic cassettes, including one with oriT, which allows plasmids to be conjugated into P. aeruginosa, and one with the stabilizing fragment (SF), which permits conversion of any plasmid into a Pseudomonas replicon. In addition, we altered a tetracycline resistance gene to be transcriptionally nonpolar in order to construct non-polar mutations within operons. All three of these constructs have been cloned into a plasmid vector that contains a palindromic multiple cloning sites (MCS) such that each of the constructs can be isolated as a cassette using a variety of restriction enzymes. For facilitating gene expression studies, we constructed lacZ transcriptional and translational fusion plasmids that can be conjugated directly into P. aeruginosa and integrate into the chromosome such that expression of a gene of interest can be studied at a single copy level. This minimizes some of the problems that are inherent with multi-copy plasmid systems, including titrating out potential regulators. Promoter-probing transposons are a powerful set of genetic tools that allow investigators to simultaneously isolate mutants and to study gene expression of the target genes. We modified a mini-Tn5 promoter probing transposon by replacing kanamycin resistance with gentamicin resistance, and thus made it amenable for use in P. aeruginosa. Finally, we constructed a tightly regulated promoter system that can be used to control the expression of a gene of interest. With this promoter system, simply by repressing the expression of the gene

of interest, one can simulate the mutant phenotype and study the consequences of the mutation. Conversely, one can induce the expression of the gene of interest and restore the wildtype phenotype. In this report, we describe the construction of these new tools that facilitate genetic manipulation of Pseudomonas.

2. Materials and methods 2.1. Media and growth Bacteria were grown in L broth (LB) or LB supplemented with appropriate antibiotics at 37 jC with aeration, unless otherwise indicated. Media were solidified with 1.5% Bacto Agar (Difco). The following antibiotic concentrations were used in this study (per milliliter): ampicillin (Ap), 100 Ag for E. coli; carbenicillin (Cb), 125 Ag for single copy insertion in the genome or 250 Ag for multicopy plasmid in P. aeruginosa; gentamicin (Gm), 20 Ag for E. coli and 100 Ag for P. aeruginosa; kanamicin (Km), 50 Ag for E. coli; tetracycline (Tc), 20 Ag for E. coli and 115 Ag for P. aeruginosa; nalidixic acid (Nal), 20 Ag for E. coli. Antibiotics were purchased from Sigma (St. Louis, MO). The chromogenic substrate X-gal was purchased from Biosynth (Naperville, IL) and used at 40 Ag/ml for E. coli and 50 Ag/ml for P. aeruginosa. 2.2. DNA manipulations, transformations, and conjugations E. coli strain DH10B was routinely used as the host strain for cloning. Cloning vector pUC1918 was obtained by deleting the aacCI gene as a SacI fragment from pUCGM1 (Schweizer, 1993a). This yielded a plasmid with the MCS of pUC19 as a palindrome with the SacI site in the middle such that the unique SacI site is symmetrically flanked by restriction enzyme cleavage sites of the pUC19 MCS (Fig. 1). Plasmids were purified with QIAprep spin miniprep columns made by Qiagen (Valencia, CA) or with the Concert Plasmid Purification system from Invitrogen (Carlsbad, CA). DNA fragments in agarose gels were purified using the Qiaex II DNA gel extraction system (Qiagen). Restriction and DNA modification enzymes were purchased from New England Biolabs (NEB, Beverly, MA). The thermostable polymerase Pfu from Strategene (La

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without aeration. Then the donor, helper, and recipient cells were mixed at 7:7:1 ratio in 1 ml of saline, centrifuged, resuspended in 25 –50 Al of saline, and spotted on a LB plate, which was incubated overnight at 30 jC. Following incubation, the cells were scraped off with a sterile cotton swab, resuspended in 2 ml of saline, and plated onto selective plates. 2.3. Cloning moriT sequence and mSF sequence

Fig. 1. Genetic cassettes. A genetic cassette carrying the non-polar TcR gene can be isolated with SacI, KpnI, SmaI, XbaI, or PstI from plasmid pSS262. The cassettes carrying either the moriT or mSF can be isolated with any of the restriction enzymes present in the MCS of pUC1918. The moriT cassette carries an additional BamHI site internal to the SacI site, while the mSF carries an additional SphI site internal to the SacI site.

Jolla, CA) was used to amplify DNA fragments for cloning, and Amplitaq polymerase from Perkin Elmer (Foster City, CA) or Taq DNA polymerase from NEB were used to screen for appropriate clones. Oligonucleotide linkers for SacI and XhoI were purchased from NEB and oligonucleotide primers for PCR amplification were purchased from Qiagen/Operon, (Alameda, CA). DNA was introduced into E. coli via by electroporation using the E. coli Gene Pulser by Bio-Rad (Hercules, CA). Electroporation of plasmids into P. aeruginosa was performed as previously described (Suh et al., 1999). For transfer of plasmids into P. aeruginosa via conjugation, tri-parental conjugation was performed as previously described (Suh et al., 1999) with following modifications. E. coli donor and the helper strains were grown to mid-log phase of growth at 37 jC with aeration. The recipient P. aeruginosa strain in stationary phase was mixed 1:1 in LB + 20 mM NaNO3 and incubated for >2 h at 42 jC

The following genetic selection and screen procedures were used to facilitate clone isolation after ligation of a DNA fragment to the pUC1918-derived vector (Schweizer, 1993a). To isolate the plasmid carrying the moriT, the ligation mixture was first electroporated into E. coli strain DH10B (SmR) and selected for ApR, the antibiotic resistance carried by the vector plasmid. The ApR colonies were then pooled and conjugated into E. coli strain DH5aF’ (NalR) via triparental mating with HB101/pRK2013 as the helper strain. The transconjugants were then selected for ApR (plasmid vector) and NalR (DH5aF’) to counter-select against donors without the moriT inserts (DH10B) and the helper strain (HB101). Plasmids were isolated from ApR NalR colonies and the presence of the moriT was verified by restriction digest and PCR amplification. To isolate plasmids carrying the mSF, the ligation mixture was first electroporated into E. coli DH10B. The resulting ApR colonies were pooled and plasmids were isolated from the pool and electroporated into P. aeruginosa to select for those that can replicate autonomously in P. aeruginosa. Plasmids were then isolated from P. aeruginosa to verify presence of the mSF via restriction digest and PCR amplification. 2.4. Construction of suicide plasmid pSS213 that carries a tightly regulated promoter system The lac promoter, Plac, in pSU38 (Martinez et al., 1988) was replaced with a tightly regulated promoter, PT7(A1/04/03), from pUHE21-2 (Lanzer and Bujard, 1998). Briefly, the unique NheI site, located upstream of the Plac in pSU38 was converted to a unique XhoI site by digesting with NheI, blunting the ends via fillin reaction with Klenow, and ligating a XhoI linker (NEB, Beverly, MA). Subsequent digest with EcoRI in the MCS of pSU38 removed Plac as a XhoI– EcoRI fragment. PT7(A1/04/03) from pUHE21-2 was then

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cloned as a XhoI –EcoRI fragment to replace Plac, while preserving the MCS of pSU38. Second, the lacIq gene, originally isolated as an EcoRI fragment from pMF54 (Franklin et al., 1994), was converted to a XhoI fragment, by blunting the ends with mung bean nuclease, and ligating the XhoI linker (NEB) to the blunted DNA fragment. Finally, the KmR carried on pSU38 was removed with NcoI and BglII, and the ends were converted to blunt ends with Klenow fragment via a fill-in reaction. Then aacCI (GmR), acquired as a SmaI fragment from pUCGM1 (Schweizer, 1993a), was cloned into the blunted former NcoI/BglII site to confer GmR to facilitate selection of the plasmid in P. aeruginosa. 2.5. Enzymatic assays and pyocyanin measurement Elastase activity was determined via the elastin congo-red hydrolysis assay using f 20– 30 Ag of culture supernatant proteins as previously described (Ohman et al., 1980). The elastase activity was calculated as increase in A495 min 1 g 1 of protein. h-galactosidase assay was performed according to Miller (1972). Pyocyanin production was determined as previously described (Suh et al., 1999).

3. Results and discussion 3.1. Construction of genetic cassettes carrying moriT, mSF, or non-polar TcR We constructed a series of genetic cassettes to facilitate use of the oriT mobilization site from plasmid RP4, the P. aeruginosa stabilizing fragment (SF) origin of replication, and the TcR selectable marker from pBR322 (Fig. 1). The corresponding sequences of interest were either directly PCR amplified as SacI fragments or PCR amplified first as blunt DNA fragments followed by ligation of SacI linkers with the sequence CGAGCTCG (NEB #S1044S, Beverly, MA). A high-fidelity thermostable DNA polymerase, Pfu polymerase, was used to minimize acquisition of mutations during the amplification. The resulting SacI DNA fragments were cloned into the unique SacI site of a pUC1918 with a palindromic MCS (Schweizer, 1993a). Thus, a sequence of interest can be isolated as a cassette with a single restriction enzyme digest using

any of the restriction enzymes present in the pUC19 MCS except for EcoRI (Fig. 1). The first cassette carries the oriT (origin of transfer) sequence of plasmid RP4 which is often used to introduce plasmids into P. aeruginosa at a high efficiency via conjugation. Introduction of DNA via conjugation is especially useful for manipulation of P. aeruginosa strains that are difficult to transform or electroporate. In addition, the labor that is involved in making the recipient to take up DNA by conjugation, as described in Materials and methods, is simple. Thus, multiple P. aeruginosa recipient strains can be prepared easily with a minimal expenditure of time and energy. A useful oriT cassette to make plasmids mobilizable is available as a 1.1-kb fragment, but it carries extraneous sequences, and is not always easy to manipulate (Selvaraj et al., 1984). To optimize the usefulness of the oriT sequence, we constructed a cassette that carries only a minimal sequence of approximately 230 nt that is sufficient to mediate transfer of plasmids (Pansegrau et al., 1988; Ziegelin et al., 1991). The 230 bp of minimal-oriT (moriT) sequence was originally PCR amplified as a DNA fragment that was flanked by BamHI sites. A SacI linker was then ligated to the DNA fragment, and the moriT was cloned as a SacI fragment into the unique SacI site of the pUC1918 derivative vector to result in pSS125. None of the restriction sites within the MCS is present in the moriT sequence and thus all of the MCS sites are available for isolating moriT. In addition, plasmids were generated that carry moriT as a unique EcoRI (pLS217) or a HindIII fragment (pLS214) in pUC18 and pUC19, respectively (Table 1). Table 1 List of plasmids carrying genetic tools described in this study Plasmid

Description

pLS214 pLS217 pSS87 pSS88 pSS124 pSS125 pSS213 pSS223

moriT as a HindIII fragment in pUC19 moriT as an EcoRI fragment in pUC18 mini-Tn5 lacZ1 GmV in pUT mini-Tn5 lacZ2 GmV in pUT mSF replicon cassette in pUC1918 moriT cassette in pUC1918 regulatable promoter/repressor system conjugative and suicidal lacZ transcriptional fusion plasmid conjugative and suicidal lacZ translational fusion plasmid TcR cassette in pUC1918

pSS231 pSS262

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The second cassette carries the P. aeruginosa Stabilizing Fragment (SF) which is a commonly used replicon for plasmid maintenance in P. aeruginosa. The SF is frequently found as a 1.9-kb PstI fragment and it has been incorporated into the pUCP series of E. coli – P. aeruginosa shuttle vectors and reporter fusion vectors (Schweizer, 1991; West et al., 1994). However, it is often desirable to clone the SF into an existing plasmid to make it replicate autonomously in P. aeruginosa. West et al. (1994) had showed that a 1.2-kb StuI– PstI fragment within the 1.9-kb PstI fragment was sufficient for the maintenance of plasmids in P. aeruginosa. Here, a cassette of SF was constructed that carried just the minimal SF (mSF) sequence. The 1.2-kb mSF sequence was PCR amplified as a DNA fragment flanked by a SphI site at each end. Then a SacI linker was ligated to the DNA fragment, and the resulting fragment was cloned into the SacI site of pUC1918, to give rise to pSS124 (Fig. 1). All of the restriction sites present in the pUC1918 MCS can be used to isolate mSF as a cassette, which permits conversion of any plasmid into a Pseudomonal replicon. Finally, we constructed a cassette carrying a transcriptionally non-polar TcR gene, which provides an excellent selectable marker in P. aeruginosa. The tet gene was PCR amplified from pBR322 as a SacI fragment and cloned into the SacI site of pUC1918 to form pSS262 (Fig. 1). The 3V-end of the tet gene included 13 nt downstream of the tet nonsense codon with no obvious transcriptional pausing or termination sites to avoid polar effects. Due to the presence of BamHI, SphI, HincII, and HindIII sites within the tet gene, the TcR cassette can be isolated as SacI, KpnI, SmaI, XbaI, or PstI fragments. This non-polar TcR cassette allows one to construct double non-polar mutations in two different operons when used in conjunction with the non-polar GmR cassette that had been previously described (Schweizer, 1993a). We have utilized all of these cassettes for various purposes but especially for mutant construction and complementation. For example, a gene of interest can be cloned into a Pseudomonal suicide vector (e.g., pUC19) and then a drug resistance cassette (either GmR or TcR) is inserted to make a knock-out allele of the gene. To efficiently introduce the mutant allele

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into P. aeruginosa via conjugation, the moriT is cloned into the plasmid carrying the mutant allele. Following mutant isolation and characterization, the mSF and moriT cassettes can be cloned into a plasmid that carries the wild-type allele, which can then be introduced into the P. aeruginosa mutant for complementation studies. Utilizing these simple procedures and genetic cassettes, we successfully isolated isogenic mutants of rpoS, vfr, gacA, algT, rhlR, rhlI, lasR, lasI, and lasB among many other genes in several different strains of P. aeruginosa to study their effects on the pathogenesis of this bacterium. 3.2. Construction of conjugative and suicidal lacZ fusion reporter vectors To facilitate study of gene expression using lacZ as a reporter in P. aeruginosa, we constructed conjugative and suicidal (i.e., integrative) lacZ transcriptional or translational fusion plasmids. Starting with the E. coli lacZ2 translational fusion plasmid pMLB1034 (Silhavy et al., 1984) which starts with codon 9 of lacZ, the moriT (HindIII fragment) was inserted as a blunt DNA fragment into the unique MscI site to generate the pSS231 (Fig. 2). This produced a conjugative lacZ translational fusion vector with the MCS (EcoRI – SmaI – BamHI) of pMLB1034 intact. To convert pSS231 into a lacZ transcriptional fusion vector, the 1.1-kb EcoRI – EcoRV fragment of pSS231 that contained the MCS and a portion of lacZ sequence was replaced with trp-lacZ1 on the 1.3-kb EcoRI – EcoRV fragment from pRS415 (Simons et al., 1987). This restored the MCS and produced pSS223 with the 5V-end of trp-lacZ found on pRS415 (Simons et al., 1987) which has the RBS and start codon of lacZ (Fig. 2). These lacZ reporter fusion plasmids cannot replicate in P. aeruginosa but can be incorporated into the genome via homologous recombination with P. aeruginosa DNA. Thus, by cloning a Pseudomonas promoter of interest to create the lacZ fusion on pSS223 or pSS231, and introducing the fusion plasmid into P. aeruginosa, one can isolate a merodiploid strain that carries both the promoter fusion as well as the wildtype copy of the gene. Incorporation of the lacZ fusion into the genome in single copy to study gene expression at a single-copy has several benefits when compared to utilizing reporter fusions on multi-copy plasmids. There is little danger of saturating out

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3.3. Modification of mini-Tn5 lacZ promoter probing transposons to gentamicin resistance The mini-Tn5 lacZ1 (transcriptional fusion) and mini-Tn5 lacZ2 (translational fusion) transposons described by De Lorenzo et al. (1990) were modified here to facilitate isolation of lacZ fusions that are locked-in and can no longer transpose. The original mini-Tn5 lacZs carry KmR for selection, to which P. aeruginosa unfortunately shows innate resistance. Thus, the Kmr cassette was removed from mini-Tn5 lacZ1 as a NotI fragment and replaced with the DNA fragment carrying the GmV cassette, forming pSS87 (Fig. 3). Briefly, NotI linkers were ligated to a GmV (SmaI fragment) from pUCGMV (Schweizer, 1993a) and cloned into the NotI site of mini-Tn5 lacZ. These modified transposons still reside on the original suicide plasmid vector plasmid pUT described by de Lorenzo et al. and they can be introduced to P. aeruginosa via conjugation. We are currently using the mini-Tn5 lacZ1 GmV construct carried on pSS87 to identify genes that are important for P. aeruginosa pathogenesis using an alfalfa seedling model of infection (Silo-Suh et al., 2002). 3.4. Construction of a regulated promoter/repressor system Fig. 2. Transcriptional and translational fusion plasmids. Plasmids pSS223 (f 6.7 kb) and pSS231 (f 6.5 kb) carry the trp-lacZ1 and lacZ2 fragments, respectively.

potential regulators of the gene, which can occur by having multiple copies of the promoter. In addition, plasmid copy number will not be affected by a mutation in a regulator. In addition, recombination of the promoter sequences at the native site in the genome yields a complete promoter for the fusion construct and so the expression of the geneDreporter fusion can be accurately measured, which is important if the complete promoter or regulatory region of the gene is unknown. Unless one clones the complete regulatory region of the gene, study of gene expression on multicopy plasmids or on a single-copy at a non-native site in the chromosome can result in artifactual or incomplete data. Similar to the integrating fusion plasmids described by Hoang et al. (2000), our lacZ fusion plasmid constructs offer a simple and valid alternative.

In order to study gene regulation in P. aeruginosa, especially by global regulators, we developed a tightly controlled promoter/repressor system. We first tested the well-known trc and ara promoters to control gene expression in P. aeruginosa. Unfortunately, while able to attain over-expression, a tight repression of the cloned gene in P. aeruginosa with those promoter

Fig. 3. MiniTn5 lacZ1 GmV. The plasmid shown carries the miniTn5 lacZ1 GmV for isolation of operon fusions. Not shown is the mini-Tn5 lacZ2 GmV for isolation of protein fusions.

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systems was not observed (Suh and Silo-Suh, unpublished results). Next, we tested pSS213, which carried GmR for selection, a p15A replicon for maintenance in E. coli, the MCS of pUC18 for cloning, the modified phage T7 early gene promoter, T7(A1/04/03) for control of gene expression, and the lacIq gene for repression of

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the T7(A1/04/03) promoter (Fig. 4). The T7(A1/04/03) promoter is reported to be repressed tightly because it contains two lac operator sites for binding of Lac repressor expressed by lacIq (Lanzer and Bujard, 1998), but addition of IPTG relieves repression and the gene of interest is expressed. In order to place the

Fig. 4. Construction of a tightly regulated promoter vector for P. aeruginosa. Plasmids are not drawn to scale. pSS213 retains all of the restriction sites of pUC18 MCS.

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Fig. 5. Placement of rhlR under the control of T7(A1/04/03) promoter in P. aeruginosa. Approximately 530 bp of the promoterless and 3Vtruncated rhlR was cloned into pSS213 to place rhlR expression under the control of T7(A1/04/03) promoter. Then the 230 bp moriT was cloned as a HindIII fragment to generate pSS222. To place rhlR under control of the T7(A1/04/03) promoter in P. aeruginosa, pSS222 was integrated into the PAO1 chromosome by homologous recombination of rhlR sequences.

gene of interest under control of the T7(A1/04/03) promoter, the promoterless 5V-end of the gene (e.g., rhlR’) is cloned into pSS213 (Fig. 5). When introduced into P. aeruginosa, recombination between homologous sequences in the genome and plasmid results in the generation of a merodiploid strain in which the native promoter for the gene transcribes a truncated and thus inactive copy of the gene, while the T7(A1/04/03) promoter transcribes the full length and thus active copy of the gene. To demonstrate efficacy of the T7(A1/04/03) promoter system in pSS213, we placed the quorum sensing gene regulator rhlR under control of the T7(A1/04/03) promoter (Fig. 5). After the rhlR’ fragment was cloned into pSS213, the moriT cassette was added to the HindIII site to allow mobilization into P. aeruginosa via conjugation. Following selection for GmR and thus recombination between rhlR’ and the native rhlR on the chromosome of PAO1, the merodiploid strain had rhlR expressed under control of the T7(A1/04/03) promoter. RhlR is a quorum sensing regulator that complexes with its cognate autoinducer for maximal production of multiple virulence factors in P. aeruginosa including elastase and pyocyanin (Brint and Ohman, 1995). In the absence of induction, expression

of rhlR under control of the T7(A1/04/03) promoter produced a phenotype that resembled that of a rhlR null mutant strain, as indicated by the lack of elastase and pyocyanin production. When 1 mM IPTG was added, rhlR under the T7(A1/04/03) promoter was expressed and the phenotype resembled that of the wild-type strain PAO1 (Table 2). Thus, the T7(A1/04/03) promoter system carried on pSS213 provides a tightly regulated promoter/repressor system for controlling gene expression in P. aeruginosa. Table 2 Expression of rhlR under the control of T7(A1/04/03) promoter in P. aeruginosa PAO1 derivatives Strain

PAO1

rhlR

T7-rhlR+

T7-rhlR+

IPTG Pyocyanina

0 0.1218 (100%)b 86.7 (100%)

0 0.006 (4.9%) 12.0 (13.8%)

0 0.0048 (3.9%) 19.9 (23%)

1mM 0.125 (103%) 102.5 (118%)

Elastasec

Strain derivatives of PAO1 used are SS165 (rhlR201DaacCI) and SS750 (T7-rhlR+). a Pyocyanin level was measured as A695. b Values in parenthesis represent relative activity = highest activity H activity  100 compared to PAO1. c Elastase activity = increase in A495 min 1 g 1 of protein.

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In this study, we presented several tools that facilitate genetic manipulation of the bacterial pathogen P. aeruginosa. Every tool described in this report is used routinely in our laboratory, from controlling gene expression to making mutants, in our pursuit of elucidating molecular mechanisms of environmental stress response and pathogenesis of P. aeruginosa.

Acknowledgements We thank Shannon Miller for sharing information about the minimal oriT sequence needed for plasmid transfer and Dr. Gail Christie for providing pUHE211. This research was supported by the Cystic Fibrosis Foundation Postdoctoral Fellowship (SUH96F0), NIH/NIAID Training Grant T32 AI-07617, and the funds from Auburn University awarded to S.-J.S.; and Public Health Service grant AI-19146 from the National Institute of Allergy and Infectious Disease, and Veterans Administrations Medical Research Funds awarded to D.E.O.

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