Pseudomonas aeruginosa AmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors

Abstract

In members of the family Enterobacteriaceae, ampC, which encodes a β-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR⁺ parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal β-lactamase levels. However, this increase was not followed by a concomitant increase in the P_ampC promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated β-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the β-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive P_ampR promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the P_lasA, P_lasI, and P_lasR expression. The reduction in the LasB activity was positively correlated with the P_rhlR expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

Pseudomonasaeruginosa is an important opportunistic pathogen causing infections in patients suffering from cystic fibrosis, chronic obstructive pulmonary disease, or severe burns and in patients in intensive care units. It poses an overwhelming threat to these individuals, leading to a high risk of morbidity and mortality. The current treatment against P. aeruginosa usually includes a combination of β-lactam antibiotics and aminoglycosides (20). However, resistance to these antibiotics is not uncommon, resulting in the failure of chemotherapy. P. aeruginosa also possesses various mechanisms to counteract antimicrobial agents. These include inducible AmpC β-lactamase (33), plasmid- and transposon-encoded acquired β-lactamases (12), multidrug efflux pumps (17), low outer membrane permeability (53), the ability to grow in biofilms (7), and hypermutability (41). The rapid emergence of resistance to β-lactam antibiotics in P. aeruginosa isolates from cystic fibrosis patients in the early 1990s has been attributed to the in vivo selection of constitutive β-lactamase-producing strains (6, 15, 16). Overproduction of β-lactamase in some of the P. aeruginosa isolates has been ascribed to mutations in ampD encoding a negative regulator of AmpC (2, 28), but genetic events underlying overproduction of AmpC β-lactamase still remain to be fully elucidated.

Many members of the Enterobacteriaceae family have genes that code for a chromosomal Ambler class C β-lactamase (38). The analysis of chromosomally mediated β-lactamases revealed that, in species possessing an ampR gene, synthesis of β-lactamase is inducible, whereas in strains without ampR, synthesis is constitutive (44, 50). The ampR genes in all the species studied thus far are located immediately upstream of ampC and are transcribed divergently (18). The short intercistronic region of Citrobacter freundii ampR-ampC accommodates two overlapping promoters (3, 31). The P. aeruginosa ampC and ampR genes have similar gene organization (32, 33). However, the mechanism of ampR regulation in P. aeruginosa is not yet clear.

To address the role of P. aeruginosa ampR, we generated an ampR mutant in the prototypic, wild-type PAO1 strain. Our analysis of PAO1 and PAOampR showed that a mutation in ampR results in a high constitutive expression of β-lactamases and that AmpR is required for AmpC induction. In addition, AmpR modulates expression of yet another chromosomally encoded β-lactamase, extracellular proteases, and pyocyanin. Subsequent analyses suggest that AmpR affects the virulence factors through quorum sensing.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media.

Bacterial strains, plasmids, and primers used in this study are shown in Table 1. Escherichia coli and P. aeruginosa were routinely cultured in Luria-Bertani medium (10 g tryptone, 5 g yeast extract, 5 g NaCl, per liter). Pseudomonas isolation agar (Difco) was used in triparental mating experiments. Antibiotics were used at the following concentrations (per milliliter) unless otherwise indicated: ampicillin at 50 μg, tetracycline at 20 μg, and gentamicin at 15 μg for E. coli and carbenicillin at 300 μg, gentamicin at 300 μg, and tetracycline at 60 μg for P. aeruginosa. For induction, 500 μg/ml of benzylpenicillin was used.

TABLE 1.

Bacterial strains, plasmids, and primers used in this study

Strain or plasmid	Genotype or properties	Reference or source
Escherichia coli
DH5α	F− φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK− mK+) phoA supE44 λ−thi-1 gyrA96 relA1	New England Biolabs
TOP10F′	F′ [lac1q Tn10(Tetr)] mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 araD139 D(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG	Invitrogen
Pseudomonas aeruginosa
PAO1	Wild type	21
PKM300	ampR::aacC1; Gmr	PAOampR; this study
PKM301	attB::PampC-lacZ; Tcr	PAO1 derivative; this study
PKM302	attB::PampR-lacZ; Tcr	PAO1 derivative; this study
PKM303	ampR::aacCI attB::PampC-lacZ; Gmr Tcr	PAOampR derivative; this study
PKM304	ampR::aacCI attB::PampR-lacZ; Gmr Tcr	PAOampR derivative; this study
PKM305	attB::promoterless lacZ; Tcr	PAO1 derivative; this study
PKM306	ampR::aacCI attB::promoterless lacZ; Gmr Tcr	PAOampR derivative; this study
Plasmids
pGEMEX-1	Apr; ColE1ori lacZα	Promega
pUCGm	Apr Gmr; pUC19 derivative containing gentamicin cassette (aacCI)	47
pEX100T	Apr; sacB oriT	46
pME6030	Tcr; oriVpVS1oriVp15AoriT	19
pRK2013	Kmr; ColE1 ori-Tra (RK2)+	11
Mini-CTX-lacZ	Tcr; integration-proficient vector for single-copy chromosomal lacZ fusion	4
pLP170	Apr; lacZ transcriptional fusion vector that contains an RNase III splice sequence positioned between the multiple cloning site and lacZ	43
pLPLA	Apr; pLP170 containing PlasA-lacZ transcriptional fusion	43
pLPLB	Apr; pLP170 containing PlasB-lacZ transcriptional fusion	43
pPCS1001	Apr; pLP170 containing PlasR-lacZ transcriptional fusion	42
pPCS1002	Apr; pLP170 containing PrhlR-lacZ transcriptional fusion	42
pLPR1	Apr; pLP170 containing PrhlI-lacZ transcriptional fusion	52
pPCS223	Apr; pLP170 containing PlasI-lacZ transcriptional fusion	52
pQF50	Apr; broad-host-range vector with promoterless lacZ	10
pSJ01	Apr; pGEMEX-1 with a 1,220-bp EcoRI-BamHI-flanked fragment containing ampR	This study
pSJ02	Kmr; pCRII-TOPO with a 2,150-bp fragment containing ampC	This study
pSJ05	Apr Gmr; pGEMEX derivative with ampR::aacCI	This study
pSJ06	Apr; pME6030 with a 1,220-bp EcoRI-BamHI-flanked fragment containing ampR	This study
pSJ07	Apr Gmr; pEX100T derivative with ampR::aacCI	This study
pSJ09	Apr; pGEMEX-1 with a 330-bp EcoRI-BamHI-flanked fragment containing ampC-ampR intergenic region	This study
pSJ10	Tcr; CTX-lacZ fused with ampC promoter, PampC	This study
pSJ11	Tcr; CTX-lacZ fused with ampR promoter, PampR	This study
pKKF0675	Kmr; pCRII-TOPO with 1,770-bp fragment containing poxAB	This study
pKKF0679	Kmr; pCRII-TOPO with DraI-deleted bla	This study
Primers
SBJ01ampRFora	5′-GGAATTCTGGCGAACAGCAGTGTGGAAGCGG-3′
SBJ02ampRReva	5′-CGGGATCCATTCCAATCACAACCCCAACGCC-3′
SBJ03ampCRFora	5′-GGAATTCTGAGGCCGCGCGGCAGACGCTTGAACA-3′
SBJ04ampCRReva	5′-CGGGATCCCATGAGGATTGGCGTCCTTTG-3′
SBJ05ampCFora	5′-GGAATTCTGAGGCCGCGCGGCAGACGCTTGAACA-3′
SBJ06ampCReva	5′-CGGGATCCAACCCCGGCGCGGTGGCCAGTCCCGCCAA-3′
KKF28poxAFor	5′-AGCTGCTTGCGCACCTGT-3′
KKF30poxARev	5′-CGGTTTCGAATTCACCGTCA-3′

^aThe italicized portion indicates a restriction site in a PCR product prepared with that primer.

DNA manipulations.

All molecular techniques were performed according to standard protocols (1).

Cloning of ampC and poxB genes.

Primers were designed to PCR amplify ampC and poxB. SBJ05ampCFor and SBJ06ampCRev were used to generate a 1,220-bp fragment with ampC, while KKF28poxBFor and KKF30poxBRev generated a 2,170-bp fragment containing poxB (Table 1). These PCR amplicons were electrophoresed and purified according to manufacturer's instructions (QIAGEN, California). The cleaned products were ligated into pCRII-TOPO and transformed into TOP10F′ (Invitrogen, California). Because we were concerned that the presence of the bla gene in the pCRII-TOPO vector might interfere with data interpretation, an internal 712-bp DraI fragment of the bla gene was deleted, and the resulting vector was transformed into E. coli DH5α. Selection was carried out using kanamycin, as this resistance marker was also present in pCRII-TOPO. The plasmids pSJ02 and pKKF675 harbored the ampC and poxB genes, respectively. The negative control, plasmid pKKF0679, contained a self-ligated pCRII-TOPO in which the bla gene was deleted (Table 1).

Insertional inactivation of the ampR gene.

A 1,220-bp ampR fragment was PCR amplified using SBJ01ampRFor and SBJ02ampRRev with flanking EcoRI and BamHI sites, respectively (Table 1). The PCR product was ligated to an EcoRI-BamHI-digested pGEMEX-1 (Promega, California), generating pSJ01 (Fig. 1). A gentamicin cassette, aacCI, was retrieved from pUCGm (47) and inserted into the PstI site of ampR (Fig. 1). This disrupted the reading frame of ampR in pSJ01. Subsequently, ampR::Gm was subcloned as an EcoRI-HindIII-cut, blunt-ended fragment into the SmaI site of pEX100T (48), which is a mobilizable suicide plasmid, creating pSJ07. This plasmid was conjugated into P. aeruginosa PAO1, with a helper strain harboring pRK2013 (11). The merodiploids, resulting from homologous recombination, were selected with pseudomonas isolation agar containing gentamicin. Gm^r colonies were then screened for gentamicin resistance and carbenicillin sensitivity by replica plating. The insertion was confirmed by PCR and restriction analysis on the PCR product (data not shown). The PAO1 isogenic strain with defective ampR, henceforth referred to as PAOampR, was named PKM300.

FIG. 1.

Physical map of ampR-ampC loci (A) and plasmids (B). (A) The restriction map of the region is based on the PAO1 genome sequence with relevant restriction sites. The 5′ end of ampC (labeled ampC') and the entire ampR open reading frames are indicated. The 1.2-kb fragment has the PAO1 genome coordinates 4594088 to 4592869. SBJ03 and SBJ04 show the position where the primers annealed for the promoter amplification. The striped box at positions 197 to 207 is where ligand-bound AmpR is postulated to bind. (B) The horizontal lines below the map show the amount of PAO1 genome DNA present in each plasmid. The restriction sites, EcoRI and BamHI, flanking the ends were introduced in the primers used for PCR (Table 1). Plasmids pSJ01 and pSJ06 are derivatives of pGEMEX (Promega) and pME6030 (19), respectively. The plasmid pSJ06 is used for complementation analysis and is referred to as pAmpR. The gentamicin cassette (▾) was inserted into the SalI site of pSJ01, creating pSJ05. The ampR::aacCI (ampR::Gm) fragment was subcloned into the suicide vector pEX100T (48) to generate pSJ07, which was introduced into PAO1 to generate PAOampR by homologous recombination. A 342-bp fragment containing the promoters was cloned in both directions, upstream of promoterless lacZ (indicated by the striped arrows) in mini-CTX-lacZ (4) to generate the transcriptional fusions for ampC (pSJ10) and ampR (pSJ11) promoters. These fusions were introduced into PAO1 and PAOampR by site-specific recombination using the attP site (4).

Construction of a plasmid with intact ampR for complementation.

Plasmid pSJ06 was generated by subcloning an EcoRI-BamHI-cut fragment from pSJ01 containing ampR into an EcoRI-BamHI-cut, broad-host-range, low-copy-number plasmid, pME6030 (19). This plasmid, pSJ06, also referred to as pAmpR, was used in all complementation analyses by conjugation into PAOampR.

Gene fusions with the lacZ gene.

A 330-bp fragment was PCR amplified with the flanking primers SBJ03ampCRFor and SBJ04ampCRRev (Table 1) and then cloned into pGEMEX-1, generating pSJ09 (Fig. 1). This fragment contained the ampC-ampR intergenic region with the putative promoters (33). To avoid PCR artifacts, pSJ09 was sequenced by using M13 primers. The fragment containing the promoters was subcloned as an EcoRI-BamHI fragment into an EcoRI-BamHI-cut, promoterless lacZ, mini-CTX-lacZ reporter plasmid (4), creating pSJ10 (P_ampC-lacZ) and pSJ11 (P_ampR-lacZ). This suicide vector contained the integration-proficient attP site, which recombines into the chromosomal attB site to generate a single-copy reporter fusion (4). The resulting clones were mobilized into PAO1 and PAOampR (Table 1). The presence of the chromosomal insertions was confirmed by PCR and restriction analysis of the product.

Pyocyanin quantitation assay.

The pyocyanin assay is based upon the absorbance of excreted pyocyanin at 520 nm in acidic solution (9). A 5-ml supernatant from a stationary-phase culture (∼16 h) in LB broth was mixed with 3 ml of chloroform. The pyocyanin from the chloroform phase was then extracted into 1 ml of 0.2 N HCl, giving it a pink to deep red color, indicating the presence of pyocyanin. The absorbance was measured at 520 nm. Concentrations, expressed as micrograms of pyocyanin produced per milliliter of culture supernatant, were determined by multiplying the optical density at 520 nm (OD₅₂₀) by 17.072 (9).

LasA staphylolytic assay.

LasA protease activity was measured by determining the ability of stationary-phase P. aeruginosa culture supernatants to lyse boiled Staphylococcus aureus (25). A 30-ml volume of an overnight S. aureus culture was boiled for 10 min and then centrifuged for 10 min at 10,000 × g. The resulting pellet was resuspended in 10 mM Na₂PO₄ (pH 4.5) to an OD₆₀₀ of approximately 0.8. A 100-μl aliquot of bacterial supernatant was added to 900 μl of S. aureus suspension, and the OD₆₀₀ was determined after 0, 5, 10, 15, 20, 30, 45, and 60 min (8, 25).

LasB elastolytic assay.

The elastolytic activity of bacterial suspension was determined with the elastin Congo red (ECR; Sigma, St. Louis, MO) assay (40). A 100-μl aliquot of bacterial supernatant of 16-h culture was added to 900 μl of ECR buffer (100 mM Tris, 1 mM CaCl₂, pH 7.5) containing 20 mg of ECR and then incubated with shaking at 37°C for 3 h. Insoluble ECR was removed by centrifugation, and the absorption of the supernatant was measured at 495 nm. LB medium was used as a negative control. Activity was expressed as change in OD₄₉₅ per μg protein.

β-Lactamase assay.

The assay of the P. aeruginosa chromosomal β-lactamase was modified based on previously published protocols (39). Briefly, stationary-phase cultures were diluted 1:100 into 10 ml of LB broth and incubated with shaking at 37°C until an OD₆₀₀ of 0.6 to 0.8 was attained. For induction (500 μg/ml benzylpenicillin), the cultures were incubated for an additional 3 hours before harvesting. The cells were washed once with 50 mM sodium phosphate buffer and were then resuspended to a final volume of 2 ml of the same buffer. Following disruption of cells on ice with sonication, the cell lysate was centrifuged at 10,000 × g for 30 min at 4°C and the supernatant was retained. A 2-μl portion of the β-lactamase-containing supernatant was added to 1 ml (final volume) of assay buffer containing nitrocefin (final concentration, 100 μM) at room temperature. The reaction was allowed to take place for 20 min before nitrocefin hydrolysis was measured spectrophotometrically at 482 nm. Concurrently, the total protein was determined with the Bradford assay using the same supernatant. One milliunit of β-lactamase is defined as 1 nanomole of nitrocefin hydrolyzed per minute per microgram of protein.

β-Galactosidase assay.

Assays for β-galactosidase in P. aeruginosa were performed as previously described (34).

Statistical analysis.

All data were analyzed with one-way analysis of variance using the statistical software package SPSS (Chicago, IL).

RESULTS AND DISCUSSION

Typically the presence of β-lactam antibiotics in a medium induces the synthesis of chromosome-encoded AmpC β-lactamase. The regulator of ampC, the divergently transcribed ampR gene product, has been identified in more than 10 species (18). P. aeruginosa AmpR has a high sequence identity with the AmpR from C. freundii and Enterobacter cloacae (58.3 and 62.5% identity, respectively) (33). Because the three species have the same ampR and ampC gene organization in the chromosome, it has been assumed that they also use the same regulatory mechanism. To date, the role of AmpR is restricted to the induction of ampC. The goal of this study was to examine the role of P. aeruginosa AmpR by generating an isogenic ampR mutant in the prototypic P. aeruginosa strain PAO1 (PAOampR).

P. aeruginosa AmpR does not autoregulate its own promoter.

AmpR is a DNA-binding protein that belongs to the LysR family of regulatory proteins (22, 29, 31). Many LysR-type transcriptional regulators act as transcriptional activators of the target genes but repress their own expression (45). In addition, the P. aeruginosa ampR-ampC intergenic region is only 159 bp long, leading to the suggestion that the AmpR expression may be autoregulated (32). To address this, we constructed strains with single copies of the ampR promoter upstream of a promoterless reporter gene, lacZ. The P_ampR-lacZ fusion was integrated into the PAO1 and the PAOampR chromosomes via attB-attP site-specific recombination, thus mimicking the chromosomal regulation (4). As a negative control, the promoterless lacZ gene was also integrated in both PAO1 and PAOampR. In the absence of the inducer, PAO1 showed a basal level of expression from the ampR promoter, which was not significantly affected by the presence of the inducer (Table 2). This finding concurs with previous C. freundii ampR studies showing that the amount of C. freundii AmpR expressed in E. coli minicells remains constant in the absence and presence of inducer (30), that the constitutive ampR expression is low with a short half-life of mRNA (22), and that the ampR transcription and the level of AmpR remain unaffected in the absence and presence of antibiotics (31). Expression of P_ampR-lacZ in the PAOampR mutant was not significantly different, suggesting that ampR does not regulate its own transcription (Table 2).

TABLE 2.

β-Galactosidase activities of ampC and ampR promoters

Strain	Relevant propertiesa	β-Galactosidase activityb
		Noninduced	Inducedc
PKM302	PAO1, PampR-lacZ	77.1 ± 8.7	123.0 ± 1.2
PKM304	PAOampR, PampR-lacZ	96.3 ± 15.2	106.0 ± 16.0
PKM301	PAO1, PampC-lacZ	124.1 ± 11.6	1,644.2 ± 33.7d
PKM303	PAOampR, PampC-lacZ	113.2 ± 7.5	122.3 ± 7.4e
PKM305	PAO1, promoterless lacZ	ND	ND
PKM306	PAOampR, promoterless lacZ	ND	ND

^aThe lacZ transcriptional reporter fusions were integrated into the attB site in the respective strains.

^bβ-Galactosidase activities were expressed in Miller units. ND, nondetectable.

^cInduction was carried out using 500 μg/ml of benzylpenicillin.

^dP value of <0.05 compared between noninduced and induced conditions within the strain.

^eP value of <0.05 compared between PAO1 and PAOampR under the same condition.

P. aeruginosa AmpR is a positive regulator of ampC expression.

Class C β-lactamase has long been implicated as the culprit in the cephalosporin resistance of P. aeruginosa (5). Based on previously published work in other systems (22, 30), we postulated that the mutation in ampR would increase the activity of the ampC promoter. To test this hypothesis, we generated a P_ampC-lacZ reporter and integrated it as a single-copy fusion into the PAO1 and the PAOampR chromosomes via attB-attP site-specific recombination, thus mimicking the chromosomal regulation (4). As a negative control, the promoterless lacZ gene was also integrated in both PAO1 and PAOampR. In PAO1, little activity was synthesized from the ampC promoter (Table 2). As expected, the presence of benzylpenicillin resulted in a significant elevation in the ampC promoter activity in PAO1 (Table 2). However, no induction of the ampC promoter was detectable in PAOampR in the absence or presence of inducer. The significant loss of transcriptional induction of the ampC promoter (Table 2) was likely due to the absence of AmpR, suggesting that AmpR is a positive regulator of ampC expression in the presence of an inducer. This is in agreement with previous studies of C. freundii and E. cloacae demonstrating that AmpR modulates ampC expression. The cloned ampC genes from C. freundii and E. cloacae are activated in E. coli only if accompanied by their ampR gene (22, 30).

AmpR exhibits differential regulation on AmpC and PoxB.

To ascertain the role of AmpR in the expression of β-lactamase, the activities in both PAO1 and the isogenic ampR mutant were compared. The parental PAO1 strain showed a low noninduced level of activity, which was increased 11-fold by addition of the β-lactam agent (Table 3). However, PAOampR expressed a high β-lactamase level, 12-fold higher than that of the parental strain in the absence of inducer (Table 3). No further induction was exhibited in the presence of a β-lactam agent. The inducible phenotype was restored in the PAOampR mutant by complementation with the plasmid pSJ06, harboring ampR.

TABLE 3.

Expression of β-lactamases in P. aeruginosa and E. coli

Strain and plasmid	Relevant property(ies)	β-Lactamase activitya
		Noninduced	Inducedb
P. aeruginosa
PAO1	Wild type	15.4 ± 7.8	162.2 ± 25.5c
PAOampR	ampR::aacC1	185.0 ± 8.9d	126.0 ± 46.0
PAOampR/pSJ06	ampR::aacC1/ampR+	13.0 ± 5.0e	169.1 ± 22.7c
E. coli
DH5α/pKKF0679		ND	ND
DH5α/pSJ06	ampR+	ND	ND
DH5α/pSJ02	ampC+	1.4 ± 0.0f	0.2 ± 0.1f
DH5α/pSJ02/pSJ06	ampC+ampR+	5.9 ± 0.5g	13.0 ± 1.6c,g
DH5α/pKKF0675	poxAB+	19.5 ± 0.6f	13.8 ± 2.1f
DH5α/pKKF0675/pSJ06	poxAB+ampR+	6.9 ± 0.8h	0.5 ± 0.2c,h

^aOne milliunit of β-lactamase is defined as 1 nanomole of nitrocefin hydrolyzed per minute per microgram of protein. ND, nondetectable.

^bInduction was carried out using 50 μg/ml and 500 μg/ml of benzylpenicillin for E. coli and P. aeruginosa, respectively.

^cP value of <0.05 compared between noninduced and induced.

^dP value of <0.05 compared between PAO1 and PAOampR.

^eP value of <0.05 compared between PAOampR and PAOampR/pSJ06.

^fP value of <0.05 in a comparison of DH5α/pKKF0679 with DH5α/pSJ02 and DH5α/pKKF0675.

^gP value of <0.05 compared between DH5α/pSJ02 and DH5α/pSJ02/pSJ06.

^hP value of <0.05 compared between DH5α/pKKF0675 and DH5α/pKKF0675/pSJ06.

However, based on the P_ampC-lacZ expression assay (Table 2), we expected the AmpC β-lactamase levels to remain the same in the absence of ampR (Table 3). This discrepancy in the biochemical and transcriptional analyses of ampC and the characterization of a P. aeruginosa ampC mutant led to the discovery of a novel chromosomally encoded class D β-lactamase, encoded by poxB (27). This class D β-lactamase was independently reported by Girlich et al., and the enzyme was named OXA-50 (13). However, our analysis of the sequence argues that PoxB is distinct from OXA enzymes (27). This discovery led us to propose that AmpR might also regulate poxB. The presence of more than one copy of β-lactamases in P. aeruginosa complicated the analysis of the effects of AmpR on each of these genes in a homologous host model. Therefore, we cloned ampC and poxB genes into E. coli and analyzed their activity in the absence and presence of the ampR gene.

In strain E. coli DH5α, AmpC expression was very low (Table 3). This is in contrast to what has been observed with C. freundii AmpC activity (30). In the absence of β-lactam antibiotic and C. freundii ampR, an increase in C. freundii AmpC β-lactamase activity in E. coli was observed, suggesting that C. freundii AmpR is a negative regulator (30). However, in our analysis, the presence of ampR is required for ampC expression.

Introduction of P. aeruginosa ampR into E. coli strain DH5α failed to induce the endogenous β-lactamase from the chromosomal ampC (Table 3). However, when P. aeruginosa ampR was introduced along with its cognate ampC into E. coli strain DH5α, the β-lactamase activity was significantly elevated both in the absence (fourfold) and in the presence (65-fold) of inducer (Table 3), further supporting the notion that ampR is a positive regulator required for ampC expression as well as induction.

In contrast to ampC, the activity of poxB was constitutively high in E. coli (Table 3). When AmpR was supplied in trans, poxB β-lactamase activity significantly dropped 65% and 96% in the absence and presence, respectively, of inducer (Table 3). These data suggest that AmpR suppresses the expression of PoxB.

It has been demonstrated that the increased resistance in clinical isolates of P. aeruginosa is also due to the derepression of β-lactamase (5, 14). Since the existence of PoxB was not known, and the biochemical assays used were not sensitive enough to distinguish these two β-lactamases, it was presumed that AmpC was overexpressed in the hyperproducing clinical isolates. A high constitutive expression of β-lactamase seen in PAOampR suggests that the clinical P. aeruginosa isolates that are hyperproducing β-lactamase may harbor poxB and a mutation in ampR. A Southern blot analysis of a few of these clinical P. aeruginosa isolates with ampR- and ampC-specific probes showed no gross changes in the ampR-ampC intergenic DNA (5). This led the authors to rule out any changes in ampR and ampC genes, although this technique is not sensitive enough to detect minor deletions or point mutations (5). In fact, one of these P. aeruginosa β-lactamase hyperproducers was shown to have a point mutation in ampR (2). It is not clear if this clinical isolate harbors the poxB gene, though we have demonstrated that poxB is fairly ubiquitous and is found in chromosomes of both clinical and environmental P. aeruginosa isolates (27).

Mutation in ampR affects pyocyanin production.

A change in virulence factor levels was first macroscopically observed when PAOampR produced a highly pigmented culture in mid-log phase, compared to the parent PAO1. Pyocyanin is a typical green pigment produced by P. aeruginosa that is involved in microbial competitiveness, the suppression of soilborne plant pathogens, and virulence in human and animal hosts (35). Two seven-gene phenazine biosynthetic operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are involved in pyocyanin production (35). Besides the presence of a LasR binding site upstream of the phzA1 operon (35), little is known about the regulation of these operons.

In the parental strain PAO1, pyocyanin production was relatively low and inducible on antibiotic challenge, albeit insignificantly (Table 4). However, pyocyanin production in the ampR mutant was significantly higher in the absence and presence of the β-lactam agent (Table 4). The high constitutive pyocyanin level was reduced by ampR in trans from the low-copy-number plasmid (Table 4). This suggests that AmpR negatively regulates pyocyanin production. It remains to be clarified if AmpR regulates one or both of these phz operons.

TABLE 4.

Effect of ampR mutation on pyocyanin production and LasA staphylolytic and LasB elastase activity

Strain	Pyocyanin productiona		LasA activityb		Elastase activityc
	Noninduced	Inducedd	Noninduced	Inducedd	Noninduced	Inducedd
PAO1	0.3 ± 0.2	2.3 ± 0.2	0.310 ± 0.065	0.317 ± 0.059	41.7 ± 1.1	105.0 ± 24.0
PAOampR	2.9 ± 0.8f	3.3 ± 0.6	1.109 ± 0.099f	0.951 ± 0.045f	32.8 ± 2.3	30.7 ± 1.7f
PAOampR/pSJ06e	0.1 ± 0.0g	1.4 ± 0.3	0.188 ± 0.055g	0.186 ± 0.019g	56.5 ± 1.5	90.5 ± 8.4g

^aPyocyanin concentrations were expressed as micrograms of pyocyanin produced per microgram of total protein.

^bLasA activity was expressed as change in OD₆₀₀ per hour per microgram of protein.

^cElastase activity was expressed as change in OD₄₉₅ per microgram of protein.

^dInduction was carried out using 500 μg/ml of benzylpenicillin.

^eThe plasmid pSJ06 is a derivative of low-copy-number plasmid pME6030 containing ampR.

^fP value of <0.05 compared between PAO1 and PAOampR.

^gP value of <0.05 compared between PAOampR and PAOampR/pSJ06.

AmpR regulates staphylolytic protease LasA and LasB elastase.

P. aeruginosa strains that overproduce pyocyanin are also known to produce other extracellular virulence factors. P. aeruginosa secretes a number of proteases, which are believed to play a major role in pathogenesis, especially during acute infections (26, 37). We determined that the mutation in ampR also affects the expression of two proteases, the LasB elastase and the LasA staphylolytic protease.

LasA staphylolytic protease is a 20-kDa zinc metalloendopeptidase belonging to the β-lytic endopeptidase family of proteases (24). The PAOampR supernatant lysed the Staphylococcus aureus cells at a significantly higher rate than PAO1 (Table 4), indicating an increased production of LasA protease in the absence of ampR. This phenotype can also be rescued by ampR expressed in trans. This suggests that AmpR negatively regulates LasA protease production. Since AmpR is a homolog of LysR, the differential expression of LasA in PAOampR could be a result of the effect of AmpR on the transcription of lasA. To address this, we introduced plasmid pLPA containing P_lasA-lacZ (43) into PAO1 and PAOampR (Table 5). In PAOampR, the P_lasA expression was significantly increased compared to PAO1, suggesting that the increased transcription may contribute to a higher production of LasA staphylolytic protease. As expected, the induction was not influenced by the presence of inducer. It is interesting that the lasA promoter is significantly up-regulated in the presence of inducer.

TABLE 5.

Transcriptional activity of lasA, lasB, lasI, lasR, rhlI, and rhlR promoters

Promoter fusiona	β-Galactosidase (Miller units)
	Noninduced		Inducedb
	PAO1	PAOampR	PAO1	PAOampR
lasA	790.0 ± 128.9	1,970.5 ± 312.6c	1,364.7 ± 67.0d	2,842.0 ± 286.4d
lasB	709.3 ± 43.7	676.0 ± 75.1	773.0 ± 112.0	650.5 ± 67.3
lasR	2081.3 ± 63.2	6,904.0 ± 256.5c	783.4 ± 136.9d	6,805.8 ± 173.6c
lasI	7,488.5 ± 193.8	12,985.3 ± 209.7c	8,168.7 ± 405.2	12,413.7 ± 179.1c
rhlR	9,188.8 ± 475.6	6,259.5 ± 26.1c	10,375.5 ± 563.6	8,711.5 ± 301.6c
rhlI	7,880.9 ± 784.1	9,624.4 ± 509.7	7,316.2 ± 796.4	9,245.8 ± 550.2
Vector	353.0 ± 33.7	316.7 ± 37.0	327.0 ± 24.8	327.7 ± 20.5

^aThe promoter-lacZ fusions are pLP170 derivatives as described in Table 1.

^bInduction was carried out using 500 μg/ml of benzylpenicillin.

^cP value of <0.05 compared between PAO1 and PAOampR under the same condition.

^dP value of <0.05 compared between noninduced and induced conditions within the same strain.

Elastase, a zinc metalloprotease encoded by lasB, is capable of inactivating a wide range of biological tissues and immunological agents (43). In the absence of inducer, there is a twofold induction of elastase activity in PAO1. However, in the presence of inducer, the ampR mutation failed to induce the production of LasB elastase. This induction could be restored with ampR expressed in trans (Table 4). This suggests that AmpR positively regulates LasB activity in the presence of inducer. To determine if the effect of AmpR is on the transcription of the lasB gene, we introduced plasmid pLPB, containing the P_lasB-lacZ fusion (43), into PAO1 and PAOampR. The P_lasB expression did not differ significantly between PAO1 and PAOampR (Table 5). This suggests that AmpR does not affect P_lasB directly and that the noninducibility of LasB protease activity is likely due to an indirect effect of AmpR.

Loss of ampR affects the expression of quorum sensing genes.

The regulation of P. aeruginosa lasA and lasB genes has been explored extensively. Both of these genes are regulated by the las and rhl quorum sensing systems (49). We wanted to determine if AmpR affects the production of protease indirectly via the quorum sensing systems. To address this, plasmids containing P_lasI-lacZ, P_lasR-lacZ, P_rhlI-lacZ, and P_rhlR-lacZ transcriptional fusions were introduced into PAO1 and PAOampR (42). The analyses of these fusions in PAOampR(pAmpR) were not investigated. However, based on all the analyses above we predict that pAmpR will restore the wild-type phenotype in the mutants.

In P. aeruginosa, there is a direct association between the production of LasA protease and the LasI-LasR quoromone system (43, 51). Since LasR positively regulates LasA expression, we postulated that the higher production of LasA protease in the PAOampR strain may also be a direct result of the higher transcription of lasI and lasR genes (Table 5). In fact, there was a significant increase in the expression of the las genes, suggesting that AmpR is also a negative regulator of lasI and lasR, adding yet another layer of complexity to the regulation of these genes (49). Similar elevation was seen in PAOampR with P_lasI-lacZ and P_lasR-lacZ fusions in the presence of the inducer (Table 5). This suggests that AmpR negatively regulates lasI and lasR transcription. In addition, the presence of inducer in PAO1 resulted in a significant (P < 0.05) decrease in P_lasR-lacZ activity. This loss of activity was recovered in the absence of ampR, which further supports the idea that AmpR is a negative regulator of lasR expression.

The rhl quorum sensing is also known to regulate the transcription of lasA and lasB genes. There was no significant change in the P_rhlI-lacZ fusion between the PAO1 and PAOampR strains (Table 5). However, in PAOampR, we saw a significant reduction of rhlR transcription compared to that in PAO1 either in the absence or in the presence of inducer (Table 5), suggesting that AmpR is a positive regulator of rhlR expression.

Though the LasB elastase was slightly reduced in the ampR mutant (Table 4), there was no concomitant decrease in the expression of the lasB promoter in PAOampR (Table 5). The expression of LasB is more complicated. It involves the coordinate regulation of three quorum sensing systems, including lasR-lasI, rhlR-rhlI, and Pseudomonas quinolone signal (PQS) (36). Thus, AmpR may act indirectly via the rhlR-rhlI and Pseudomonas quinolone signal (PQS) quorum sensing systems. It appears that the reduction of LasB activity seen in PAOampR is directly correlated with the decrease of rhlR expression. These results suggest that AmpR positively regulates rhlR expression and LasB activity.

Concluding remarks. (i) P. aeruginosa AmpR is a global regulator.

E. coli naturally lacks ampR, and its native ampC gene is not inducible by β-lactam antibiotics (22). Thus, most of the studies address the role of ampR in E. coli using heterologous systems in a multicopy plasmid background. The role of AmpR as a transcriptional activator of β-lactamase production, and also as a global regulator of other virulence factors, was a serendipitous conjecture drawn from the direct observation of PAOampR on LB plates and liquid culture that appeared to synthesize higher levels of pyocyanin. In fact, our studies comparing PAO1 with its isogenic ampR mutant revealed that AmpR positively regulates the expression of ampC and rhlR and the activities of AmpC β-lactamase and LasB. In addition AmpR negatively regulates the expression of poxB, lasA, lasI, and lasR and the production of pyocyanin, PoxB β-lactamase, and LasA. In fact this is typical of LysR-type transcriptional regulators that are known for activating divergent transcription of linked target genes or unlinked regulons encoding extremely diverse functions (45). However, this is the first report linking AmpR to many diverse functions.

(ii) Mechanism of AmpR action.

As in other members of the LysR family of regulatory proteins, the DNA-binding motif is found in the N terminus of the AmpR protein (22, 29, 31). The binding of P. aeruginosa AmpR to the P. aeruginosa ampC promoter was suggested by the ability of P. aeruginosa AmpR in the E. coli extract to immobilize the ampC promoter-containing DNA fragment in a gel shift assay (33). The LysR-type proteins generally bind to inverted repeat motifs, including the T-N(11)-A motif (45). However, the binding motifs of C. freundii and E. cloacae AmpR have been determined in the ampR-ampC intergenic regions (31). Using this consensus sequence (5′-TCTGCTGCTAAATTT), we found a strong conserved sequence (5′-TCTGCTCCAAATTT-3′) within the ampR-ampC intergenic region of PAO1.

Since AmpR appears to regulate also other genes, we analyzed the intergenic regions of all the AmpR-regulated genes for the presence of its binding site. Since the transcription start sites for many of these genes have not been determined, we searched for a potential AmpR binding sequence 200 bp immediately upstream from their open reading frames, both manually and using computer software such as AlignACE (23). No AmpR binding site was found upstream of phzA1, rhlI, and rhlR. However, the putative sites in other genes are relatively weak. A detailed promoter analysis, followed by footprinting with purified protein, is necessary to further elucidate the significance of these sequences and the mechanism of regulation.

Although much is known about the ubiquity of ampC-ampR, little is understood about the regulation mechanism of β-lactamase. The study presented here provides new insights into the role of P. aeruginosa AmpR as a global regulator involved in antibiotic resistance and the expression of many virulence factors. The combination of increased resistance to antibiotics and hyperproduction of pyocyanin and LasA protease would provide a mechanism of protection and survival in the harsh lung environment.