AmrZ Modulates Pseudomonas aeruginosa Biofilm Architecture byDirectly Repressing Transcription of the psl Operon

Christopher J. Jones,a,b,c Cynthia R. Ryder,c,e Ethan E. Mann,a,c Daniel J. Wozniaka,b,c,d

Department of Microbial Infection and Immunity,a Integrated Biomedical Sciences Graduate Program,b Center for Microbial Interface Biology,c and Department ofMicrobiology,d Ohio State University, Columbus, Ohio, USA; Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NorthCarolina, USAe

Pseudomonas aeruginosa strains recovered from chronic pulmonary infections in cystic fibrosis patients are frequently mucoid.Such strains express elevated levels of alginate but reduced levels of the aggregative polysaccharide Psl; however, the mechanisticbasis for this regulation is not completely understood. Elevated pslA expression was observed in an amrZ null mutant and instrains expressing a DNA-binding-deficient AmrZ. AmrZ is a transcription factor that positively regulates twitching motility andalginate synthesis, two phenotypes involved in P. aeruginosa biofilm development. AmrZ bound directly to the pslA promoter invitro, and molecular analyses indicate that AmrZ represses psl expression by binding to a site overlapping the promoter. Alteredexpression of amrZ in nonmucoid strains impacted biofilm structure and architecture, as structured microcolonies were ob-served with low AmrZ production and flat biofilms with amrZ overexpression. These biofilm phenotypes correlated with Psllevels, since we observed elevated Psl production in amrZ mutants and lower Psl production in amrZ-overexpressing strains.These observations support the hypothesis that AmrZ is a multifunctional regulator mediating transition of P. aeruginosa bio-film infections from colonizing to chronic biofilms through repression of the psl operon while activating the algD operon.

Pseudomonas aeruginosa is a Gram-negative opportunisticpathogen that is a major burden on the health care industry.

Up to 10% of nosocomial infections are attributed to P. aerugi-nosa, with mortality rates approaching 40% in patients with P.aeruginosa bacteremia (1, 2). This bacterium is often a causativeagent of sepsis and of acute and chronic infections of burnwounds, skin, and patients harboring medical devices such ascatheters. Patients with the genetic disease cystic fibrosis (CF) areparticularly susceptible to chronic P. aeruginosa infections thatcan last decades (2, 3), leading to decreased lung function (4–6).The persistence of P. aeruginosa is enhanced by the formation ofbiofilms, which can be composed of the exopolysaccharides Psl,Pel, and alginate (7–13).

Overproduction of alginate, termed mucoidy, is a major viru-lence factor in P. aeruginosa CF infections (4, 8, 14). Alginateoverproduction is most often caused by a mutation of the anti-sigma factor-encoding gene mucA. In nonmucoid P. aeruginosa,MucA sequesters the alternative sigma factor AlgT (also calledAlgU) to the membrane, preventing it from activating transcrip-tion of its regulon, including the alginate operon (15, 16). Consti-tutively unsequestered AlgT leads to activation of the alginate bio-synthetic pathway ultimately aiding biofilm persistence, antibioticresistance, evasion of phagocytosis, and scavenging of toxic oxy-gen radicals (4, 8, 17). The mucoid phenotype is dependent onincreased local cyclic-di-GMP (ci-di-GMP) produced by MucR,indicating that mucoid conversion is interconnected with manyother phenotypes through second messengers (18). The ribbon-helix-helix transcription factor AmrZ is a member of the AlgTregulon and activates alginate production (19) and twitching mo-tility (20) while downregulating genes essential for the productionof flagella (21). The impact AmrZ has on biofilms as a result ofthese diverse regulatory functions has not been previously re-ported.

In many nonmucoid P. aeruginosa strains, biofilm formationrelies on the exopolysaccharide Psl, which promotes adherence

and cell-cell interactions (11, 13, 22, 23). Colonization of suscep-tible tissue, such as the airway in the lungs of a CF patient, likelyrequires aggregative components conferring the abilities to initi-ate biofilms and to endure initial insults by the host immune sys-tem. It stands to reason that Psl also plays an important role dur-ing this process based on its adherence and aggregation functions.Moreover, recent studies indicate potential roles for Psl in pro-moting immune evasion (24), and passive immunization studiesshow that antibodies generated against Psl can provide protectionagainst P. aeruginosa infection (25).

Since nonmucoid P. aeruginosa bacteria are proposed to ini-tially infect the lungs of CF patients (5) and Psl enhances cellaggregation and adherence (11, 13, 22, 23), Psl is likely involved inprimary biofilm-related colonization (11, 17, 26). While somestudies have provided insights into expression and regulation ofPsl in mucoid P. aeruginosa strains (23, 27), there are few reportsdescribing transcriptional control. Our work shows that once mu-coid conversion occurs, the AlgT-responsive transcription factorAmrZ directly represses transcription of the psl operon and acti-vates alginate operon expression. Collectively, this work revealsthe mechanism of inverse control of alginate and Psl in P. aerugi-nosa biofilm infections.

Received 3 December 2012 Accepted 18 January 2013

Published ahead of print 25 January 2013

Address correspondence to Daniel J. Wozniak, daniel.wozniak@osumc.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.02190-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.02190-12

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MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used alongwith their genotypes are provided in Table S1 in the supplemental mate-rial. Strains were inoculated in LBNS (10 g tryptone/liter, 5 g yeast extract/liter [pH 7.5]) at 37°C overnight under shaking conditions unless other-wise noted. Strains were grown at 37°C on LANS (LBNS with 1.5% agar)or Pseudomonas isolation agar (Difco, Detroit, MI) agar plates to deter-mine mucoidy.

Immunoblots and ELISAs. Immunoblotting and enzyme-linked im-munosorbent assays (ELISAs) were performed as previously described(28). Briefly, Psl was extracted from 1.0 ml of a culture with an opticaldensity at 600 nm (OD600) of 1.0 (1.0 OD600 culture) to normalize for cellnumber. Results are reported as micrograms of Psl per 1.0 OD600 culture.Psl was extracted from the cell pellet in boiling 0.5 M EDTA, and the Pslprotein was treated with proteinase K. The samples were diluted 1:10 inTBST (Tris-buffered saline with Tween 20) (20 mM Tris, 137 mM NaCl,0.1% Tween 20 [pH 7.6]) and spotted onto nitrocellulose membranes forimmunoblotting. Immunoblots were blocked in 10% skim milk and thenincubated with Psl-specific rabbit antibody (1:25,000 in phosphate-buff-ered saline [PBS]). After the immunoblot was washed, the secondary an-tibody (donkey anti-rabbit antibody; GE Healthcare) was added at a1:10,000 dilution. After a last wash, the blot was treated with SuperSignalWest Dura extended-duration substrate per the manufacturer’s instruc-tion (Pierce). Blots were visualized using the Bio-Rad Chemidoc system.

ELISA plates were coated with the 1:100 dilution of sample and astandard curve from 50 to 0.05 �g/ml of purified Psl overnight at 4°C. Theplates were blocked with PBS plus 10% newborn calf serum and thenwashed in PBST (PBS plus 0.05% Tween 20). The plates were incubatedwith Psl-specific rabbit antibody (1:25,000 in PBS). After the plates werewashed, donkey anti-rabbit secondary antibody (1:10,000 in PBS) wasadded, and then the plates were washed again. 3,3=,5,5=-Tetramethyl-benzidine (TMB) was added to each well at room temperature (RT) for 30min, and then 50 �l of 2 N H2SO4 was added to each well. The plates werethen read at 450 nm with a Molecular Devices SpectraMax plate reader. AllELISAs were performed in triplicate.

Carbazole assay. Alginate was purified as previously described (29).One milliliter of a 1.0 OD600 culture was centrifuged (12,000 � g for 30min) to pellet cells and normalize polysaccharide extraction to cell num-ber. Supernatants were treated with 2% cetyl pyridinium chloride, andprecipitated alginate was collected by centrifugation. Alginate was resus-pended in 1 M NaCl, precipitated with cold isopropanol, and resuspendedin saline. Alginate quantification was determined via the carbazole assay(30) adapted to a 96-well format (31). Purified alginate was heated to100°C for 15 min with borate-sulfuric acid reagent (10 mM H3BO3 inconcentrated H2SO4). Carbazole (0.1%) was added and heated to 100°Cfor 10 min. Absorbance was determined at 550 nm on a Molecular DevicesSpectraMax plate reader.

Western blot. Western blots for AmrZ were performed as previouslydescribed (32). Strains were grown as described above to an OD600 of 1.0.Cells were pelleted and boiled for 5 min, then cleared via centrifugation.Supernatants were resolved on a 12% SDS-polyacrylamide gel and thentransferred to a nitrocellulose membrane in a semidry manner. Blots wereprobed with AmrZ-specific serum and horseradish peroxidase (HRP)-conjugated secondary antibody and then examined for chemilumines-cence detection using a Bio-Rad GelDoc XR� system. The indicated load-ing control is a nonspecific cross-reacting band visible at approximately37 kDa.

EMSA. Wild-type and R22A-binding-deficient AmrZ proteins werepurified as described previously (33). PCR amplification of the desired pslpromoter regions or annealing of complementary oligonucleotides wasused to produce DNA fragments. DNA binding was performed as previ-ously published (33). Unless otherwise noted, 5 ng of protein was com-bined with 150 fmol of DNA fragment in electrophoretic mobility shiftassay (EMSA) binding buffer (20 mM Tris-HCl [pH 8.0], 200 mM NaCl,20 mM MgCl2, 20% glycerol) and 750 ng poly(dI-dC) (Sigma) and al-

lowed to bind for 10 min at room temperature. The samples were resolvedon a 4% polyacrylamide native gel. Gels in Fig. 4A and B utilized 5= 6-car-boxyfluorescein (FAM)-labeled probes, while the gel in Fig. 4C wasstained using SYBR green EMSA stain (catalog no. E33075; Invitrogen) bythe protocol provided by the manufacturer and imaged on a Typhoonfluorescent scanner with 488-nm excitation and high sensitivity.

Quantitative reverse transcription real-time PCR. RNA isolationwas performed from log-phase cells grown to an OD600 of 1.0 by using theRNeasy Plus minikit (Qiagen), and then cDNA was synthesized with Su-perscript III (Invitrogen) using the supplied protocols. Real-time PCRwas performed using a Bio-Rad CFX1000 thermal cycler and Bio-RadCFX96 real-time imager with primer pairs in Table S2 in the supplementalmaterial and iQ SYBR green supermix (Bio-Rad). Results are from threeindependent experiments performed in triplicate. All samples were nor-malized to the expression of the housekeeping gene rpoD via the Pfafflmethod (34).

Flow cell biofilm study. Inoculation of flow cells was done by normal-izing overnight cultures to an optical density of 0.5 and injecting the cellsinto an Ibidi �-Slide VI 0.4 (catalog no. 80601; Ibidi). To seed the flow cellsurface, the flow of medium was suspended and the bacteria were allowedto adhere at room temperature for 3 h. The flow of 5% (vol/vol) LBNSwith 0.5% arabinose was initiated at a rate of 0.15 ml · min�1 and contin-ued for 24 h. Following the biofilm growth period, the flow was termi-nated, and the biofilms were fixed with 4% paraformaldehyde. The sam-ples were blocked overnight at 4°C in PBF (PBS with 10% heat-inactivatedfetal bovine serum and 0.5% bovine serum albumin). Polyclonal anti-Pslantibody was diluted in PBF (1:20,000) and added to the flow cell for staticincubation for 30 min at room temperature. The samples were washedthree times in 200 �l of PBF and then incubated for 30 min with AlexaFluor 647-labeled goat anti-rabbit IgG (catalog no. A21244; Invitrogen)and Syto9 (catalog no. S34854; Invitrogen) diluted in PBF to a concentra-tion of 0.2 �g/ml and 2.5 nM, respectively. After three washes in 200 �l ofPBF, confocal images were taken at the Ohio State University CampusMicroscopy and Imaging Facility on an Olympus Fluoview 1000 laserscanning confocal microscope. Images were obtained with a 20� oil im-mersion objective and processed using the Olympus FV1000 Viewer soft-ware. Quantitative analyses were performed using the COMSTAT soft-ware package (35).

Rapid-attachment assay. Rapid-attachment assays were performed aspreviously described (36) with the following modifications. One hundredmicroliters of culture (OD600 of 0.5) was added to 96-well polyvinyl chlo-ride (PVC) microtiter dishes and incubated at 25°C for 1 h statically. Theplates were then washed vigorously by immersion in water. Biomass wasstained with 0.1% crystal violet for 30 min and then vigorously washed.The remaining crystal violet was solubilized in 100 �l of 95% ethanol for30 min and transferred to a fresh 96-well plate. Absorbance was deter-mined at 540 nm.

RESULTS AND DISCUSSIONMucoid strains express reduced levels of Psl compared with iso-genic nonmucoid pairs. To determine the levels of Psl producedby mucoid P. aeruginosa, we utilized the Psl antibody previouslydescribed (28) to perform immunoblotting and ELISAs on twoisogenic mucoid/nonmucoid strain pairs. Two parental lineageswere tested: PAO1, a clinical nonmucoid isolate, and FRD1, aclinical mucoid isolate derived from a CF patient. Mucoid andnonmucoid isogenic strains were also tested (see Table S1 in thesupplemental material). Introduction of a mucA22 mutation in P.aeruginosa PAO1 results in alginate overproduction (15), whiledeletion of the alternative sigma factor algT in the mucoid clinicalisolate FRD1 yields a nonmucoid phenotype (37). Both the ELISAand immunoblot show that the FRD1 and PAO1 mucA22 mucoidstrains produce less Psl than their respective nonmucoid variants,FRD1 �algT mutant and PAO1, respectively (Fig. 1). Concomi-

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tantly, alginate assays of these strains confirm that mucoid strainsproduce elevated alginate levels compared to their isogenic non-mucoid pairs (Fig. 1). In support of prior studies (23, 27), thesedata show that Psl and alginate are inversely produced, suggestingsome mechanism of common regulation.

The inverse regulation of algD and pslA operons is mediatedby AmrZ. To determine the factors involved in repression of psl inmucoid cells, we first considered transcriptional regulators knownto be highly expressed in mucoid strains; AlgT, AlgB, AlgR, andAmrZ (Fig. 2) (19, 21, 32, 38, 39). Recent work suggested that lowPsl expression in mucoid strains is AlgT dependent but did notidentify the mechanism of repression (23). We reasoned that ifone of these AlgT-dependent regulators was involved in directly orindirectly repressing psl expression, mutations should alleviatethis repression, resulting in increased Psl production. Psl produc-tion was evaluated from FRD1-derived strains possessing muta-

tions in each of these regulators. Both the algT and amrZ mutantstrains produced greater Psl than the parental FRD1 strain did(Fig. 2). Since deletion of algT also results in loss of AmrZ, thisindicates that AmrZ is likely a regulator of psl expression. Psl pro-duction was also greater in a strain containing an AmrZ R22Asubstitution (Fig. 2). The R22 residue is required for maximalAmrZ DNA binding activity (20, 40); thus, the increase in Psl inthis strain suggests that DNA binding by AmrZ is required for pslrepression. Importantly, we also observed that the level of Psl doesnot increase when algD is mutated, indicating that the reductionof Psl in mucoid P. aeruginosa is not a result of interference ofalginate in the immunoassays. Mutating algD does not affect theupstream regulatory machinery but merely stops the cell fromgenerating GDP-mannuronic acid, a precursor in the alginatepathway (14). Additionally, the low production of Psl in the algDmutant supports the hypothesis that transcriptional regulation ofthe psl operon is an important mechanism of inverse regulation ofPsl and alginate, while not excluding the possibility of modulationof sugar-nucleotide precursors as an additional mechanism. Thisis documented further in Fig. 3 (see below).

AmrZ exhibits transcriptional control of the algD and pslAoperons. The inverse transcriptional regulation of algD and pslA isdemonstrated using quantitative reverse transcription real-timePCR (Fig. 3). Western blots indicate that the mucoid strains,PAO1 mucA22 and FRD1 strains, express elevated amounts ofAmrZ compared to their nonmucoid counterparts (Fig. 3A). Withthe exception of the algD mutant, reciprocal levels of algD andpslA transcription were observed in each strain tested, supportingthe hypothesis that AmrZ activates transcription of algD whilerepressing that of pslA. Additionally, pslA expression is elevated inthe �algT, �amrZ, and R22A amrZ strains, implicating AmrZ asthe regulator responsible for the AlgT-dependent repression of psloperon expression. There are several differences between the algDand pslA promoters, which may account for the different magni-tude of the responses. First, AmrZ is an activator of algD (19) anda repressor of pslA. Second, both the binding affinity and distancein which AmrZ binds relative to the algD and pslA promotersdiffer (40), and this may account for differences in transcriptionalactivity. Finally, in mucoid cells, the algD promoter activity is

FIG 1 Inverse levels of alginate and Psl in mucoid and nonmucoid strains. Polysaccharides were extracted from 1.0 ml of a 1.0 OD600 culture to normalize forcell number. Results are reported as micrograms of target polysaccharide per milliliter of OD600 culture � standard deviation. Alginate levels were determinedvia a carbazole assay (see Materials and Methods). Psl concentrations were determined via an ELISA and visualized with an immunoblot. Concentrations are theaverage of four independent assays performed in triplicate. P. aeruginosa PAO1 and FRD1 �algT strains are nonmucoid; PAO1 mucA22 and FRD1 strains aretheir respective isogenic mucoid counterpart. Comparisons of polysaccharide production were determined via Student’s t test. �-Psl, anti-Psl antibody.

FIG 2 Deletion of algT and amrZ leads to an increase of Psl in P. aeruginosaFRD1. Psl was extracted from 1.0 ml of a 1.0 OD600 culture to normalize for cellnumber. Results are reported as �g of target polysaccharide per ml of OD600

culture. All strains are derivatives of the mucoid clinical isolate FRD1. Pslconcentrations are the averages of four independent assays performed in trip-licate, with the standard deviations represented by error bars. A one-way anal-ysis of variance (ANOVA) with Tukey post hoc test was used to determinestatistical significance. All values are compared to the values for FRD1 (��, P �0.01; ���, P � 0.001).

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affected by the binding of other regulators in addition to AmrZ(19). These regulators enhance algD transcription but do not ap-pear to act at the pslA promoter. It should also be noted that algDtranscription was detected in the algD mutant because the reversetranscription real-time PCR primers chosen for algD analyseswere located upstream of the sequences deleted within algD.

AmrZ binds and directly represses the pslA promoter. Oncewe established that AmrZ is involved in psl repression, we soughtto determine whether AmrZ acts directly or indirectly on psl tran-scription. To test this, we employed electrophoretic mobility shiftassays (EMSAs) with purified AmrZ or variants as shown in Fig. 4.As previously documented, AmrZ binds specifically to the algDpromoter region, and this binding requires the R22 residue (19).Similarly, AmrZ binds to the psl promoter region (Fig. 4A) in anR22-dependent manner, and this likely leads to repression of Pslproduction in mucoid strains. The multiple banding patterns ob-served in Fig. 4A are common with this assay, as AmrZ formsoligomers, resulting in multiple AmrZ-DNA complexes observed

FIG 3 AmrZ correlates with inverse transcript levels of algD and pslA. (A)AmrZ Western blot of whole-cell lysates. Nonspecific cross-reactive band wereincluded as a loading control. (B) Observation of pslA and algD mRNA levelsvia quantitative reverse transcription real-time PCR. All samples were normal-ized to the level of expression in the mucoid isolate FRD1. The graph repre-sents mean expression of three independent experiments performed in tripli-cate. Error bars represent standard deviations. (C) A one-way ANOVA withTukey post hoc test was used to determine statistical differences in Fig. 3B. Allcomparisons are between the indicated strain and FRD1, and statistical signif-icance of the differences is indicated as follows: �, P � 0.05; ��, P � 0.01; ���,P � 0.001; ns, not significant.

FIG 4 AmrZ binds to the psl promoter region. (A) Increasing amounts ofpurified AmrZ or AmrZ R22A were incubated with fluorescently labeled frag-ments of DNA overlapping the algD or psl promoter. Lanes: 1, free DNA; 2, 4.0nM wild-type (WT) AmrZ; 3, 2.8 nM WT AmrZ; 4, 1.1 nM WT AmrZ; 5, 0.6nM WT AmrZ; 6, 0.3 nM WT AmrZ; 7, 4.0 nM AmrZ R22A; 8, 0.3 nM AmrZR22A. The position of free DNA is indicated by an arrow, while DNA bound byAmrZ is indicated by a bracket. (B) AmrZ binding fragments containing the pslpromoter from 400 bp to 110 bp. Lanes contain either free DNA of the frag-ment size noted (�) or DNA of the size indicated and 4.0 nM WT AmrZprotein (�). (C) Sequence of the 200-bp region upstream of the pslA codingsequence. The ATG codon of pslA is located at bases 201 to 203. The putativeAmrZ binding site is indicated in red (40, 43). Gray and black bars indicate thesequences of fragment 1 and fragment 2, respectively. These fragments wereused in an EMSA to verify AmrZ binding. Irie et al. (42) identified the tran-scription start site (�1) at base 53. Overhage et al. (41) mapped an alternativetranscription start site (�1) at base 160. Sequence alignment of the consensusAmrZ binding site (40, 43) to binding sites of fragment 1 (red sequence) andmutant fragment 2 of the pslA promoter is shown. DNA fragments used in Fig.4A and B were produced utilizing 5=-FAM-labeled fragments, while those inFig. 4C utilized unlabeled fragments that were stained with the SYBR greenEMSA stain.

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in this native gel condition (33, 40). To further define the AmrZbinding site at the psl promoter, a series of increasingly smallerDNA fragments were incubated with AmrZ. Binding to psl wasseen with a fragment as small as 110 bp (blue-colored fragment inFig. 4B). The lack of AmrZ R22A binding to any pslA sequencealong with the observation that AmrZ fails to bind an immediatelyadjacent 50-bp sequence (red-colored fragment in Fig. 4B), as wellas our previously published data (33, 40) indicates that AmrZbinds specifically to pslA.

There have been two reports regarding mapping of the psloperon transcription start site (41, 42). Overhage et al. (41) iden-tified a 70 promoter with atypical spacing from the start of tran-scription beginning 41 bp upstream of the pslA ATG (Fig. 4C). Irieet al. (42) described an alternative RpoS-dependent promoterwith a transcription start site 148 bp upstream of the pslA ATG,which was significantly more active than the promoter identifiedby Overhage (Fig. 4C). Each psl fragment found to bind AmrZincludes the region immediately upstream of the transcriptionalstart site described by Overhage et al. (41) (Fig. 4C) and that re-ported by Irie et al. (42) (Fig. 4C). This suggests that AmrZ mayblock RNA polymerase binding to the psl promoter or may inter-fere with efficient transcription initiation (see below).

Inspection of these sequences revealed a potential AmrZ bind-ing site (red sequence in Fig. 4C) based on similarity to the pub-lished consensus AmrZ binding site (sequences at the bottom ofFig. 4C) (40, 43). Two overlapping fragments were generated, andEMSA was performed to determine whether AmrZ binds to thissite (located in fragment 1 [Fig. 4C]). Wild-type AmrZ, but notDNA-binding-deficient R22A AmrZ bound to fragment 1, but notfragment 2 (Fig. 4C). Additionally, the AmrZ binding site wasreplaced with a scrambled sequence to which AmrZ would not bepredicted to bind (40), and this was confirmed in DNA bindingstudies (Fig. 4C, gel). Thus, we conclude that the bona fide AmrZbinding site regulating psl expression is 5=-GCCACTATCGACGAA-3= (Fig. 4C). This would be centered 13 bp downstream of thetranscription start site reported by Irie et al. (42) (Fig. 4C) but 96bp upstream of the promoter mapped by Overhage et al. (41) (Fig.4C). Further work will be needed to determine how AmrZ bindingimpacts transcription from one or both of the reported start sitesin Fig. 4C (41, 42).

AmrZ-mediated modulation of Psl and alginate productionaffects biofilm structure. Biofilm growth generates gradients ofnutrients and waste products that promote heterogeneous popu-lations. Elimination of structure and decreased heterogeneity canalter susceptibility of P. aeruginosa to antimicrobial agents (44–48). Expression of the psl operon in a biofilm is limited to theinitially adhering cells and vertical biofilm structures, called mi-crocolonies (41). Recently, it was shown that elimination of Psl inmucoid strains leads to an abolishment of microcolony structure(23). To investigate whether AmrZ affects Psl-dependent biofilmstructure, we compared biofilms using nonmucoid strains engi-neered with arabinose-responsive amrZ and psl expression. Con-sistent with recently published reports (23, 27), we observed thatwild-type P. aeruginosa PAO1 produces typical mushroom-shaped microcolonies, while the �ppsL mutant forms a flat bio-film devoid of structured towers (Fig. 5A, center panel). Arabi-nose-induced Psl overexpression produced taller towers thanthose produced by strain PAO1, indicating that Psl enhancestower formation (Fig. 5A, bottom left panel vs. center panel). TheamrZ mutant, which expresses large amounts of Psl, also exhibited

tall microcolonies (Fig. 5A, top left panel). However, when AmrZwas overexpressed, the biofilm was thin and completely lackedsuch structures (Fig. 5A, top right panel). Interestingly, the micro-colony structures of the amrZ mutants corroborated the molecu-lar observations that AmrZ represses psl expression (Fig. 5B).COMSTAT analysis of biofilm images confirms that �amrZ mu-tants produce biofilms with biomass and microcolony volumesimilar to the biofilms of strain PAO1, while the AmrZ-overex-pressing strain formed a biofilm with significantly less biomassand microcolony volume (see Fig. S1 in the supplemental mate-rial). The differences observed in biofilms are likely due to en-hanced attachment of �amrZ mutants, as determined via rapid-attachment assays (Fig. S2). These data are consistent withincreased attachment in Psl-overproducing strains (11, 13, 22, 23,28, 41).

Antibody staining of biofilms (Fig. 5A, red) indicates that Psl isproduced in patterns consistent with those observed elsewhereusing Psl-specific lectin staining (22). Here, Psl polysaccharideaccumulated around the periphery of the microcolony (Fig. 5A,red staining). Additionally, Psl production appears to be elevatedin the �amrZ mutant (Fig. 5A, top left panel) but was not detectedin the AmrZ-overexpressing strain (Fig. 5A, top right panel), fur-ther supporting the hypothesis that AmrZ-mediated transcrip-tional regulation of the psl and algD operons is a major mecha-nism for inverse alginate and Psl regulation. Collectively, thesedata support the model that the AlgT-responsive transcriptionalregulator AmrZ directly represses psl operon transcription andthus results in changes in the biofilm architecture and structure(Fig. 5B).

Biofilm formation is a critical component of P. aeruginosa in-fections, and polysaccharides provide much of the attachment andstructure for these communities (11, 49). In this study, we exam-ined the inverse relationship between alginate and Psl production.The need for inverse production could be explained in severalways. First, the functions of Psl and alginate may be overlapping.Both polysaccharides form a scaffold for the biofilm and promoteattachment, but strains producing only one polysaccharide arestrikingly unique. Strains producing Psl are better able to achieveinitial attachment, while the ability of alginate to protect againsthost defenses is well documented (4, 8, 17). It may be that Pslexpression is advantageous in early colonizing stages to allow thebacterium to efficiently attach and aggregate, while alginate isneeded at later stages in order to protect against the immune re-sponse.

Both Psl and alginate require mannose and ci-di-GMP for bio-synthesis (12, 50–53). Reduced production of Psl in mucoidstrains could free up these precursors for production of alginate.The results shown here identify a transcriptional regulatory mech-anism for biofilm polysaccharides. Early in infection, it may beadvantageous for the bacteria to aggregate and initiate biofilms(22, 54). At this time, inflammation would likely be low. Psl maybe produced at a basal level until alginate production occurs dueto a particular stress, leading to mucA mutations induced in asubpopulation via neutrophil-mediated oxidative damage (55).The prevalence of mucoid conversion in CF patients implies aselective advantage for alginate producers at a given stage inchronic infection, likely as a resistance mechanism to the neutro-phil influx and elevated oxidative molecules associated withchronic lung infections (15, 55). We propose that AmrZ operatesas a transcriptional switch to alleviate demand on common pre-

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cursors by repressing expression of the Psl biosynthetic machinerywhile activating transcription of the alginate genes (Fig. 5B). Thisscenario efficiently switches the cell from Psl production to alg-inate production by using a bifunctional protein to activate ex-pression of one polysaccharide while repressing a competing one.In this way, the bacterium is able to adjust to the changing envi-ronment of the lungs of CF patients and better survive.

Collectively, these data support the hypothesis that AmrZ re-presses psl expression. Mucoid strains produce large amounts ofAmrZ and alginate; however, they produce small amounts of Psl.We demonstrate that AmrZ binds directly and specifically to a site(5=-GCCACTATCGACGAA-3=) located in the promoter regionof the psl operon, leading to reduced Psl expression. This effect isdependent on AmrZ DNA binding activity, as the DNA-binding-deficient mutant R22A AmrZ is unable to repress the psl operonand fails to bind to this site. Finally, we report that AmrZ-medi-ated repression of psl abrogates biofilm tower formation, withpotential effects on treatment strategies. AmrZ repression of Pslproduction may be an important aspect of biofilm formation andmucoidy in chronic P. aeruginosa infections. With further under-standing of this interaction, AmrZ and Psl may emerge as thera-peutic targets to prevent biofilm formation and establishment ofchronic infections that are recalcitrant to treatments.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants AI061396 andHL058334 (D.J.W.), and fellowships from the College of Medicine Sys-

tems and Integrated Biology Training Program at Ohio State University(C.J.J.) and American Heart Association Great Rivers Affiliate PredoctoralFellowship (C.J.J.). Images presented in this report were generated usingthe instruments and services at the Campus Microscopy and ImagingFacility of Ohio State University. DNA sequences were obtained from thePseudomonas Genome Database (56, 57).

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FIG 5 Modulation of AmrZ and Psl production impacts biofilm structure. (A) Confocal scanning laser microscopy of 24-h biofilms grown in flow cell chambers.Top down images (top left of image) and orthogonal views (right side and bottom of image) allow visualization of biofilm thickness and structure. Red scale barsrepresent 20 �m. Green fluorescence is Syto9 stain to indicate biomass, while the red staining represents antibody detection of Psl. (B) Model of the AmrZ-mediated effect of Psl regulation on biofilms. Elevated AlgT leads to high AmrZ, which represses Psl. Biofilms formed by Psl-deficient bacteria are thin, flat, anddevoid of tall microcolonies compared to wild-type biofilms.

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