INFECTION AND IMMUNITY,0019-9567/00/$04.0010

May 2000, p. 2493–2502 Vol. 68, No. 5

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

RpmA Is Required for Nonopsonic Phagocytosisof Pseudomonas aeruginosa

DAVID A. SIMPSON1* AND DAVID P. SPEERT1,2

Division of Infectious and Immunological Diseases, Department of Pediatrics, and theCanadian Bacterial Diseases Network,1 and Department of Microbiology and Immunology,2

University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4

Received 6 December 1999/Returned for modification 7 January 2000/Accepted 19 January 2000

Pseudomonas aeruginosa causes severe respiratory tract infections in patients with cystic fibrosis (CF). Wehave been examining nonopsonic phagocytosis of P. aeruginosa by macrophages. To study the P. aeruginosa-macrophage interaction at the molecular level, we have constructed a transposon Tn5G bank in a clinicalisolate of P. aeruginosa (strain 4020) and identified mutants resistant to nonopsonic phagocytosis. Phagocy-tosis-resistant mutants were enriched by passaging the transposon bank over 18 macrophage monolayers. Of900 individual mutants isolated from this enriched pool in a nonopsonic phagocytosis assay, we identified 85putative mutants that were resistant to phagocytosis. In this study, we have characterized one of thesetransposon mutants, P. aeruginosa 4020 H27A, which was poorly ingested. H27A possessed a Tn5G insertionin a gene encoding a protein with homology to the MotA proteins of several species of bacteria. We have calledthis gene rpmA for required for phagocytosis by macrophages. RpmA is one of two MotA paralogs in P.aeruginosa. This rpmA::Tn5G mutant was motile both on agar plates and in visual examination of wet mounts.The phagocytosis defect was partially complemented by providing the rpmA gene in trans and fully comple-mented when both rpmA and rpmB were provided. A rpmA null mutant was ingested by macrophages similarto the H27A transposon mutant. These data suggest that the rpmA and rpmB gene products are required forthe efficient ingestion of P. aeruginosa by macrophages.

Pseudomonas aeruginosa is an opportunistic pathogen thatcauses diverse infections in humans. It produces a wide arrayof both cell-associated and extracellular components that mayact as virulence factors. Lung infections of cystic fibrosis (CF)patients by P. aeruginosa are particularly problematic. Onceacquired, it is extremely difficult to eradicate the organismfrom the lungs. Colonization of the host involves initial adher-ence of the bacteria to host receptors on either cells or mucins.This adhesion is mediated by pili (12, 13, 43, 63), outer mem-brane components (9), and flagellar components (3, 50). Initialinteraction of P. aeruginosa with the host immune system in thelung environment probably occurs under nonopsonic condi-tions. Opsonophagocytosis may be inefficient in the lungs ofchronically colonized CF patients, since both host and bacterialproteases cleave complement and phagocytosis receptors (59).Initial colonizing isolates of P. aeruginosa are predominantlyserum resistant, but P. aeruginosa strains recovered fromchronically colonized CF patients are serum sensitive (18),indicating that both complement-mediated lysis and op-sonophagocytic clearance are defective in the lungs of thesepatients (52). Nonopsonic phagocytosis of P. aeruginosa is areceptor-mediated event, and evidence suggests that multiplebacterial ligands are involved in the interaction of bacteria withmacrophages. Several studies have demonstrated that the bac-terial pilus is involved in the ingestion of P. aeruginosa bymacrophages (25, 35, 54). Our laboratory has been investigat-ing the interaction of P. aeruginosa and macrophages. Thebacterium-macrophage interaction occurs in two steps. Thebacteria adhere to the macrophage, followed by a signal thattriggers the phagocytosis of the bacteria (53). Using isogenic

mutants, we have shown that pili are involved in the interactionof P. aeruginosa with macrophages, but they are not sufficientfor triggering phagocytosis (30). The flagella of P. aeruginosa,or a component associated with the flagellar filament, appearto be necessary for phagocytosis (30). Nonmotile P. aeruginosaisolates are frequently isolated from chronically colonized CFpatients (28, 31), and these nonmotile strains are resistant tononopsonic phagocytosis (31). Motility defects in these strainshave been shown to be due to defects in either rpoN or algTgenes (16, 31). The nonmotile phenotype may enable the bac-teria to evade phagocytosis and persist in the lung. Approxi-mately 50 genes are involved in the production and function offlagella in Escherichia coli or Salmonella enterica serovar Ty-phimurium (5, 29). Proteins encoded by these genes includestructural components of the filament, flagellar motor compo-nents, chemotaxis components, and the export apparatus re-quired for flagellar synthesis. Some of the genes involved inflagellar biosynthesis and function of P. aeruginosa flagellahave been described (3, 24, 42, 50, 55, 60). A type III secretionsystem has been identified in P. aeruginosa (15, 64). Recently,it has been demonstrated that P. aeruginosa kills macrophagesand macrophage-like cells via this type III secretion system (10,20, 45). Interaction of P. aeruginosa with the macrophage isrequired for type III secretion system-mediated effects.

Identification of the bacterial ligands mediating the interac-tion of P. aeruginosa with macrophages may allow us to mod-ulate this interaction and allow the host to eradicate P. aerugi-nosa more readily. In this study, we have created a transposonmutant bank in a strain of P. aeruginosa isolated from a pedi-atric CF patient, and we have isolated mutants resistant tononopsonic phagocytosis. We have characterized a mutant inwhich the transposon had inserted into the P. aeruginosa rpmAgene. RpmA is a paralog of the MotA proteins described inenteric bacteria which comprise one component of the flagellarmotor (46). Both MotA and MotB are required for torque

* Corresponding author. Mailing address: B.C. Research Institutefor Children’s and Women’s Health, 950 W 28th Ave., Rm. 381, Van-couver, BC, Canada V5Z 4H4. Phone: (604) 875-2469. Fax: (604)875-2226. E-mail: dsimpson@cbdn.ca.

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generation (6, 8). MotA and MotB are cytoplasmic membraneproteins (41) that interact to provide conductance of protonsacross the membrane (7, 56). We have demonstrated that thisP. aeruginosa rpmA mutant retained motility but was resistantto nonopsonic phagocytosis mediated by thioglycolate-elicitedmouse peritoneal macrophages.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. P. aeruginosa 4020 is the initial strainisolated from a pediatric CF patient. Randomly amplified polymorphic DNA(RAPD) analysis (32) of subsequent isolates from this patient indicated that thisstrain chronically colonized this patient for several years (data not shown). Allbacterial strains and plasmids are listed in Table 1. All bacterial stocks weremaintained at 270°C in Luria broth (L broth) plus 8% (vol/vol) dimethyl sul-foxide (DMSO). Antibiotics were used at the following concentrations: for P.aeruginosa gentamicin (Gm) was used at 75 mg/ml for selection and at 20 mg/mlfor maintenance, carbenicillin (Cb) was used at 150 mg/ml, and tetracycline (Tc)was used at 100 mg/ml and for E. coli Gm was used at 10 mg/ml, Tc was used at20 mg/ml, ampicillin (Ap) was used at 100 mg/ml, spectinomycin was used at 50mg/ml, and kanamycin (Km) was used at 25 mg/ml. Bacterial were routinelygrown in either L broth or Minimal A salts medium (11) supplemented with 50mM monosodium glutamate and 1% (vol/vol) glycerol. Motility was determinedon L broth–0.3% agar plates.

Enzymes and chemicals. All restriction enzymes and T4 DNA ligase werepurchased from New England Biolabs. All cell culture reagents were purchased

from Gibco-BRL. The random primed labeling kit was purchased from Boehr-inger-Mannheim Canada.

DNA manipulations. Plasmid DNA was prepared by the method of Birnboimand Doly (4). Agarose gel electrophoresis, DNA restriction digests, DNA liga-tions, and Southern blots (under moderate to high stringency) were performedessentially as previously described (44). Chromosomal DNA was isolated fromP. aeruginosa as described earlier (33). P. aeruginosa was electroporated using aBio-Rad Gene Pulser (Bio-Rad, Mississauga, Ontario, Canada) by previouslydescribed methods (51).

Transposon mutagenesis of P. aeruginosa 4020. The transposon Tn5G (36) wasused to mutagenize P. aeruginosa 4020 as follows: P. aeruginosa 4020 was grownin L broth and E. coli DH5a(pRK2013::Tn5G) was grown in L broth plus Gmand Km overnight at 37°C. Prior to use in filter matings, the P. aeruginosa culturewas heated to 50°C for 3 min. Aliquots (150 ml) of both donor and recipient wereadded to 5 ml of 10 mM MgSO4 and filtered onto 0.45-mm (pore size) nitrocel-lulose disks. The filters were placed on L agar and incubated overnight at 37°C.The filters were then removed from the L agar, bacteria were resuspended in 5ml of 10 mM MgSO4, and aliquots were plated onto minimal agar containing 75mg of Gm per ml. The colonies after overnight growth at 37°C were scraped fromthe plates and frozen in aliquots at 270°C in L broth plus 8% (vol/vol) DMSO.Three separate filter matings were used to derive approximately 25,000 P. aerugi-nosa 4020 mutants.

Enrichment for P. aeruginosa 4020 mutants resistant to nonopsonic phagocy-tosis. The bank of transposon mutants was grown overnight in L broth plus 75 mgof Gm ml21 in two conditions: either with shaking at 150 rpm or statically. Thepellicle from the static culture was removed with a pipette and added to L-Gmbroth and adjusted to an optical density at 600 nm (OD600) of approximately 0.6.Similarly, the OD of the shaken culture was adjusted to approximately 0.6. Each

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Relevant genotype or phenotype Source or reference

StrainsP. aeruginosa

4020 Wild-type clinical isolate This study4020 H27A rpmA::Tn5G This study4020-27 rpmA::Tc This study4020-27Sc Single-crossover cointegrate of pDS27Tc in 4020 This studyPAK Wild-type laboratory strain D. BradleyPAK-N1G PAK rpoN::Gmr 23

E. coliDH5a hsdR recA lacZYA F80 lacZ DM15 Gibco-BRLXL1-Blue MRF9 D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F9

proAB lacIqZDM15 Tn10 (Tcr)]Stratagene

Cosmid (SuperCos1) Cosmid cloning vector Stratagene

PlasmidspRK2013::Tn5G mob1 tra1, Kmr Gmr S. LorypBluescript II SK and KS Cloning vectors, Apr StratagenepGEM5Zf(1) Cloning vector PromegapUCP19 Broad-host-range cloning vector, Apr 47pUC19mob pUC19 with mob region of broad-host-range plasmid pRP4, can be mobilized into P.

aeruginosa but will not replicate37

pPC110 Gm cassette 61pDSH27A 7.5-kb SalI fragment from 4020 H27A, cloned into SalI-restricted pBluescript II SK(1) This studypDS27.1 0.85-kb XhoI fragment from pDSH27A, cloned into XhoI restricted pBluescript II KS(2)

contains end of transposon plus flanking DNAThis study

pDSA4H10 SuperCos1 cosmid vector with ;33-kb insert containing rpmA and rpmB This studypDS27.5 3.3-kb SalI fragment from pDSA4H10 cloned into SalI-restricted pUCP19 rpmA1 This studypDS27.6 Insert opposite orientation to pDS27.5 rpmA1 This studypDS27B6 6-kb EcoRV fragment from pDSA4H10 cloned into SmaI-restricted pUCP rpmA1 rpmB1 This studypDS27B7 Insert opposite orientation from pDS27B6 rpmA1 rpmB1 This studypDS290 2-kb SacI fragment from pDS27B6 cloned into SacI-restricted pUCP19 This studypDS291 Insert opposite orientation to pDS290 This studypDS297 2.8-kb SacI fragment from pDS27B7 cloned into SacI-restricted pUCP19 rpmB1 This studypDS298 Insert opposite orientation to pDS297 rpmB1 This studypDS27Tc SmaI-restricted Tc cassette from miniTn5Tc cloned into unique SmaI site of rpmA gene

in pDS27.9This study

pDS27Tcmob 3.5-kb PstI fragment from pDS27Tc cloned into PstI-restricted pUC19mob This study

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of these pools was used in parallel in the enrichment procedure described below.Thioglycolate-elicited mouse peritoneal macrophages were harvested by perito-neal lavage as described previously (31), and approximately 3.5 3 105 macro-phages were added to glass coverslips in a 24-well tissue culture dish. Afterovernight incubation, the wells were washed three times with 2.5 ml of phos-phate-buffered saline, followed by the addition of 490 ml of phagocytic medium(138 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 1 mM MgCl2,0.6 mM CaCl2; pH 7.4) plus 10 mM glucose. Bacteria were added to macrophagemonolayers at a multiplicity of infection (MOI) of approximately 1:10 bacteria/macrophage. The 24-well plate was then centrifuged at 2,000 3 g for 10 min tofacilitate interaction of the bacteria with the macrophage monolayers. After 1 hof incubation at 37°C, the supernatant containing uningested organisms wasremoved and added to the next uninfected macrophage monolayer. After threeto five consecutive passages, depending upon the round of enrichment, thesupernatant was removed and plated on Minimal A salts medium plus 75 mg ofGm per ml and grown overnight at 37°C. Colonies from each enrichment werescraped off the plates with a glass rod into L broth; DMSO was added to 8%(vol/vol), and the pooled bacteria were frozen in aliquots at 270°C. Each en-richment was performed with both shaken and statically grown cultures. Aliquotsfrom each of the primary enrichments were inoculated into L-Gm broth andgrown overnight either shaken or statically, consistent with the growth conditionsprior to the primary enrichment. This enrichment procedure was repeated fivetimes so that the uningested organisms at the end of this selection process hadbeen passaged over 18 macrophage monolayers. Each enrichment pool, fromboth the shaken and the static cultures, was then plated out so individual mutantscould be tested in the phagocytosis assay.

Identification of transposon mutants resistant to phagocytosis. Individualmutants were grown with shaking at 150 rpm at 37°C overnight in L broth plus20 mg of Gm per ml. Bacteria were diluted to an OD600 of 0.6, and 50-ml aliquotswere added to macrophages adhered to glass coverslips in 24-well plates to givean MOI of approximately 100:1 bacteria/macrophage. The phagocytosis assaywas performed essentially as described previously (31), except the wash volumeswere increased to 2 ml. Initially, the mutants were scored visually for defects inphagocytosis. Potential mutants were then tested in triplicate and counted. Allstatistical analyses were performed using the Student’s t test.

Identification of transposon insertions. Chromosomal DNA isolated fromputative transposon mutants was digested with restriction enzymes and electro-phoretically separated in 0.8% agarose gels. Southern analysis was performedusing a probe derived from the gentamicin cassette of plasmid pPC110. Allprobes were labeled with [a-32P]dGTP (NEN Life Science Products, MandelScientific, Guelph, Ontario, Canada) using a random primer labeling kit (Boehr-inger-Mannheim Canada), according to the manufacturer’s instructions. Probeswere hybridized at moderate stringency. Chromosomal DNA from 4020 H27Awas restricted with SalI and ligated to SalI-restricted pBluescript II, and Gm-resistant colonies were isolated. Plasmid pDSH27A contains a 7.5-kb SalI frag-ment containing the entire transposon plus approximately 3.2-kb flanking DNA.

Cloning of rpmA and rpmB from P. aeruginosa 4020. P. aeruginosa 4020 chro-mosomal DNA was partially restricted with Sau3AI and ligated into the BamHIsite of the cosmid vector SuperCos1. Cosmids were packaged using a GigaPackGold kit (Stratagene). E. coli XL1-Blue MRF9 were transfected with the pack-aged cosmids. The average insert size was determined to be approximately 30 kb.Southern analysis of colony blots of the cosmid bank using a 0.85-kb XhoIfragment of DNA from pDSH27A containing the end of the transposon plusflanking DNA as a probe allowed us to identify several probe-reactive cosmids.Restriction of these cosmids followed by Southern blotting indicated that a3.3-kb SalI fragment from cosmid pDSA4H10 hybridized with DNA flanking thetransposon. This SalI fragment containing the rpmA gene was cloned into SalIrestricted pUCP19 in both orientations for use in complementation experiments.The fragment was also cloned into SalI-restricted pGEM5Zf(1) to formpDS27.9 for use in sequencing. In addition, a 6-kb EcoRV fragment frompDSA4H10 was subcloned into SmaI-restricted pUCP19 in both orientations toform pDS27B6 and pDS27B7. This construct contained both the rpmA and rpmBgenes. A 2.8-kb SacI fragment containing the rpmB gene from pDS27B6 wascloned into SacI-restricted pUCP19 in both orientations to form pDS297 andpDS298. Upstream sequences were cloned as a 1.7-kb SacI fragment into SacI-restricted pUCP19 in both orientations to form pDS290 and pDS291.

Sequence analysis. A series of unidirectional nested deletions of pDS27.9 werecreated using the Erase-a-Base System kit (Promega). Subclones of pDS27.9using mapped restriction sites were constructed in pBluescript vectors and alsoused for sequencing. All plasmid DNA used for sequencing was purified using aQiagen plasmid kit (Qiagen). Sequencing was performed by automated PCRsequencing (Applied Biosystems 377 Automated DNA Sequencer and AmpliTaqDyeDeoxy Terminator Cycle Sequencing). Sequence data was assembled andanalyzed by computer software (Lasergene for Windows; DNASTAR, Inc.).Homologs of putative proteins were identified using the BLAST algorithm (2) atthe National Center for Biotechnology Information.

Construction of a rpmA null mutant. A tetracycline cassette from miniTn5-Tcwas isolated as a SmaI fragment and ligated into the unique SmaI site in therpmA gene to form pDS27Tc. A 5.5-kb PstI fragment from pDS27Tc was clonedinto pUC19mob (37) to form pDS27Tcmob. This plasmid was used for homol-ogous recombination. P. aeruginosa 4020 was electroporated with pDS27Tcmob,and cointegrates were isolated on L-Tc plates. Putative rpmA mutants were

isolated as Cb-sensitive, Tc-resistant colonies. Southern blots of chromosomalDNA of putative mutants were performed using a radiolabeled DNA fragmentinternal to the rpmA gene as a probe. Both a single crossover cointegrate termedP. aeruginosa 4020-27Sc and a rpmA::Tc mutant termed P. aeruginosa 4020-27were isolated.

Detection of flagellin and pilin proteins. Bacteria were grown overnight in Lbroth (plus appropriate antibiotics) with tumbling, pelleted by centrifugation,and washed one time in GET buffer (10 mM Tris-Cl, 50 mM glucose, 10 mMEDTA; pH 8.0). The cells were pelleted and resuspended in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer andboiled for 5 min prior to electrophoresis through SDS–12% PAGE gels (27).Proteins in the gel were transferred to nitrocellulose (62), and pilin protein wasvisualized using polyclonal antipilin antiserum (developed against P. aeruginosaPAK pilin [57]). Flagellin was visualized with monoclonal antiflagellin (devel-oped against P. aeruginosa PAK flagellin) (49). Horseradish peroxidase-labeledlabeled secondary antibody, followed by color development with 4-chloro-1-napthol, was used to identify immunoreactive components.

Pilin production was also monitored using phage PO4 sensitivity. Phage PO4causes a lytic infection of P. aeruginosa upon binding to functional pilus or-ganelles. Bacteria were streaked out on agar plates, and approximately 2 3 107

PFU PO4 particles in a 5-ml aliquot were spotted onto the streaked bacteria. Azone of bacterial lysis in the infected area indicated that the bacteria wereproducing twitching pili.

Nucleotide sequence accession number. The sequence of pDS27.5 containingthe entire rpmA gene and the first 423 nucleotides of the rpmB gene has beensubmitted to GenBank under accession number AF136403.

RESULTS

Identification of P. aeruginosa 4020::Tn5G mutants resistantto nonopsonic phagocytosis. A transposon bank was con-structed in P. aeruginosa 4020, the initial P. aeruginosa isolaterecovered from a pediatric CF patient. RAPD analysis (32)indicated that this same strain colonized this patient for severalyears (data not shown). To facilitate identification of phago-cytosis-resistant mutants, we grew the transposon bank eitherwith shaking or statically, passaged each of these transposonbanks in parallel over macrophages, and recovered nonphago-cytosed bacteria. Initially, approximately 2% of the inoculumwas recovered as noningested organisms from the phagocyticmedium over the macrophage monolayers; however, after fiveseparate enrichments, the amount recovered increased to ap-proximately 20% of the inoculum. After this final enrichment,bacteria were plated to isolate individual mutants. Approxi-mately 950 of these mutants (400 from the shaken pool and 550from the statically grown pool) were examined individually ina nonopsonic phagocytosis assay. Of these mutants, 85 had aphagocytosis defect, 59 isolated from the statically grown pooland 26 isolated from the shaken pool.

After the initial screen, Southern blots were performed withchromosomal DNA isolated and restricted from these mutants,using the Gm cassette as a probe. Several mutants were iden-tified as siblings since they possessed the same probe-reactivefragments in the Southern blots when the DNA was restrictedwith multiple enzymes. In one case the same mutant was iso-lated from both the shaking and the statically grown pools(T25B and H7F). Subclones containing the transposon andflanking DNA on SalI fragments from several mutants wereconstructed in the pBluescript vectors. Table 2 lists some ofthese mutants and their phenotypes. Figure 1 illustrates thelevels of nonopsonic phagocytosis of several P. aeruginosa4020::Tn5G mutants by thioglycolate-elicited mouse peritonealmacrophages. All of these mutants possessed a significant re-sistance to nonopsonic phagocytosis compared to the parentalstrain (P , 0.05).

Since flagella and pili are involved in the interaction of P.aeruginosa with macrophages, we examined both the motilityand the pilus production of the parental strain and selectedmutants. Motility of the mutants was determined on 0.3% agar,and pilus production was monitored by phage PO4 sensitivity.The normal pattern of motility, in which bacteria swim through

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the agar matrix, results in the formation of concentric circlescentered about the inoculation site. P. aeruginosa PAK exhibitsthis pattern (Fig. 2A). Motility exhibited by P. aeruginosa 4020initially resembled this phenotype, but after the colony of bac-teria reached approximately 15 mm in diameter (which oc-curred after 14 h of incubation at 37°C), rapid spreading upon

the surface of the agar commenced (Fig. 2B to D). This phe-notype was lost after repeated subcultures of P. aeruginosa4020 on L agar. Several nonmotile mutants, such as T13H, andmutants that exhibited a normal motility pattern, such asH39F, were isolated, but the majority of mutants exhibited theunusual motility pattern. The majority of phagocytosis-resis-tant mutants were phage PO4 sensitive, indicating that theyproduced an intact functional pilus. We isolated several phagePO4-resistant mutants, although further molecular character-ization indicated that many of these were siblings. We selectedone of the phagocytosis-deficient transposon mutants, P. aeru-ginosa 4020 H27A for further study.

Characterization of P. aeruginosa 4020 H27A. P. aeruginosa4020 H27A was identified as the most resistant to nonopsonicphagocytosis of the mutants that were isolated using our screen(Fig. 1). This mutant was ingested at a level similar to that ofan RpoN mutant (PAK-N1G). The ingestion of H27A wassimilar whether or not the bacteria had been centrifuged ontothe monolayer (compare Fig. 1, where bacteria were centri-fuged, versus Fig. 5, where bacteria were not centrifuged).H27A exhibited the parental motility phenotype (Fig. 2E), andit was also sensitive to phage PO4 infection. Visual examina-tion of wet mounts of P. aeruginosa 4020 H27A also indicatedthat the mutant was motile. Western blots of both whole cellsand sheared extracts from cells indicated that H27A producedboth flagellin (Fig. 3A) and pilin proteins (Fig. 3B).

Complementation of the H27A mutation. The transposonplus flanking DNA was cloned as a 7.5-kb SalI fragment intopBluescript II SK(1), giving pDSH27A. A 0.85-kb XhoI frag-ment from pDSH27A encompassing the end of Tn5G plusapproximately 400 bp of flanking DNA (termed pDS27.1) wasused as a probe to screen a P. aeruginosa 4020 cosmid bank.Five probe-positive cosmids were identified. Cosmid DNA wasrestricted with several enzymes, and a Southern blot was per-formed using the 0.85-kb XhoI fragment from pDS27.1 as a

FIG. 1. Nonopsonic phagocytosis of P. aeruginosa 4020 and selected Tn5G mutants by mouse thioglycolate-elicited murine peritoneal macrophages. Phagocytosisassays were performed as described in Materials and Methods. The y axis represents the mean number of bacteria ingested per macrophage. Error bars represent thestandard error of three separate experiments. An asterisk indicates that the number of bacteria phagocytosed was significantly lower than with P. aeruginosa 4020(P , 0.05).

TABLE 2. Phenotype of selected P. aeruginosa4020::Tn5G phagocytosis-deficient mutants

MutantaCharacteristics

Ingestionb Motilityc Phage PO4d

H27A* (H24F) Low 1 sT3B* (T5C) Low 1 sT25B* (H7F, H8H) Low 1 sT15C* (T29F, T41F) Low 1 sT36B Low-moderate 1 sT7C Low-moderate 1 sT41B Low-moderate 1 sH39F* (H18F, H33F) Low-moderate 1 rH5E Low-moderate 1 rT13H Low-moderate 2 sT32F Low-moderate 1 sT49C Moderate 1 sH49E Moderate 1 sT2E Moderate 2 sPAK-N1G (rpoN::Gm) Low 2 r

a An asterisk indicates that the transposon plus flanking DNA has been clonedfrom this mutant. Mutants in parentheses are siblings of the respective mutants(Southern blots). Mutants with the H designation were isolated from a shakingpool; mutants with the T designation were isolated from a static pool.

b The initial screen of ingestion was graded: low, 25% of the parental P.aeruginosa 4020 strain; low-moderate, 25 to 40% of the parental strain; andmoderate, ;50% of the parental strain.

c Motility was determined on L broth–0.3% agar plates.d s, sensitive to PO4 infection; r, resistant to PO4.

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probe. Several probe-reactive fragments were cloned from thecosmid into pUCP19 (50), which can be electroporated intoP. aeruginosa. A 3.3-kb SalI fragment from the cosmid wascloned into pUCP19 in both orientations to form pDS27.5 andpDS27.6, respectively (Fig. 4, only pDS27.5 is shown). P. aeru-ginosa 4020 H27A containing either pDS27.5 or pDS27.6 wasphagocytosed at a level approximately half that of the parentbut twofold greater than the mutant H27A (Fig. 5). This issignificantly higher than either H27A alone or H27A carryingthe vector (pUCP19) (P , 0.05). Since the orientation ofthe fragment played no role in complementation, the genespresent in the insert were likely being transcribed from theirown promoters. A 6-kb EcoRV probe-reactive fragment fromthe cosmid was cloned into pUCP19 in both orientations toform pDS27B6 (Fig. 4) and pDS27B7, and H27A containingeither of these constructs were phagocytosed at a level similarto that of the parental P. aeruginosa 4020 strain (Fig. 5). Thus,genes present on pDS27B6 were required in trans to fullycomplement the phagocytosis defect of H27A. Plasmids pDS290(Fig. 4) and pDS291, which contain a portion of the pDS27.5sequence plus upstream sequences, did not complement thephagocytosis defect when conjugated into H27A (Fig. 5) (P ,0.005). Similarly, plasmids pDS297 (Fig. 4) and pDS298, whichcontain a small portion of pDS27.5 sequence and downstreamsequences, had no effect on the H27A phagocytosis defect (Fig.5) (P , 0.005).

Genes identified as rpmA and rpmB. The site of insertion ofthe transposon in H27A was identified by sequencing pDS27.1.

Sequence analysis indicated that the DNA flanking the trans-poson encoded a paralog of the MotA proteins present in anumber of species of bacteria. To identify the entire gene,plasmid pDS27.5 was sequenced in its entirety using a series ofnested deletions. The insert was 3,315 nucleotides in lengthand contained an open reading frame (ORF) encoding a para-log of the MotA protein; we have called this gene rpmA forrequired for phagocytosis by macrophages. The putative ATGinitiation and stop codons for rpmA are present at nucleotidepositions 2007 and 2858, respectively (data not shown). ThisORF would encode a 283-amino-acid protein. There are sev-eral other potential initiation sites that would produce smallerproteins. A consensus s28 recognition site (TAA-N15-GCCGATAA) was located 58 bases upstream from the putative ATGinitiation codon (at position 2007) of the rpmA gene. There are20 nucleotides separating the stop codon of rpmA from theATG initiation codon of a putative rpmB gene. The first 423nucleotides of rpmB, which encode the first 141 amino acids ofthe RpmB protein, are present on pDS27.5. Partial comple-mentation of phagocytosis of H27A was observed when onlythe rpmA gene was present in trans. Both the rpmA and rpmBgenes were present on the plasmid pDS27B6 which, whenintroduced in trans, restored phagocytosis to parental levels.There were two ORFs present on pDS27.5 upstream of therpmA gene. Plasmid pDS290, which contained these genes, didnot complement the phagocytosis defect. The gene immedi-ately upstream of rpmA has been termed orfX since it pos-sessed little homology to any component in the databases,

FIG. 2. Motility of P. aeruginosa 4020 and phagocytosis-deficient mutants. P. aeruginosa 4020 strains were stabbed into 0.3% L agar plates and incubated for theindicated times at 37°C. Panels: A, P. aeruginosa PAK, 18 h of incubation; B, C, and D, and P. aeruginosa 4020 after 14, 16, or 18 h of incubation, respectively; E,P. aeruginosa 4020 H27A after 16 h of incubation at 37°C.

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while a partial cysD homolog encoding an O-acetylhomoserinesulfhydrylase was present downstream of the orfX gene (Fig.4). Plasmid pDS297, which contained the entire rpmB gene aswell as a gene encoding a homolog of the YjeQ protein fromE. coli, had no effect on phagocytosis of H27A when providedin trans. This arrangement of genes is in agreement with that ofthe P. aeruginosa PAO1 genome (http://www.pseudomonas.com). Sequencing of pDS27.1 indicated that the transposonhad inserted between nucleotides 2397 and 2398 of pDS27.5corresponding to insertion between R130 and I131 of the pu-tative RpmA protein.

Construction of a rpmA null mutant. A Tc cassette frommini-Tn5Tc was ligated into a unique SmaI site in the rpmAgene. This was mobilized into P. aeruginosa 4020 as a constructin pUC19mob. A mutant in the rpmA gene was isolated (4020-27) and utilized in a nonopsonic phagocytosis assay. The4020-27 mutant was as resistant as H27A to nonopsonic phago-cytosis (Fig. 6). A single crossover cointegrate (4020-27Sc) wasphagocytosed as well as the parental strain.

Analysis of RpmA. The molecular mass of the predicted 283amino acid RpmA protein is 30,743 Da. The P. aeruginosa 4020RpmA protein is 95% identical to the predicted PAO1 RpmAprotein. Analysis of the predicted amino acid sequence indi-cates that P. aeruginosa RpmA likely has six putative trans-membrane helices. Overall, there was 41% identity and 64%similarity between P. aeruginosa RpmA and its most closelyrelated homolog, S. enterica serovar Typhimurium MotA.Other closely related homologs include: E. coli MotA, Vibrio

parahaemolyticus LafT, Agrobacterium tumefaciens MotA, andSinorhizobium meliloti MotA.

DISCUSSION

The interaction of P. aeruginosa with macrophages in theabsence of opsonins is multifactorial. Both the bacterial flagel-lar filament and the pilus have been shown to be required forthe initial interaction of the bacterium with the macrophage. Inthis study, we have mutagenized a clinical strain of P. aerugi-nosa and identified mutants with defects in nonopsonic phago-cytosis mediated by mouse peritoneal macrophages. Since bac-terial organelles involved in motility are implicated in thebacterium-macrophage interaction, we devised specific selec-tion and enrichment procedures to decrease the likelihood ofisolating these types of mutations. The rationale for growingthe mutant pool under static conditions and utilizing the pel-licle as a source of organisms was to decrease our chances thatpilin and flagellin mutants would be isolated. These classes ofmutants would not be well represented in the pellicle of a staticculture. After testing 950 individual mutants, we identified 85mutants that had a phagocytic defect. Several mutants had anumber of readily identifiable phenotypic defects in addition tothe phagocytosis defect. Despite the screening procedures, wedid identify mutants in both pilus and flagellar biosyntheticpathways, including flagellin-deficient mutants (T13H) andphage PO4-resistant mutants (pilus defective; H39F).

The parental strain used for the mutagenesis exhibited amotility pattern unusual for P. aeruginosa. It resembled theswarming phenotype exhibited by other bacteria, including E.coli, Serratia marcescens, and S. enterica serovar Typhimurium(1, 19). This swarming phenotype has also recently been de-scribed in Pseudomonas syringae B728a (26). Swarming motilityexhibited by P. aeruginosa has recently been observed in otherlaboratories (48). We are currently characterizing the nature ofthis swarming phenotype, but it appears that it is not requiredfor nonopsonic phagocytosis since H27A displays the samemotility pattern as the parent.

We characterized in depth one P. aeruginosa mutant whichhad a transposon insertion in a gene that encoded a paralog ofenteric MotA proteins, which we have called rpmA. This mu-tant was poorly ingested compared to the parental strain. Withthe sequencing of the P. aeruginosa PAO1 genome it is appar-ent that P. aeruginosa possesses two MotA homologs in sepa-rate regions of the chromosome. Recently, Kato et al. (24)constructed a mutant in orf1 of P. aeruginosa PAO1 whichencodes a MotA homolog and demonstrated that this mutant(PM1) does not swim in agar motility plates but is motile whenviewed microscopically. This suggests that orf1 likely encodesthe functional equivalent of enteric MotA proteins. Further-more, the arrangement of genes surrounding orf1 includes anumber of chemotaxis genes, a finding which is similar to theclusters of chemotaxis genes surrounding enteric motA genes.The orf1 gene would encode a 246-amino-acid protein of apredicted molecular mass of 25,962 Da. In this study, we haveidentified the other paralog of MotA, RpmA. Analysis of theregion surrounding the P. aeruginosa 4020 rpmA gene revealeda high degree of identity with the PAO1 genome. A MotBparalog termed RpmB is immediately downstream of RpmA.There are no apparent chemotaxis or motility genes surround-ing rpmA or rpmB. This contrasts with other MotA homologs.The predicted PAO1 Orf1/MotA and 4020 RpmA proteins are24.7% identical and 59.6% similar over a 246-amino-acid over-lap, which suggests that they have some structural similaritybut are likely functionally distinct. RpmA is longer than PAO1Orf1/MotA (283 versus 246 amino acids) and is structurally

FIG. 3. Detection of flagellin or pilin in selected mutants. Strains are labeledat the top of each lane. PAK-N1G (rpoN::Gm) was used as a negative control(i.e., no pilin, no flagellin). Samples were electrophoresed in SDS–12% PAGEgels and transferred to nitrocellulose. Immunoreactive components were visual-ized with either monoclonal anti-PAK flagellin (A) or anti-PAK pilin polyclonalantibody (B), followed by horseradish peroxidase-labeled secondary antibody.The asterisk in panel A indicates flagellin. Numbers on the left refer to themolecular mass in kilodaltons.

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more similar to enteric MotA proteins than to PAO1 MotA.The only other organisms that have had two MotA homologsdescribed are Bacillus subtilis and B. megaterium (17, 21). In B.subtilis, one of these homologs termed MotA plays a role inmotility (34), while the function of the second homolog(ORF1) is unknown but unrelated to motility (17, 22). ThisORF1 is present downstream of the ccpA gene, which is in-volved in catabolite repression, and immediately upstream of aMotB homolog, ORF2.

The phagocytic defect of H27A (rpmA::Tn5G) was only par-tially complemented when the rpmA gene was provided intrans, possibly due to a dominant-negative effect of the rpmAmutation. Multiple copies of MotA form a complex with otherproteins to form the flagellar motor (56, 58). It is possible thatRpmA also forms a complex with other components. It is morelikely, however, that the transposon insertion into the rpmAgene in H27A had a polar effect on the rpmB gene, sinceproviding both genes in trans fully abrogated the ingestiondefect. Furthermore, a rpmA::Tc mutant (4020-27) was as re-sistant as the transposon mutant H27A to nonopsonic phago-cytosis.

Our results suggest that RpmA of P. aeruginosa may havedifferent or additional functions than the MotA of other spe-cies. E. coli and S. enterica serovar Typhimurium motA andmotB null mutants produce flagella that are paralyzed and sodo not swim in motility plates. P. aeruginosa H27A is motile in

the absence of a functional RpmA (and possibly RpmB), andthis motility was indistinguishable from that of the parentalstrain. This indicated that RpmA was not involved in the un-usual motility exhibited by P. aeruginosa 4020. This swarmingmotility is not involved in nonopsonic phagocytosis, since thephagocytosis-resistant mutant H27A (rpmA::Tn5G) exhibitsthe same motility as the parental strain. Flagellar rotation in P.aeruginosa 4020 appears to be unrelated to RpmA function,since H27A remains motile. However, it was possible that theunusual motility expressed by P. aeruginosa 4020 masks ourability to detect a defect in flagellar motility of the rpmA strain.

A consensus s28 recognition site upstream of the rpmA genesuggests that this gene is regulated via the flagellum regulatoryhierarchy. Since rpmA was not involved in the unusual motilitypattern, it is possible that a second flagellum-dependent mo-tility system involving RpmA exists. The phagocytosis defectexhibited by H27A (rpmA mutant phenotype) suggests thatsome subtle aspect of genes regulated by the flagellar cascadeare involved in phagocytosis. It has been well established thatmotility is involved in bacterial interactions with cells (re-viewed in reference 39). Flagella have been shown to play arole in certain types of infections caused by P. aeruginosa (14).Flagellum-mediated motility is involved in the formation ofbiofilms on abiotic surfaces by both P. aeruginosa and E. coli(38, 40). The flagellar cap protein, FliD, interacts with mucin(3), and other flagellar components are involved in the inter-

FIG. 4. Mapping of the rpmA region. Plasmids are indicated in boldface. Only one orientation of each insert is shown. Upstream and in an opposite orientationto the rpmA gene are orfX, which codes for a protein of unknown function, and the 59 region of a cysD homolog encoding a putative N-acetylhomocysteine sulfhydrylase.The yjeQ gene and a partial yjeR (orn) gene are present on pDS27B6 downstream of the rpmB gene. Various subclones of pDS27B6 described in the text are shown.Restriction enzyme abbreviations: P, PstI; S, SalI; Sc, SacI; Sm, SmaI; V, EcoRV; X, XhoI. The map is drawn to scale (bar 5 1 kb).

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FIG. 5. Phagocytosis of P. aeruginosa 4020 H27A containing rpmA and rpmB derivatives in trans. Phagocytosis assays were performed as described in the text.Bacteria were not centrifuged onto the monolayer. Plasmid-encoded proteins were induced with 0.5 mM IPTG (isopropyl-b-D-thiogalactopyranoside). The numbersrepresent the mean, and the error bars represent the standard error for triplicate experiments. p, Number of bacteria phagocytosed significantly lower than with P.aeruginosa 4020 H27A (pDS27.6) (P , 0.05); pp, number of bacteria phagocytosed significantly higher than with P. aeruginosa 4020 H27A (pDS27.6) (P , 0.01); #,number of bacteria phagocytosed significantly lower than with P. aeruginosa 4020 (P , 0.001).

FIG. 6. Nonopsonic phagocytosis of a P. aeruginosa 4020 rpmA::Tc (4020-27) mutant by mouse peritoneal thioglycolate-elicited macrophages. Phagocytosis assayswere performed as described in Materials and Methods. The numbers represent the mean plus the standard error for triplicate experiments. An asterisk indicates thenumber of bacteria phagocytosed was significantly lower than with P. aeruginosa 4020 (P , 0.0001).

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action of P. aeruginosa with eukaryotic cells (50). We believethat it is unlikely that the RpmA protein is directly involved inthe interaction of P. aeruginosa with macrophages, but thefunction of RpmA is required for ingestion.

This study indicates that a functional RpmA protein (and toa lesser extent RpmB) is required for efficient phagocytosis ofP. aeruginosa 4020 by thioglycolate-elicited murine peritonealmacrophages. Two paralogs of enteric MotA proteins exist inP. aeruginosa, and these appear to be functionally distinct. P.aeruginosa PAO1 Orf1/MotA mutants are not motile in agarswim plates (24), while P. aeruginosa 4020 rpmA mutants arestill motile. Thus, the function of RpmA in P. aeruginosa isdifferent from that of MotA in enteric bacteria. Ongoing char-acterization of RpmA should allow us to delineate the preciserole it plays in nonopsonic phagocytosis.

ACKNOWLEDGMENTS

We thank Eshwar Mahenthiralingam and members of the Speert labfor helpful comments, Jacqueline Chung for technical assistance, Mar-guerite Cervin for careful review of the manuscript, and Steve Lory forstrains.

This work was supported by a grant to D.P.S. from the MedicalResearch Council of Canada.

REFERENCES

1. Alberti, L., and R. M. Harshey. 1990. Differentiation of Serratia marcescens274 into swimmer and swarmer cells. J. Bacteriol. 172:4322–4328.

2. Altschul, S. F., T. F. Madden, A. A. Schaffer, J. Zhang, W. Miller, and D. J.Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of pro-tein database search programs. Nucleic Acids Res. 25:3389–3402.

3. Arora, S. K., B. W. Ritchings, E. C. Almira, S. Lory, and R. Ramphal. 1998.The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible formucin adhesion. Infect. Immun. 66:1000–1007.

4. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure forscreening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523.

5. Blair, D. F. 1995. How bacteria sense and swim. Annu. Rev. Microbiol.49:489–522.

6. Blair, D. F., and H. C. Berg. 1988. Restoration of torque in defective flagellarmotors. Science 242:1678–1681.

7. Blair, D. F., and H. C. Berg. 1990. The MotA protein of Escherichia coli is aproton-conducting component of the flagellar motor. Cell 60:439–449.

8. Block, S. M., and H. C. Berg. 1984. Successive incorporation of force gen-erating units in the bacterial rotary motor. Nature 309:470–472.

9. Carnoy, C., A. Scharfman, B. E. Van, G. Lamblin, R. Ramphal, and P.Roussel. 1994. Pseudomonas aeruginosa outer membrane adhesins for hu-man respiratory mucus glycoproteins. Infect. Immun. 62:1896–1900.

10. Coburn, J., and D. W. Frank. 1999. Macrophages and epithelial cells responddifferently to the Pseudomonas aeruginosa type III secretion system. Infect.Immun. 67:3151–3154.

11. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiringvitamin B-12. J. Bacteriol. 60:17–28.

12. Doig, P., T. Todd, P. A. Sastry, K. K. Lee, R. S. Hodges, W. Paranchych, andR. T. Irvin. 1988. Role of pili in adhesion of Pseudomonas aeruginosa tohuman respiratory epithelial cells. Infect. Immun. 56:1641–1646.

13. Doig, P., P. A. Sastry, R. S. Hodges, K. K. Lee, W. Paranchych, and R. T.Irvin. 1990. Inhibition of pilus-mediated adhesion of Pseudomonas aerugi-nosa to human buccal epithelial cells by monoclonal antibodies directedagainst pili. Infect. Immun. 58:124–130.

14. Drake, D., and T. C. Montie. 1988. Flagella, motility, and invasive virulenceof Pseudomonas aeruginosa. J. Gen. Microbiol. 134:43–52.

15. Frank, D. 1997. The exoenzyme S regulon of Pseudomonas aeruginosa. Mol.Microbiol. 26:621–629.

16. Garrett, E. S., D. Perlegas, and D. J. Wozniak. 1999. Negative control offlagellum synthesis in Pseudomonas aeruginosa is modulated by the alterna-tive sigma factor AlgT (AlgU). J. Bacteriol. 181:7401–7404.

17. Grundy, F. J., D. A. Waters, T. Y. Takova, and T. M. Henkin. 1993. Identi-fication of genes involved in utilization of acetate and acetoin in Bacillussubtilis. Mol. Microbiol. 10:259–271.

18. Hancock, R. E. W., L. M. Mutharia, L. Chan, R. P. Darveau, D. P. Speert,and G. B. Pier. 1983. Pseudomonas aeruginosa isolates from patients withcystic fibrosis: a class of serum sensitive, nontypable strains deficient inlipopolysaccharide O side chains. Infect. Immun. 42:170–177.

19. Harshey, R. M., and T. Matsuyama. 1994. Dimorphic transition in Esche-richia coli and Salmonella typhimurium: surface-induced differentiation intohyperflagellate swarmer cells. Proc. Natl. Acad. Sci. USA 91:8631–8635.

20. Hauser, A. R., and J. E. Engel. 1999. Pseudomonas aeruginosa induces type-

III-secretion-mediated apoptosis of macrophages and epithelial cells. Infect.Immun. 67:5530–5537.

21. Hueck, C. J., A. Kraus, and W. Hillen. 1994. Sequences of ccpA and twodownstream Bacillus megaterium genes with homology to the motAB operonfrom Bacillus subtilis. Gene 143:147–148.

22. Hueck, C. J., A. Kraus, D. Schmiedel, and W. Hillen. 1995. Cloning, expres-sion and functional analyses of the catabolite control protein CcpA fromBacillus megaterium. Mol. Microbiol. 16:855–864.

23. Ishimoto, K. S., and S. Lory. 1992. Identification of pilR, which encodes atranscriptional activator of the Pseudomonas aeruginosa pilin gene. J. Bac-teriol. 174:3514–3521.

24. Kato, J., T. Nakamura, A. Kuroda, and H. Ohtake. 1999. Cloning andcharacterization of chemotaxis genes in Pseudomonas aeruginosa. Biosci.Biotechnol. Biochem. 63:155–161.

25. Kelly, N. M., J. Kluftinger, B. L. Pasloske, W. Paranchych, and R. E. W.Hancock. 1989. Pseudomonas aeruginosa pili as ligands for nonopsonicphagocytosis by fibronectin-stimulated macrophages. Infect. Immun. 57:3841–3845.

26. Kinscherf, T. G., and D. K. Willis. 1999. Swarming by Pseudomonas syringaeB728a requires gacS (lemA) and gacA but not the acyl-homoserine lactonebiosynthetic gene ahlI. J. Bacteriol. 181:4133–4136.

27. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

28. Luzar, M. A., M. J. Thomassen, and T. C. Montie. 1985. Flagella and motilityalterations in Pseudomonas aeruginosa strains from patients with cystic fi-brosis: relationship to patient clinical condition. Infect. Immun. 50:577–582.

29. Macnab, R. M. 1992. Genetics and biogenesis of bacterial flagella. Annu.Rev. Genet. 26:131–158.

30. Mahenthiralingam, E., and D. P. Speert. 1995. Nonopsonic phagocytosis ofPseudomonas aeruginosa by macrophages and polymorphonuclear leuko-cytes requires the presence of the bacterial flagellum. Infect. Immun. 63:4519–4523.

31. Mahenthiralingam, E., M. E. Campbell, and D. P. Speert. 1994. Nonmotilityand phagocytic resistance of Pseudomonas aeruginosa isolates from chroni-cally colonized patients with cystic fibrosis. Infect. Immun. 62:596–605.

32. Mahenthiralingam, E., M. E. Campbell, J. Foster, J. S. Lam, and D. P.Speert. 1996. Random amplified polymorphic DNA typing of Pseudomonasaeruginosa isolates recovered from patients with cystic fibrosis. J. Clin. Mi-crobiol. 34:1129–1135.

33. Mahenthiralingam, E., D. A. Simpson, and D. P. Speert. 1997. Identificationand characterization of a novel DNA marker associated with epidemic Burk-holderia cepacia strains recovered from patients with cystic fibrosis. J. Clin.Microbiol. 35:808–816.

34. Mirel, D. B., V. M. Lustre, and M. J. Chamberlin. 1992. An operon ofBacillus subtilis motility genes transcribed by the sD form of RNA polymer-ase. J. Bacteriol. 174:4197–4204.

35. Mork, T., and R. E. W. Hancock. 1993. Mechanisms of nonopsonic phago-cytosis of Pseudomonas aeruginosa. Infect. Immun. 61:3287–3293.

36. Nunn, D. N., and S. Lory. 1992. Components of the protein-excretion appa-ratus of Pseudomonas aeruginosa are processed by the type IV prepilinpeptidase. Proc. Natl. Acad. Sci. USA 89:47–51.

37. Nunn, D. N., S. Bergman, and S. Lory. 1990. Product of the three accessorygenes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonasaeruginosa pili. J. Bacteriol. 172:2911–2919.

38. O’Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility arenecessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol.30:295–304.

39. Ottemann, K. M., and J. F. Miller. 1997. Roles for motility in bacterial-hostinteractions. Mol. Microbiol. 24:1109–1117.

40. Pratt, L. A., and R. Kolter. 1998. Genetic analysis of Escherichia coli biofilmformation: roles of flagella, motility, chemotaxis and type I pili. Mol. Micro-biol. 30:285–293.

41. Ridgway, H. F., M. Silverman, and M. I. Simon. 1977. Localization ofproteins controlling motility and chemotaxis in Escherichia coli. J. Bacteriol.132:657–665.

42. Ritchings, B. W., E. C. Almira, S. Lory, and R. Ramphal. 1995. Cloning andphenotypic characterization of fleS and fleR, new response regulators ofPseudomonas aeruginosa which regulate motility and adhesion to mucin.Infect. Immun. 63:4868–4876.

43. Saiman, L., K. Ishimoto, S. Lory, and A. Prince. 1990. The effect of piliationand exoproduct expression on the adherence of Pseudomonas aeruginosa torespiratory epithelial monolayers. J. Infect. Dis. 161:541–548.

44. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor,N.Y.

45. Sawa, T., T. L. Yahr, M. Ohara, K. Kurahashi, M. A. Gropper, J. P. Wiener-Kronish, and D. W. Frank. 1999. Active and passive immunization with thePseudomonas V antigen protects against type III intoxication and lung injury.Nat. Med. 5:392–398.

46. Schuster, S. C., and S. Khan. 1994. The bacterial flagellar motor. Annu. Rev.Biophys. Biomol. Struct. 23:509–539.

VOL. 68, 2000 NONOPSONIC PHAGOCYTOSIS OF P. AERUGINOSA MUTANTS 2501

on March 5, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

47. Schweizer, H. P. 1991. Escherichia-Pseudomonas shuttle vectors derived frompUC18/19. Gene 97:109–121.

48. Semmler, A. B., C. B. Whitchurch, and J. S. Mattick. 1999. A re-examinationof twitching motility in Pseudomonas aeruginosa. Microbiology 145:2863–2873.

49. Simpson, D. A., R. Ramphal, and S. Lory. 1992. Genetic analysis of Pseudo-monas aeruginosa adherence: distinct genetic loci control attachment toepithelial cells and mucins. Infect. Immun. 60:3771–3779.

50. Simpson, D. A., R. Ramphal, and S. Lory. 1995. Characterization of Pseudo-monas aeruginosa fliO, a gene involved in flagellar biosynthesis and adher-ence. Infect. Immun. 63:2950–2957.

51. Smith, A. W., and B. H. Iglewski. 1989. Transformation of Pseudomonasaeruginosa by electroporation. Nucleic Acids Res. 17:10509.

52. Speert, D. P. 1994. Pseudomonas aeruginosa infections in patients with cysticfibrosis, p. 183–235. In A. L. Baltch and R. P. Smith (ed.), Pseudomonasaeruginosa infections and treatment. Marcel Dekker, Inc., New York, N.Y.

53. Speert, D. P., and S. Gordon. 1992. Phagocytosis of unopsonized Pseudomo-nas aeruginosa by murine macrophages is a two-step process requiring glu-cose. J. Clin. Investig. 90:1085–1092.

54. Speert, D. P., B. A. Loh, D. A. Cabral, and I. E. Salit. 1986. Nonopsonicphagocytosis of nonmucoid Pseudomonas aeruginosa by human neutrophilsand monocyte-derived macrophages is correlated with bacterial piliation andhydrophobicity. Infect. Immun. 53:207–212.

55. Starnbach, M. N., and S. Lory. 1992. The fliA (rpoF) gene of Pseudomonasaeruginosa encodes an alternative sigma factor required for flagellin synthe-sis. Mol. Microbiol. 6:459–469.

56. Stolz, B., and H. C. Berg. 1991. Evidence for interactions between MotA andMotB, torque-generating elements of the flagellar motor of Escherichia coli.J. Bacteriol. 173:7033–7037.

57. Strom, M. S., and S. Lory. 1986. Cloning and expression of the pilin gene ofPseudomonas aeruginosa PAK in Escherichia coli. J. Bacteriol. 165:367–372.

58. Tang, H., T. F. Braun, and D. F. Blair. 1996. Motility protein complexes inthe bacterial flagellar motor. J. Mol. Biol. 261:209–221.

59. Tosi, M. F., H. Zakem, and M. Berger. 1988. Neutrophil elastase cleavesC3Bi on opsonized Pseudomonas as well as CR1 on neutrophils to create afunctionally important opsonin receptor mismatch. J. Clin. Investig. 86:300–308.

60. Totten, P. A., and S. Lory. 1990. Characterization of the type a flagellin genefrom Pseudomonas aeruginosa PAK. J. Bacteriol. 172:7188–7199.

61. Totten, P. A., J. C. Lara, and S. Lory. 1990. The rpoN gene product ofPseudomonas aeruginosa is required for expression of diverse genes, includ-ing the flagellin gene. J. Bacteriol. 172:389–396.

62. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.

63. Woods, D. E., D. C. Straus, W. J. Johanson, V. K. Berry, and J. A. Bass. 1980.Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccalepithelial cells. Infect. Immun. 29:1146–1151.

64. Yahr, T. L., J. T. Barbieri, and D. W. Frank. 1996. Exoenzyme S of Pseudo-monas aeruginosa is secreted by a type III pathway. Mol. Microbiol. 22:991–1003.

Editor: E. I. Tuomanen

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