Moraxella catarrhalis Synthesizes an Autotransporter That ... · Moraxella catarrhalis has...

JOURNAL OF BACTERIOLOGY, Feb. 2008, p. 1459–1472 Vol. 190, No. 40021-9193/08/$08.00�0 doi:10.1128/JB.01688-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Moraxella catarrhalis Synthesizes an Autotransporter That Isan Acid Phosphatase�

Todd C. Hoopman,1,2 Wei Wang,1 Chad A. Brautigam,3 Jennifer L. Sedillo,1Thomas J. Reilly,4 and Eric J. Hansen1*

Department of Microbiology,1 Department of Internal Medicine, Pulmonary and Critical Care Medicine,2 and Department ofBiochemistry,3 University of Texas Southwestern Medical Center, Dallas, Texas 75390, and Department of

Veterinary Pathobiology and Veterinary, Medical Diagnostic Laboratory, University ofMissouri, Columbia, Missouri 652114

Received 19 October 2007/Accepted 21 November 2007

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acidphosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA)was most similar (the BLAST probability score was 10�10) to bacterial class A nonspecific acid phosphatases.The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins,whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransport-ers. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acidphosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membraneof M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhaliscells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocationdomain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporterconfirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strainO35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be comple-mented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M.catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directedmutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domainin MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first descriptionof an autotransporter protein that expresses acid phosphatase activity.

Initially thought to be a harmless commensal organism,Moraxella catarrhalis has gradually gained repute as an etio-logic agent of at least two significant diseases in humans. Thisgram-negative, unencapsulated bacterium has been shown tocolonize the upper airways of infants and very young children(14, 15) and is one of the three most prominent causes of otitismedia (45). Additionally, adults with chronic obstructive pul-monary disease are at risk for infectious exacerbations causedby M. catarrhalis (45, 66). A recent study indicates that eachyear in the United States, as many as four million chronicobstructive pulmonary disease exacerbations may be attributedto M. catarrhalis (46).

The secretion of proteins by gram-negative bacteria is afunction necessary for numerous metabolic and physiologicprocesses. Five different secretion systems have been well char-acterized in bacteria (13), and a sixth has recently been de-scribed (44, 56). The type V secretion system has receivedincreased attention in recent years (11, 27, 35, 38). The ab-sence of a requirement for energy coupling or accessory factorsfor successful protein secretion has resulted in this class ofproteins being described as autotransporters. In gram-negativebacteria, autotransporters make up the largest family of outer

membrane porins involved in protein translocation (12). Theautotransporter secretion system was first described for theimmunoglobulin A1 protease of Neisseria gonorrhoeae (54, 55),and subsequently, numerous autotransporters have beendescribed for other gram-negative bacteria (25, 27). A three-domain model for type V secretion systems has emerged, com-prising (i) an amino-terminal leader peptide or signal se-quence, (ii) the secreted mature protein (or passengerdomain), and (iii) a C-terminal translocation domain respon-sible for the formation of a pore in the outer membrane toallow passage of the passenger domain to the cell surface (26).

The passenger domains of previously described autotrans-porter systems have been shown to have widely different func-tions in gram-negative bacteria, including but not limited toproteolytic, adhesive, and cytotoxic activities (27). M. catarrhalishas been shown to synthesize at least three proteins (i.e., UspA1,UspA2, and Hag) that have been classified as trimeric autotrans-porters and one additional protein that is considered a conven-tional autotransporter (i.e., McaP) (for reviews, see references 11,19, and 35). These four previously characterized M. catarrhalisautotransporters have been shown to be involved in adherence(1), serum resistance (6), binding of immunoglobulin D (18),autoaggregation (51), and lipolysis (68).

Acid phosphatases catalyze the hydrolysis of phosphomo-noesters at an acidic pH (9). Bacterial nonspecific acid phos-phatases (NSAP) are subdivided into three classes (for a re-view, see reference 67). Class A acid phosphatases typically aresecreted enzymes with broad substrate specificity and usually

* Corresponding author. Mailing address: Department of Microbi-ology, University of Texas Southwestern Medical Center, 5323 HarryHines Boulevard, Dallas, TX 75390-9048. Phone: (214) 648-5974. Fax:(214) 648-5905. E-mail: [email protected].

� Published ahead of print on 7 December 2007.

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consist of relatively small (25 kDa to 27 kDa) polypeptides thatmay occur as monomers or oligomers. In contrast, class B acidphosphatases are polymeric metalloproteins that also demon-strate broad substrate specificity. Class C acid phosphatasesare the most recently identified and are lipoproteins that bearsome resemblance to class B acid phosphatases. Typically, theenzymatic activity of an acid phosphatase helps to catalyze thetwo-step hydrolysis of phosphomonoesters. The enzymes uti-lize a covalent inorganic phosphoenzyme complex as a short-lived intermediate (71).

The biological function of these bacterial nonspecific acidphosphatases has been defined in only a few instances. It hasbeen assumed that these enzymes are involved in cleavingorganic phosphoesters, such as nucleotides and sugar phos-phates, into inorganic phosphate and a dephosphorylatedproduct that can be transported across the cytoplasmic mem-brane (63). In addition, it has been suggested that intracellularpathogens can use acid phosphatases to affect signaling path-ways controlling the respiratory burst (58, 64). In Salmonellaenterica, the periplasmic AphA acid phosphatase removesphosphate from the nicotinamide mononucleotide so that theresultant nicotinamide ribonucleoside can be taken up by atransport system in the cytoplasmic membrane (21). The Hae-mophilus influenzae lipoprotein e (P4) has been reported toutilize NADP as a substrate (57) and has been studied in greatdetail (34, 49, 59, 61). A Legionella pneumophila acid phos-phatase was shown to be secreted by a type II secretion systembut was reported to not be essential for intracellular replica-tion of L. pneumophila in macrophages (4). Most recently, arespiratory burst-inhibiting acid phosphatase from Francisellatularensis has been crystallized and studied in considerabledetail (7, 17, 17, 58, 60).

In the present report, we describe the identification andcharacterization of a novel M. catarrhalis autotransporter(MapA) that exhibits acid phosphatase activity. When ex-pressed in recombinant form in Escherichia coli, the mapAgene product retained its acid phosphatase activity. Site-di-rected mutagenesis was used to confirm the identity of theactive site in the acid phosphatase domain, and the functionalability of the C-terminal translocation domain was proven ex-perimentally. We demonstrated that this autotransporter pro-tein is localized to the M. catarrhalis outer membrane and ishighly conserved among strains of this pathogen. To the best ofour knowledge, this is the first detailed description of an au-totransporter protein that expresses acid phosphatase activity.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. The bacterial strains andplasmids used in this study are listed in Table 1. M. catarrhalis strains were grownas described previously (5) using brain heart infusion (BHI) broth (Difco/BectonDickinson, Sparks, MD); this medium was supplemented with spectinomycin (15�g/ml) when appropriate. E. coli strains were grown on Luria-Bertani (LB)medium as described previously (65); this medium was supplemented with eitherampicillin (100 �g/ml) or spectinomycin (100 �g/ml) when required.

Phosphatase activity indicator plates. LB agar was supplemented with phe-nolphthalein diphosphate (1 mg/ml) and methyl green (50 �g/ml) as an indicatorof phosphatase activity (62). Phosphatase activity was evidenced by the appear-ance of green colonies on this medium, whereas phosphatase-negative coloniesappeared yellow or colorless.

Production of a MAb against MapA. The predicted amino acid (aa) sequenceof the MapA protein from M. catarrhalis strain O35E was used to design a smalloligopeptide for immunization purposes. A 20-aa sequence (KAYDGISHIYQ

DIETTTQDK; aa 378 to 397 from the O35E MapA protein) was synthesizedwith a cysteine residue added to the N terminus of this peptide. This peptide wascovalently coupled to Imject maleimide-activated mariculture keyhole limpethemocyanin (Pierce, Rockford, IL) and used to immunize mice. The hybridomafusion procedure was carried out by the Monoclonal Antibody Center at UTSouthwestern Medical Center. The MapA-reactive monoclonal antibody (MAb)1H12 was identified by screening hybridoma culture supernatant fluids in anenzyme-linked immunosorbent assay using the MapA-derived peptide describedabove as the antigen.

Preparation of bacterial samples and Western blot analysis. Agar plate-grownM. catarrhalis and E. coli cells were used to prepare whole-cell lysates as de-scribed previously (37). Outer membrane vesicles were prepared from broth-grown M. catarrhalis cells as described previously (47). Concentrated culturesupernatant fluids were prepared from overnight broth cultures of M. catarrhalisas described previously (73), except that the Amicon Ultra centrifugal filterdevice (Millipore Corp., Billerica, MA) used in this procedure had a nominalcutoff of 10,000 Da. Western blot analysis was performed as described previously(5) by using MAb 1H12 to detect MapA and MAb 10F3 to detect the CopB outermembrane protein (22). The lipooligosaccharide (LOS) present in proteinaseK-treated whole-cell lysates was resolved by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) and stained with silver (69) or probed byWestern blot analysis with the M. catarrhalis LOS-specific MAb 8E7 as describedpreviously (6).

Assay for phosphatase activity. Acid phosphatase activity was measured byusing p-nitrophenyl phosphate (PnPP) (Sigma Chemical, St. Louis, MO) as thesubstrate in an assay procedure modified from that described previously by Reillyand colleagues (60). Recombinant E. coli strains were grown overnight in LBbroth supplemented with ampicillin. M. catarrhalis strains were grown overnightin BHI broth. The bacterial suspensions were subjected to centrifugation at 7,600 �g for 8 min. The bacterial cell pellet was suspended in 0.4 M sodium acetatebuffer (pH 6.0) to obtain a final suspension that yielded a reading of 300 Klett Uon a Klett-Summerson colorimeter (VWR Scientific). A 5-ml portion of thissuspension was then subjected to centrifugation at 1,860 � g for 5 min. The finalcell pellet was suspended in 1 ml of the sodium acetate buffer. Portions (100 �l)of this cell suspension and serial 10-fold dilutions were added in duplicate intoindividual wells in a 96-well, clear-bottom, black-sided microtiter plate (CorningInternational, Corning, NY). The standard assay mixture consisted of 100 �l ofbacterial suspension, 80 �l H2O, and 20 �l of the PnPP substrate (20 mM stocksolution). The reaction was allowed to proceed for 30 min at 37°C. The microtiterplate was then subjected to centrifugation at 96 � g for 5 min. A 100-�l portionof the resultant supernatant fluid was then transferred to an empty well, and a100-�l volume of 0.5 M glycine (pH 10.0) was added to the well to stop residualenzyme activity. The absorbance was then measured at 405 nm in a SpectraFluorPlus microplate reader (Tecan, Research Triangle Park, NC). Analysis of vari-ance methods were used for statistical analysis of enzyme activity levels (36).

Preparation of bacterial cell fractions. A 500-ml overnight culture of M.catarrhalis cells was harvested by centrifugation and suspended in 6 ml of phos-phate-buffered saline (PBS). This very dense suspension was sonicated using aBranson sonifier (model 450; Branson Ultransonics, Danbury, CT) for three1-min cycles at an output of 50% with a duty cycle of 5. The sonicate was thensubjected to centrifugation at 12,000 � g for 10 min at 4°C to remove whole cellsand large debris, and the resultant supernatant fluid was transferred to anothercentrifuge tube. This supernatant fluid was subjected to centrifugation at 100,000 �g for 90 min at 4°C. The final supernatant fluid was carefully removed bypipetting without disturbing the pellet; this fluid represented the soluble cyto-plasmic fraction. The pellet was resuspended in PBS and represented the cellenvelope fraction. The well-established method of Murphy and Loeb (47) wasused to prepare outer membrane vesicles from broth-grown M. catarrhalis cells.

Recombinant DNA techniques. Standard molecular biology and recombinantDNA techniques were performed as described previously (65). ExTaq DNApolymerase (PanVera, Madison, WI) was used for PCR-based amplification ofDNA fragments for cloning purposes and for overlapping extension PCR (28).Taq DNA polymerase (New England Biolabs) was used for colony PCRs. TheEasy-DNA kit (Invitrogen, Carlsbad, CA) was used to prepare chromosomalDNA from M. catarrhalis. The Qiaprep Spin Miniprep kit (Qiagen, Valencia,CA) was used to purify plasmid DNA from both E. coli and M. catarrhalis.

Cloning of the mapA gene. The oligonucleotide primers AT01 (5�-ATAGGATCCGCACCAGCCTCATCAAAT-3�, with the BamHI site underlined) andAT02 (5�-AATGGATCCTTGTGCCAGTGCCATTT-3�, with the BamHI siteunderlined) were used in PCR with chromosomal DNA from M. catarrhalisO35E to obtain a 3.5-kb fragment containing the mapA gene, which was ligatedinto plasmid pCR2.1 (Invitrogen, Carlsbad, CA) and used to transform E. coliTOP10 cells (Invitrogen). An ampicillin-resistant transformant was shown to

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contain the desired recombinant plasmid, which was designated pKW04. As anegative control, a 108-nucleotide (nt) fragment of M. catarrhalis O12E chro-mosomal DNA, carrying part of the promoter region of the uspA2 gene and partof the uspA2 open reading frame (ORF), was cloned into pCR2.1 and designatedpTH10. Similarly, the mapA genes from four other M. catarrhalis strains (7169,ETSU-9, O12E.44, and V1120) were amplified by PCR for nucleotide sequenceanalysis.

Construction of an M. catarrhalis mapA deletion mutant. The primers TH22(5�-TTGGATCCGACCTGCCAGCACGATCAAG-3�, with the BamHI site un-derlined) and TH20 (5�-TGA CTTGTCACGCCCGGGCATCAAGATGTTGATACC-3�, with the SmaI site underlined) were used to amplify a 1.2-kb fragmentcontaining the extreme 5� end of the O35E mapA gene and upstream flankingDNA, whereas primers TH21 (5�-GGGCGTGACAAGTCAAATCAA-3�, with apartial SmaI site underlined) and TH23 (5�-TACCGAGCTCGATGATAACGGGCGTGTA-3�, with the SacI site underlined) were used to obtain a 0.8-kbfragment containing the extreme 3� end of the mapA ORF and downstreamflanking DNA. These two amplicons were mixed and used as the templates in

overlapping extension PCR (28, 29) with primers TH22 and TH23 to generate anapproximately 2.0-kb fragment that was cloned into pCR2.1 and used to trans-form E. coli TOP10; recombinant clones were identified by blue-white screeningon LB agar containing X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyrano-side) (30 �g/ml). Colony PCR was used to confirm the presence of the 2-kbamplicon, and one of these recombinant plasmids, designated pTH03, was se-lected for further use. This plasmid was digested with SmaI, and the spectino-mycin resistance cassette from pSPECR (74) was blunt end ligated into this siteto obtain plasmid pTH04. Plasmid pTH04 was used to transform the wild-type M.catarrhalis strain O35E as described previously (52), and transformants wereselected for spectinomycin resistance. One of these transformants, designatedO35E�mapA, was selected for further study. Additional mapA deletion mutantswere constructed in M. catarrhalis strains 7169 and ETSU-9 by the same method.

Construction of an M. catarrhalis mapA deletion mutant lacking an antibioticresistance cassette. The use of plasmid pWW115 (72) for complementationanalysis requires spectinomycin as the selective marker. This necessitated thereconstruction of the M. catarrhalis O35E�mapA mutant to eliminate the spec-

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Genotype or description Reference or source

StrainsM. catarrhalis

O35E Wild-type disease isolate 27169 Wild-type disease isolate 40ETSU-9 Wild-type disease isolate Steven BerkATCC 43617 Wild-type disease isolate American Type Culture

CollectionO12E.44 Wild-type disease isolate 37V1120 Nasopharyngeal isolate Frederick HendersonFIN2265 Wild-type disease isolate Merja HelminenFIN2406 Wild-type disease isolate Merja HelminenFIN2344 Wild-type disease isolate Merja HelminenV1145 Nasopharyngeal isolate Frederick HendersonV1156 Nasopharyngeal isolate Frederick HendersonFR3221 Wild-type disease isolate Richard WallaceFR2213 Wild-type disease isolate Richard WallaceFR2336 Wild-type disease isolate Richard WallaceB59911 Nasopharyngeal isolate David GoldblattB59504 Nasopharyngeal isolate David GoldblattETSU-17 Wild-type disease isolate Steven BerkETSU-5 Wild-type disease isolate Steven BerkO35E�mapA mapA deletion mutant, spectinomycin resistant This studyO35E�mapA-9 mapA deletion mutant, spectinomycin sensitive This studyO35E�mapA-9(pTH13) mapA deletion mutant containing pTH13, expresses MapA This studyO35E�mapA-9(pWW115) mapA deletion mutant containing the vector pWW115 This studyO35E.M mcaP mutant of O35E 39O35E.M(pTH34) O35E.M mutant containing pTH34, expresses the McaP-MapA fusion

proteinThis study

O35E.M(pWW115) O35E.M mutant containing the vector pWW115 This studyETSU-9�mapA mapA deletion mutant, spectinomycin resistant This study7169�mapA mapA deletion mutant, spectinomycin resistant This study

E. coliDH5� Host strain for cloning experiments 65TOP10 Host strain for cloning experiments Invitrogen

PlasmidspWW115 M. catarrhalis cloning vector, Specr 72pTH13 pWW115 containing the M. catarrhalis O35E mapA gene This studypTH34 pWW115 containing the M. catarrhalis O35E mcaP-mapA fusion construct This studypCR 2.1 Cloning vector, Kanr Ampr InvitrogenpKW04 pCR2.1 containing the M. catarrhalis O35E mapA gene This studypTH24 pKW04 with the H233A mutation This studypTH26 pKW04 with the H110A mutation This studypTH10 pCR2.1 containing a 108-nt fragment from the M. catarrhalis O12E uspA2

geneThis study

pTH03 pCR2.1 containing the 3� and 5� ends of the O35E mapA gene andflanking DNA linked through a SmaI site

This study

pSPECR Source of the spectinomycin resistance cassette 74pTH04 pTH03 with the spectinomycin resistance cassette in the SmaI site This study

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tinomycin resistance cassette. M. catarrhalis O35E�mapA was used in the platetransformation system (52) with the 2-kb amplicon described above (i.e., thatobtained with primers TH22 and TH23), which had the large mapA deletion.Potential transformants were patched onto both BHI agar and BHI-spectinomy-cin agar to identify those transformants in which allelic exchange had resulted inthe removal of the spectinomycin resistance cassette. Subsequent nucleotidesequence analysis of the relevant chromosomal region from one of these trans-formants, designated O35E�mapA-9, confirmed the successful removal of thespectinomycin resistance cassette from the disrupted mapA gene.

Construction of an M. catarrhalis plasmid containing the mapA gene. Chro-mosomal DNA from M. catarrhalis strain O35E was used in PCR with primersAT01 and TH31 (5�-CAAGAGCTCATCTATCGGCAGATGCTTGAAT-3�,with the SacI site underlined) to obtain a 3.4-kb amplicon containing the entiremapA gene. This amplicon was digested with both BamHI and SacI and thenligated into pWW115 (72), which had also been digested with the same twoenzymes. This ligation reaction mixture was used to electroporate M. catarrhalisO35E, and transformants were selected on BHI-spectinomycin agar. A plasmidfrom one of these transformants was shown to contain the mapA amplicon andwas designated pTH13.

Complementation of the mapA mutation. M. catarrhalis O35E�mapA-9 waselectroporated with either pTH13 or pWW115 (i.e., vector-only control). Spec-tinomycin-resistant recombinant clones were selected on BHI-spectinomycinagar. Nucleotide sequence analysis of the plasmid recovered from this recombi-nant strain [M. catarrhalis O35E�mapA-9(pTH13)] confirmed that pTH13 wassuccessfully cloned into this mutant. Additional nucleotide sequence analysis ofthe relevant chromosomal region of this complemented mutant confirmed thepresence of the mapA deletion.

Construction of an M. catarrhalis McaP-MapA fusion protein. Approximately1.5 kb of DNA encoding the leader peptide, predicted passenger (lipase) do-main, and 17 residues of the predicted �-helical linker region of the McaPprotein (39) of M. catarrhalis strain O35E was amplified by PCR using theprimers TH75 (5�-GCGCGGATCCCATTGCGGTAACT-3�, with the BamHIsite underlined) and TH77 (5�-GCTTTGTTGACCATGTTTAATCAGA-3�),with O35E chromosomal DNA as the template. A second PCR utilizing primersTH78 (5�-CATGGTCAACAAAGCATGGGAAGCTTGTATACATTA-3�) andTH76 (5�-GCGCGAGCTCCGCAGACACAGAA-3�, with the SacI site under-lined) was performed to amplify the nucleotide sequence (approximately 1.1 kb)encoding the entire predicted translocation module (i.e., autotransporter do-main) of the O35E MapA protein, together with 10 aa immediately upstream ofthis module. These two amplicons were mixed and used as the templates inoverlapping extension PCR with primers TH75 and TH76 to generate an ap-proximately 2.6-kb fragment. This amplicon was subsequently digested with bothSacI and BamHI and ligated into pWW115 (72). This ligation mixture was usedto electroporate the M. catarrhalis mcaP mutant O35E.M (39), and transfor-mants were selected on BHI-spectinomycin agar. A plasmid isolated from one ofthese transformants was shown to contain the desired fusion product and wasdesignated pTH34. Nucleotide sequence analysis of the 2.6-kb DNA insert in thepTH34 plasmid indicated a few nucleotide changes, one of which caused a singlepredicted amino acid change (i.e., Y105H) in the McaP passenger domain. Therewere no predicted amino acid changes in the MapA autotransporter domain.

Detection of surface localization of McaP by flow cytometry. Detection of theMcaP passenger domain on the surface of the recombinant strain M. catarrhalisO35E.M(pTH34) was accomplished by using polyclonal murine antibodies to aa51 to 650 of the O35E McaP protein (39) as the primary antibody in flowcytometry. Briefly, bacterial cells were grown overnight on BHI agar plates andsuspended in PBS to an optical density at 600 nm of 0.35. Portions (100 �l) ofthese suspensions were subjected to centrifugation at 18,500 � g and resus-pended in 100 �l of PBS containing 1% (wt/vol) bovine serum albumin (PBS-BSA) in which the polyclonal antibody to the McaP protein had been diluted1:60. These tubes were incubated at room temperature for 20 min and thenwashed three times with 500 �l of PBS-BSA. The bacteria were then incubatedin the dark with 1 �g of fluorescein isothiocyanate-conjugated goat anti-mouseantibody (Abcam, Cambridge, MA) for 20 min at room temperature. The bac-teria were next washed three times with 500 �l PBS-BSA, resuspended in 1 ml of

filter-sterilized PBS, and analyzed by flow cytometry utilizing a FACScan flowcytometer (Becton Dickinson).

Site-directed mutagenesis of the predicted active site in the acid phosphatasedomain in MapA. The QuikChange II site-directed mutagenesis kit (Stratagene,La Jolla, CA) was utilized to generate a specific mutation in the putative activesite of the M. catarrhalis MapA protein. By using pKW04 as the template, weused primers TH48 (5�-AGCCGAGTCATTGTGGGTGCGGCTTTTCCAACAGATACCATGACTTCT-3�, with altered bases underlined) and TH49 (5�-AGAAGTCATGGTATCTGTTGGAAAAGCCGCACCCACAATGACTCGGCT-3�, with altered bases underlined) in a PCR to change the histidine at position233 in the MapA protein to an alanine. The plasmid encoding the H233Amutation was designated pTH24, and nucleotide sequence analysis confirmedthat this was the only change. In addition, as a control, a second histidine wasmutated at a position predicted to be unlikely to be involved in the enzymaticactivity of the MapA protein. The H110A change was accomplished by usingpKW04 as the template, together with primers TH52 (5�-CCTTAAAGGAGTTTGCTCCGGCTATAACTGATGAACAGTTTGTAAATATC-3�, with alteredbases underlined) and TH53 (5�-GATATTTACAAACTGTTCATCAGTTATAGCCGGAGCAAACTCCTTTAAGG-3�, with altered bases underlined). Theplasmid encoding the H110A mutation was designated pTH26.

Nucleotide sequence accession numbers. The nucleotide sequences of thesemapA genes were deposited in GenBank and assigned the following accessionnumbers: EF186006 (for O35E), EF192600 (for 7169), EF192601 (for ETSU-9),EF192599 (for O12E.44), and EF192602 (for V1120).

RESULTS

Identification of a gene encoding a predicted autotrans-porter protein. Previous efforts in this laboratory involved theM. catarrhalis Hag protein (51, 52), which is encoded by a genelocated in contig 32 (GenBank accession number AX067457)from the sequenced genome of M. catarrhalis ATCC 43617. Anexamination of the chromosomal loci flanking the hag generevealed the presence of an ORF located approximately 2 kbupstream from hag, which encoded a predicted autotrans-porter protein (Fig. 1). More extensive BLAST-based analysesrevealed that the N-terminal region of this predicted auto-transporter protein resembled several bacterial nonspecificacid phosphatases (described in detail below). We tentativelydesignated this protein as M. catarrhalis acid phosphatase A(MapA).

Characterization of the mapA gene and its encoded proteinproduct. The mapA ORF contained 2,820 nt encoding a pre-dicted protein with 940 aa. The mapA gene was flanked up-stream by an ORF, designated prmA, encoding a predictedribosomal protein L11 methyltransferase (70) and downstreamby a gene encoding a predicted penicillin-binding protein 1B(Fig. 1). The mapA gene appears not to be transcriptionallylinked to any other ORFs. Nucleotide sequence analysis of themapA gene from five additional M. catarrhalis strains showedthat the encoded proteins were very highly conserved with 97%identity (Fig. 2). Hydrophobicity analysis of the N-terminalregion and the use of the SignalP predictor program (Sig-nalP V2.0.b2 [www.cbs.dtu.dk/services/SignalP/]) showedthe likely presence of a signal peptide with a predicted signalpeptidase I cleavage site between residues 23 and 24 (Fig.

FIG. 1. Schematic representation of the M. catarrhalis ATCC 43617 locus containing the mapA gene and adjacent genes, including hag.


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2). The calculated mass of the predicted mature MapAprotein was 103 kDa.

Cloning and expression of the mapA gene in E. coli. Toconfirm the presence of acid phosphatase activity in theMapA protein, the mapA gene from M. catarrhalis O35E wascloned into E. coli, yielding the recombinant plasmidpKW04. When E. coli DH5�(pKW04) was streaked ontophenolphthalein-methyl green agar, this recombinant strain

produced the deep green color associated with phosphataseactivity (Fig. 3A, lane 1). In contrast, when a control straincontaining pCR2.1 with an irrelevant M. catarrhalis DNAinsert (i.e., pTH10) was streaked onto the same medium, itdid not produce a color change (Fig. 3A, lane 2). Additionalenzymatic analysis using a liquid-phase assay with wholecells of E. coli DH5�(pKW04) (Fig. 3B, bar 1) showed thepresence of acid phosphatase activity at levels at least 10-

FIG. 2. Alignment of the predicted amino acid sequence of the MapA proteins from M. catarrhalis strains 7169, ATCC 43617, ETSU-9,O12E.44, O35E, and V1120. The arrow indicates the predicted signal peptidase I cleavage site between aa 23 and 24. The asterisk indicates theposition of the histidine residue (aa 233) located in the active site of the acid phosphatase domain (described in Results).


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fold greater than those obtained with the negative controlstrain (Fig. 3B, bar 2) (P � 0.0001).

Construction of an M. catarrhalis mapA mutant. To facilitateinvestigation of the expression of MapA in its native back-ground, we constructed the O35E�mapA deletion mutant asdescribed in Materials and Methods. PCR and nucleotide se-quence analysis were used to confirm the presence of thedesired deletion mutation (Fig. 4A). Western blot analysisusing the MapA-specific MAb 1H12 showed that the wild-typeparent strain (Fig. 4B, lane 1) expressed an antigen that boundthis MAb and migrated just below the 105-kDa marker,whereas the O35E�mapA mutant (Fig. 4B, lane 2) did not. Useof the liquid-phase enzymatic assay with whole cells of thesetwo strains showed that the wild-type M. catarrhalis parentstrain (Fig. 4C, bar 1) had levels of acid phosphatase activitythat were higher than those expressed by the O35E�mapAmutant (Fig. 4C, bar 2) (P � 0.0001). The protein compositionsof outer membrane vesicles obtained from both the wild-typeparent strain and the O35E�mapA mutant appeared to be thesame, as determined by SDS-PAGE, followed by Coomassieblue staining. It must be noted that the MapA protein is ex-pressed at a level that could not be detected by staining withCoomassie blue (data not shown). Both silver staining andWestern blot analysis with the M. catarrhalis LOS-reactiveMAb 8E7 (30) showed no apparent difference in the LOSexpressed by these two strains (data not shown). Similarly,there were no apparent differences in the growth rates of thewild-type parent strain and this mutant in BHI broth (data notshown).

Complementation of the mapA mutation. The wild-typemapA gene from M. catarrhalis O35E was cloned into

pWW115 to obtain pTH13. However, this recombinant plas-mid containing the mapA gene could not be used with theO35E�mapA mutant because this mutant contained the spec-tinomycin resistance cassette. Therefore, transformation andallelic exchange were used to excise the spectinomycin resis-tance cassette from O35E�mapA, resulting in a new mapAdeletion mutant designated O35E�mapA-9. When pTH13 wasintroduced into O35E�mapA-9, the resultant recombinant M.catarrhalis strain expressed the MapA protein (Fig. 4B, lane 3)at a level much greater than that expressed by the wild-typeparent strain (Fig. 4B, lane 1). A control construct in which thepWW115 plasmid was introduced into the O35E�mapA-9 mu-tant did not express any MapA protein (Fig. 4B, lane 4). Asexpected, acid phosphatase activity expressed by the recombi-nant strain containing the cloned mapA gene in trans (Fig. 4C,bar 3) was much greater than that expressed by the samemutant containing the empty plasmid vector (Fig. 4C, bar 4)(P � 0.0001).

Detection of the MapA protein in other M. catarrhalisstrains. MAb 1H12 was used to detect the presence of MapAin other M. catarrhalis isolates. The MapA protein was readilydetectable in whole-cell lysates of these 14 additional strains(Fig. 5A, lanes 3 to 16), which included both strains isolatedfrom patients with disease and strains isolated from the naso-pharynxes of healthy individuals. The positive and negativecontrols in this screen included the wild-type M. catarrhalisstrain O35E (Fig. 5A, lane 1) and the O35E�mapA mutant(Fig. 5A, lane 2).

Localization of the MapA protein. When whole-cell lysatesand cell envelope preparations of M. catarrhalis O35E and theM. catarrhalis O35E�mapA mutant were probed by Western

FIG. 3. Expression of acid phosphatase activity by recombinant E. coli cells. (A) Appearance of recombinant E. coli strains grown overnighton phenolphthalein-methyl green agar. Lanes: 1, E. coli DH5�(pKW04); 2, E. coli DH5�(pTH10); 3, E. coli DH5�(pTH24) with the H233Amutation; 4, E. coli DH5�(pTH26) with the H110A mutation. (B) Acid phosphatase assay using PnPP as the substrate with whole cells (2.5 � 106

CFU) of E. coli DH5�(pKW04) (bar 1), E. coli DH5�(pTH10) (bar 2), E. coli DH5�(pTH24) (bar 3), and E. coli DH5�(pTH26) (bar 4). The datarepresent the means from three independent experiments, with error bars showing the standard deviations of the means. OD405, optical densityat 405 nm.


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blot analysis with MAb 1H12, the MapA antigen was readilydetectable in the wild-type parent strain (Fig. 5B, lanes 1 and3), but not in the mapA deletion mutant (Fig. 5B, lanes 2 and4). We were unable to detect the presence of the MapA pro-tein either in the soluble cytoplasmic fraction derived from thewild-type parent strain (Fig. 5B, lane 5) or in the concentratedculture supernatant fluid of the same strain (Fig. 5B, lane 7).

Detection of the MapA protein in outer membrane vesiclepreparations derived from M. catarrhalis. The localization ofthe MapA protein to the cell envelope fraction (Fig. 5B, lane3), together with its predicted autotransporter domain (de-scribed above), indicated that this protein should be present inthe outer membrane of M. catarrhalis. To address this local-ization issue directly, outer membrane vesicles were extractedfrom three wild-type M. catarrhalis strains (O35E, ETSU-9,

and 7169) and their corresponding mapA deletion mutants asdescribed in Materials and Methods. Western blot analysiswith MAb 1H12 showed that the MapA protein was present inthe outer membrane vesicles from wild-type O35E (Fig. 6, lane1), ETSU-9 (Fig. 6, lane 3), and 7169 (Fig. 6, lane 5) andmissing from outer membrane vesicles of the mapA deletionmutants (Fig. 6, lanes 2, 4, and 6, respectively) constructedfrom these three wild-type strains.

Construction and characterization of an McaP-MapA fu-sion protein. To prove directly that MapA was an autotrans-porter protein, the predicted MapA translocation domain(designated AT [Fig. 7A]) was used to replace the predictedtranslocation domain of the McaP protein (39) as describedin Materials and Methods. The resultant mcaP-mapA fusionconstruct, encoding the McaP passenger domain (designated

FIG. 4. Construction and characterization of an M. catarrhalis mapA mutant. (A) Schematic showing the mapA deletion in the chromosome ofthe M. catarrhalis O35E�mapA mutant, together with the relevant primers used for PCR (with wild-type O35E chromosomal DNA) andoverlapping extension PCR. (B) Western blot analysis of MapA expression by whole-cell lysates of O35E (lane 1), O35E�mapA (lane 2),O35E�mapA-9(pTH13) (lane 3), and O35E�mapA-9(pWW115) (lane 4). All four samples were run on the same gel; exposure times different fromthose used for lanes 1 and 2 were used for lanes 3 and 4. Molecular mass position markers (in kilodaltons) are presented on the left side of thesepanels. (C) Acid phosphatase assay using PnPP as the substrate with whole cells (5 � 108 CFU) of wild-type M. catarrhalis O35E (bar 1) and theO35E�mapA mutant (bar 2) and with whole cells (5 � 106 CFU) of M. catarrhalis O35E�mapA-9(pTH13) (bar 3) and the O35E�mapA-9(pWW115) (bar 4). The data in panel C represent the means from three independent experiments, with error bars showing the standard deviationsof the means. OD405, optical density at 405 nm.


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lipase [Fig. 7A]) and the MapA translocation domain, wascloned into pWW115 to obtain pTH34 and expressed in the M.catarrhalis mcaP mutant O35E.M. When probed by Westernblot analysis with a polyclonal antiserum previously shown tobind surface epitopes of McaP (39), the wild-type O35E strain(Fig. 7B, lane 1) expressed a readily detectable McaP protein.Neither the mcaP mutant (Fig. 7B, lane 2) nor the mcaPmutant containing the vector pWW115 (Fig. 7B, lane 3) ex-pressed any detectable protein reactive with this antiserum. Incontrast, the recombinant M. catarrhalis strain O35E.M(pTH34)(Fig. 7B, lane 4) expressed an McaP-MapA fusion protein ofthe predicted size. To confirm that the McaP passenger do-main of this fusion protein had been translocated to the sur-face of M. catarrhalis by the MapA translocation domain, thesame four strains were subjected to flow cytometry using theMcaP polyclonal antiserum as the primary antibody. Boththe wild-type strain O35E (Fig. 7C, panel 1) and the mcaP mutantexpressing the McaP-MapA fusion protein (Fig. 7C, panel 4)showed similar levels of positive reactivity with these poly-

clonal antibodies, whereas the mcaP mutant (Fig. 7C, panel 2)and the mcaP mutant containing the vector pWW115 (Fig. 7C,panel 3) exhibited very little reactivity with the same antibod-ies.

Comparison of MapA with other bacterial acid phosphata-ses. An analysis of the deduced amino acid sequence of MapAfrom M. catarrhalis ATCC 43617 using BLAST (3) revealedthat the N-terminal 273 aa of this protein have homology withseveral bacterial orthologs previously identified as class ANSAP. Of the top eight homologous sequences, BLAST prob-ability scores ranged from 8 � 1010 for the Pseudomonasfluorescens ortholog (27% amino acid identity over 237 resi-dues) to 4 � 1011 for the Enterobacter aerogenes ortholog(30% amino acid identity over 213 residues). Included in thelist of MapA orthologs is the nonspecific class A acid phos-phatase from Escherichia blattae (29% amino acid identity over213 residues; the BLAST probability score was 6 � 1010), anenzyme for which the three-dimensional structure (31) hasbeen elucidated (Protein Data Bank [PDB] code 1D2T). Re-sults from ClustalW sequence alignment of the acid phos-phatase portion of MapA with these eight orthologs are shownin Fig. 8, together with the signature motif of the NSAP classA family as described previously by Thaller and colleagues(67).

Site-directed mutagenesis of the predicted active site in theMapA protein. The aforementioned signature motif for acidphosphatases (67) and other related phosphatases has twoconserved histidine residues (Fig. 8). The second correspondsto H233 of the MapA protein. By analogy to other enzymes(i.e., glucose 6-phosphatase and vanadium-containing chlo-roperoxidase) that contain this signature sequence, H233 pu-tatively serves to attack a phosphorylated substrate, thereafterforming a covalent phosphoenzyme intermediate (24, 50). In

FIG. 5. Detection of the MapA protein in other M. catarrhalis strains and in different cell fractions. (A) Western blot analysis using theMapA-directed MAb 1H12 to probe whole-cell lysates of the following M. catarrhalis strains: O35E (lane 1), the O35E�mapA mutant (lane 2),FIN2265 (lane 3), FIN2406 (lane 4), FIN2344 (lane 5), V1145 (lane 6), V1156 (lane 7), V1120 (lane 8), FR3221 (lane 9), FR2213 (lane 10), FR2336(lane 11), B59911 (lane 12), B59504 (lane 13), ETSU-17 (lane 14), ETSU-5 (lane 15), and ATCC 43617 (lane 16). (B) Western blot analysis usingthe MapA-directed MAb 1H12 to probe whole-cell lysates (lanes 1 and 2), cell envelopes (lanes 3 and 4), cytoplasmic proteins (lanes 5 and 6), andconcentrated culture supernatant fluid (lanes 7 and 8) prepared from M. catarrhalis O35E (lanes 1, 3, 5, and 7) and the O35E�mapA mutant (lanes2, 4, 6, and 8). Protein loads in each pair of lanes 3 to 8 were standardized by means of the Bradford protein assay. Molecular mass position markers(in kilodaltons) are present on the left side of the figure.

FIG. 6. Detection of MapA in outer membrane vesicles from threeM. catarrhalis strains. Proteins present in outer membrane vesiclesprepared from O35E (lane 1), the O35E�mapA mutant (lane 2),ETSU-9 (lane 3), the ETSU-9�mapA mutant (lane 4), 7169 (lane 5),and the 7169�mapA (lane 6) mutant were resolved by SDS-PAGE andprobed by Western blot analysis with MAb 1H12. The Bradfordmethod was used to standardize protein amounts. Molecular massposition markers (in kilodaltons) are present on the left side of thefigure.


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the acid phosphatase PiACP from Prevotella intermedia, theanalogous histidine (H209) is essential for catalytic activity(10). To determine whether H233 of MapA was involved inacid phosphatase activity, site-directed mutagenesis was usedto convert this histidine to an alanine. The resultant recombi-nant MapA (H233A) protein expressed by pTH24 (Fig. 3A,lane 3, and B, bar 3) had little or no phosphatase activitycompared to that expressed by the wild-type protein encodedby pKW04 (Fig. 3A, lane 1, and B, bar 1) (P � 0.0001).

Alteration of a different histidine residue (H110) outsidethe predicted active site in pTH26 had no apparent effect onphosphatase activity as measured by the indicator plate as-say (Fig. 3A, lane 4). However, the level of enzymatic ac-tivity obtained with pTH26 (Fig. 3B, bar 4) was slightly lessthan that obtained with the wild-type enzyme expressed bypKW04 (Fig. 3B, bar 1) (P � 0.0104). These data indicatethat H233 is essential for the phosphatase activity of the M.catarrhalis MapA protein.

FIG. 7. Construction and characterization of an McaP-MapA fusion protein. (A) Schematic of the predicted domains used to produce theMcaP-MapA fusion protein expressed by pTH34. AP, acid phosphatase domain in MapA; P, pertactin domain in MapA; AT, translocation domainin both MapA and McaP; lipase, passenger domain in McaP. This passenger portion of the McaP protein also contained 17 residues from thepredicted �-helical linker domain (39). The translocation domain of the MapA protein used in this fusion construct also contained 10 aa locatedimmediately upstream of this domain. (B) Western blot analysis of protein present in whole-cell lysates from O35E (lane 1), the mcaP mutantO35E.M (lane 2), O35E.M(pWW115) (lane 3), and O35E.M(pTH34) (lane 4) as determined by using polyclonal McaP antiserum as the primaryantibody (upper panel). The arrow indicates the position of the wild-type McaP protein in lane 1 and the McaP-MapA fusion protein in lane 4.Molecular mass position markers (in kilodaltons) are present on the left side of the panel. The CopB-specific MAb 10F3 (22) was used to probethese whole-cell lysates to ensure equivalent loading of these samples (lower panel). (C) Flow cytometry-based detection of polyclonal McaPantibodies binding to the surfaces of O35E (panel 1), the mcaP mutant O35E.M (panel 2), O35E.M(pWW115) (panel 3), and O35E.M(pTH34)(panel 4).


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Computer-based structural modeling of the MapA protein.The N-terminal portion of the MapA protein (aa 24 to 279[Fig. 7A]) is most similar to class A NSAP molecules as de-scribed above. Sequence alignment of this region of the MapA

protein with the NSAP of E. blattae demonstrated that the twoproteins share about 23% identity over this region. It thereforeappears to be likely that the MapA amino terminus will besignificantly structurally similar to the NSAP of E. blattae. The

FIG. 8. ClustalW-derived alignment of eight nonspecific class A acid phosphatases with the N-terminal portion of the MapA protein from M.catarrhalis ATCC 43617. Results from ESPript are shown (20), and they contain the signature motif of the NSAP class A family as describedpreviously by Thaller and colleagues (67). This motif, KX6RP-(X12–54)-PSGH-(X31–54)-SRX5HX3D, along with other highly conserved residues,is indicated in red or white letters and extends from aa 133 to aa 211 (relative to the E. blattae numbering sequence). The active site histidine (23),shown at position 207 (relative to the E. blattae numbering sequence), is present in all of these orthologs near the N terminus of helix 12 and isindicated by the black arrows. Abbreviations: E.bla, E. blattae (GenBank accession number BAA84942); M.cat, M. catarrhalis ATCC 43617(derived from contig 32 [GenBank accession number AX067457]); E.aer, Enterobacter aerogenes (GenBank accession number BAB18917); K.pne,Klebsiella pneumoniae (GenBank accession number CAB59725); D.psy, Desulfotalea psychrophila LSv54 (GenBank accession number YP_066091);R.pla, Raoultella planticola (GenBank accession number BAB18918); Uncul, uncultured bacterium from environmental sample (GenBankaccession number ABC24660); P.flu, Pseudomonas fluorescens Pf-5 (GenBank accession number YP_262073); G.bet, Granulibacter bethesdenisCGDNIH1 (GenBank accession number YP_745642).


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latter is a compact, all-�-helical protein (Fig. 9A), and it is theonly NSAP for which a crystal structure is available. The struc-ture of the NSAP of E. blattae demonstrated that the afore-mentioned signature sequence contains residues that are cru-cial to the functioning of the enzyme’s active site. MapA alsocontains the class A NSAP signature sequence with the crucialhistidine residue located at position 233.

The middle portion of the MapA polypeptide does not har-bor strong sequence homology to proteins in the data banks.However, there is a region (aa 414 to 639 [Fig. 7A]) thatdemonstrates limited homology to pertactin-like passenger do-mains described for other autotransporter proteins (41). The Evalue (42) of this homology is 0.003; for comparison, the muchstronger homology described above for the acid phosphataseregion has an E value of 7 � 1024. Pertactin-like domains canhave a variety of activities, including proteolysis and cell ad-hesion. However, no protease or cell-adhesion signature se-quences were located in the MapA sequence. The pertactin-like region of MapA may act as a linker between the acidphosphatase domain and the carboxy-terminal translocationdomain.

The carboxy-terminal domain (aa 672 to 940 [Fig. 7A]) ofMapA is homologous to the � domains of conventional auto-transporters (E value of 6 � 1010). A PROMALS-generatedalignment showed strong correspondence between the pre-dicted �-strands (33) of MapA (Fig. 9B) and the experimen-tally observed �-strands of the crystalline NalP autotransporterdomain (48).

DISCUSSION

The identification of an autotransporter-associated acidphosphatase in M. catarrhalis is a novel finding. Previous workfrom this laboratory and others has demonstrated that M.catarrhalis synthesizes at least four other autotransporters (1,6, 51, 68). The MapA protein appears to be most similar toconventional autotransporters. The prototypical signal se-quence for a bacterial autotransporter usually consists of 18 to26 aa; the predicted signal peptide for MapA has 23 residues(Fig. 2). The passenger domain of the typical autotransportermolecule contains the region responsible for the biologicalfunction of the translocated protein and can exceed 100 kDa(11, 27, 35). In the M. catarrhalis MapA protein, the predictedpassenger domain (Fig. 7A) has similarity to the pertactinfamily of passenger domains but has no readily identifiablefunctional activity. Instead, it may serve to link the acid phos-phatase domain to the MapA translocation domain, such thatthe passenger component of MapA includes both the acidphosphatase domain and the pertactin-like domain.

Based on their translocation domains, autotransporters canbe divided into two types. The C-terminal region of conven-tional autotransporters, including MapA, usually consists of250 to 300 aa and forms a �-barrel structure with transmem-brane �-strands (11, 12, 27). A trimeric autotransporter alsohas a �-barrel structure, but it comprises three sets of four�-strands. In both cases, the �-barrel secondary structure, onceinserted into the outer membrane, allows for the movement of

FIG. 9. Computer-based structural modeling of the MapA protein. (A) Sequence identity between the NSAP of E. blattae and the amino-terminal domain of MapA mapped onto the structure of the NSAP of E. blattae. A ribbon-type drawing of the NSAP of E. blattae (PDB code1D2T) is shown. In cyan are residues that are identical between the two sequences when aligned. All other amino acids are colored purple. Theactive site of the NSAP of E. blattae is marked by a sulfate anion (yellow and red spheres) that resides there. (B) Predicted �-strands of theautotransporter domain of MapA mapped onto the structure of the autotransporter domain of NalP. A ribbons-type diagram of the structure ofthe autotransporter domain of NalP (PDB code 1UYN) is shown. The approximate boundaries of the outer membrane (OM) are shown as blacklines, with the extracellular region (E) and periplasmic space (P) noted. The sequences of the two proteins were aligned (data not shown). Shownin pink are those residues that are aligned to MapA residues that are predicted to be �-strands. Residues were colored pink only if included ina predicted �-strand of six or more residues. Residues aligned with MapA residues that do not meet the above criteria are colored blue. PanelsA and B were generated in PyMOL (www.pymol.org) and rendered using POV-Ray (www.povray.org).


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the effector portion to the bacterial cell surface (12). Initially,autotransporter proteins had been predicted to contain 14membrane-spanning �-strands arranged as a barrel with a cen-tral pore (12). However, more recently, a crystal structure ofthe �-domain of the NalP autotransporter from Neisseria men-ingitidis (48) showed that this protein comprises only 12�-strands, with a single �-helix residing in the pore formed bythe strands. This result was controversial because the NalP�-domain protein used for crystallization had to be solubilizedand refolded from inclusion bodies. However, a subsequentcrystal structure of the �-domain of a natively folded trimericautotransporter, that of Hia from Haemophilus influenzae, hasbeen determined (43). This structure confirmed the 12-stranded organization of the �-domain of autotransporters.

We aligned the amino acid sequences of the �-domain of theM. catarrhalis MapA protein and NalP, which is the only con-ventional autotransporter whose structure has been deter-mined by X-ray crystallography (48). The alignment revealedthat these domains have only limited identity (about 9%).However, the alignment algorithm that was used (PROMALS)employs a new hidden Markov model that scores aligned po-sitions based on both amino acid similarity and the correspon-dence of secondary structures predicted by PSIPRED (33, 53).Based on the available crystal structures, the sequential ho-mologies, and the putative structural homologies, we concludethat the �-domain of MapA is likely to be a 12-stranded mem-brane-spanning pore with an �-helix occupying the center ofthe pore. Proof that this domain was functional in transloca-tion of a passenger domain to the M. catarrhalis cell surfacewas obtained by using the McaP-MapA fusion protein (Fig. 7).

BLAST analysis revealed the presence of four putative au-totransporters in the databases with E scores of less than 8 �1017 that also have a class A acid phosphatase-like domainnear their N termini. These include predicted proteins fromPseudomonas syringae pv. phaseolicola 1448A (GenBank acces-sion number AAZ35404) (32), P. syringae pv. syringae B728A(GenBank accession number YP_233446) (16), P. syringae pv.tomato DC3000 (GenBank accession number NP_794931) (8),and Bradyrhizobium sp. strain BTAi1 (GenBank accessionnumber ZP_00861929). No descriptions of these proteins havebeen published to date.

The conservation of the MapA protein (97% identity)among the six M. catarrhalis strains used in this study was veryhigh (Fig. 2). In addition, Western blot analysis of 13 addi-tional M. catarrhalis isolates with the MapA-reactive MAb1H12 (Fig. 5A) showed that at least the MapA epitope boundby this MAb is present in all of these strains. Our experimentsindicate that the MapA protein is localized to the outer mem-brane and, by analogy with other autotransporters, is likelyexposed on the surface of the bacterium. The identification ofthe putative active site of the MapA acid phosphatase domain(Fig. 8) and the subsequent mutagenesis of its critical histidineresidue served to confirm the enzymatic activity of this protein(Fig. 3). The fact that MapA is expressed at a relatively lowlevel in comparison to other M. catarrhalis autotransporters(e.g., UspA2) may be related to the fact that its enzymaticfunction is catalytic and does not require abundant proteinexpression. The cleavage and release of phosphate from or-ganic sources by this acid phosphatase may be necessary for theuptake of an essential nutrient. The wild-type parent strain and

the mapA deletion mutant had similar growth rates in vitro, butthese conditions clearly do not mimic those encountered in thenasopharynx, where certain nutrients and other environmentalfactors may be limiting in the absence of MapA activity. Takentogether, these data suggest that the mapA gene product islikely important to M. catarrhalis, although the specific biolog-ical function of this acid phosphatase, like that of most otherbacterial nonspecific acid phosphatases, remains to be deter-mined.

ACKNOWLEDGMENTS

This study was supported by U.S. Public Health Service grant no.AI36344 to E.J.H.

We thank John Nelson, Anthony Campagnari, David Goldblatt,Steven Berk, Frederick Henderson, and Merja Helminen for the iso-lates of M. catarrhalis used in this study and Jason Huntley for assis-tance with bioinformatics. We also thank Eric Lafontaine for providingthe mcaP mutant O35E.M and the McaP polyclonal antiserum.

REFERENCES

1. Aebi, C., E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H.McCracken, Jr., and E. J. Hansen. 1998. Phenotypic effect of isogenic uspA1and uspA2 mutations on Moraxella catarrhalis O35E. Infect. Immun. 66:3113–3119.

2. Aebi, C., I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas,G. H. McCracken, Jr., and E. J. Hansen. 1997. A protective epitope ofMoraxella catarrhalis is encoded by two different genes. Infect. Immun. 65:4367–4377.

3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

4. Aragon, V., S. Kurtz, and N. P. Cianciotto. 2001. Legionella pneumophilamajor acid phosphatase and its role in intracellular infection. Infect. Immun.69:177–185.

5. Attia, A. S., and E. J. Hansen. 2006. A conserved tetranucleotide repeat isnecessary for wild-type expression of the Moraxella catarrhalis UspA2 pro-tein. J. Bacteriol. 188:7840–7852.

6. Attia, A. S., E. R. Lafontaine, J. L. Latimer, C. Aebi, G. A. Syrogiannopoulos,and E. J. Hansen. 2005. The UspA2 protein of Moraxella catarrhalis isdirectly involved in the expression of serum resistance. Infect. Immun. 73:2400–2410.

7. Baron, G. S., T. J. Reilly, and F. E. Nano. 1999. The respiratory burst-inhibiting acid phosphatase AcpA is not essential for the intramacrophagegrowth or virulence of Francisella novicida. FEMS Microbiol. Lett.176:85–90.

8. Buell, C. R., V. Joardar, M. Lindeberg, J. Selengut, I. T. Paulsen, M. L.Gwinn, R. J. Dodson, R. T. Deboy, A. S. Durkin, J. F. Kolonay, R. Madupu,S. Daugherty, L. Brinkac, M. J. Beanan, D. H. Haft, W. C. Nelson, T.Davidsen, N. Zafar, L. Zhou, J. Liu, Q. Yuan, H. Khouri, N. Fedorova, B.Tran, D. Russell, K. Berry, T. Utterback, S. E. Van Aken, T. V. Feldblyum,M. D’Ascenzo, W. L. Deng, A. R. Ramos, J. R. Alfano, S. Cartinhour, A. K.Chatterjee, T. P. Delaney, S. G. Lazarowitz, G. B. Martin, D. J. Schneider,X. Tang, C. L. Bender, O. White, C. M. Fraser, and A. Collmer. 2003. Thecomplete genome sequence of the Arabidopsis and tomato pathogen Pseudo-monas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. USA 100:10181–10186.

9. Bull, H., P. G. Murray, D. Thomas, A. M. Fraser, and P. N. Nelson. 2002.Acid phosphatases. Mol. Pathol. 55:65–72.

10. Chen, X., T. Ansai, S. Barik, and T. Takehara. 2003. Kinetic and site-directedmutagenesis studies of Prevotella intermedia acid phosphatase. Protein Pept.Lett. 10:53–59.

11. Cotter, S. E., N. K. Surana, and J. W. St. Geme III. 2005. Trimeric auto-transporters: a distinct subfamily of autotransporter proteins. Trends Micro-biol. 13:199–205.

12. Desvaux, M., N. J. Parham, and I. R. Henderson. 2004. The autotransportersecretion system. Res. Microbiol. 155:53–60.

13. Economou, A., P. J. Christie, R. C. Fernandez, T. Palmer, G. V. Plano, andA. P. Pugsley. 2006. Secretion by numbers: protein traffic in prokaryotes.Mol. Microbiol. 62:308–319.

14. Faden, H. S., L. Duffy, R. Wasielewski, J. Wolf, D. A. Krystofik, Y. Tung, etal. 1997. Relationship between nasopharyngeal colonization and the devel-opment of otitis media in children. J. Infect. Dis. 175:1440–1445.

15. Faden, H. S., Y. Harabuchi, J. J. Hong, et al. 1994. Epidemiology of Morax-ella catarrhalis in children during the first 2 years of life: relationship to otitismedia. J. Infect. Dis. 169:1312–1317.

16. Feil, H., W. S. Feil, P. Chain, F. Larimer, G. DiBartolo, A. Copeland, A.


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Lykidis, S. Trong, M. Nolan, E. Goltsman, J. Thiel, S. Malfatti, J. E. Loper,A. Lapidus, J. C. Detter, M. Land, P. M. Richardson, N. C. Kyrpides, N.Ivanova, and S. E. Lindow. 2005. Comparison of the complete genomesequences of Pseudomonas syringae pv. syringae B728a and pv. tomatoDC3000. Proc. Natl. Acad. Sci. USA 102:11064–11069.

17. Felts, R. L., T. J. Reilly, and J. J. Tanner. 2006. Structure of Francisellatularensis AcpA: prototype of a unique superfamily of acid phosphatases andphospholipases C. J. Biol. Chem. 281:30289–30298.

18. Forsgren, A., M. Brant, A. Mollenkvist, A. Muyombwe, H. Janson, N. Woin,and K. Riesbeck. 2001. Isolation and characterization of a novel IgD-bindingprotein from Moraxella catarrhalis. J. Immunol. 167:2112–2120.

19. Girard, V., and M. Mourez. 2006. Adhesion mediated by autotransporters ofgram-negative bacteria: structural and functional features. Res. Microbiol.157:407–416.

20. Gouet, P., E. Courcelle, D. I. Stuart, and F. Metoz. 1999. ESPript: analysis ofmultiple sequence alignments in PostScript. Bioinformatics 15:305–308.

21. Grose, J. H., U. Bergthorsson, Y. Xu, J. Sterneckert, B. Khodaverdian, andJ. R. Roth. 2005. Assimilation of nicotinamide mononucleotide requiresperiplasmic AphA phosphatase in Salmonella enterica. J. Bacteriol. 187:4521–4530.

22. Helminen, M. E., I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken,Jr., and E. J. Hansen. 1993. A major outer membrane protein of Moraxellacatarrhalis is a target for antibodies that enhance pulmonary clearance of thepathogen in an animal model. Infect. Immun. 61:2003–2010.

23. Hemrika, W., R. Renirie, H. L. Dekker, P. Barnett, and R. Wever. 1997.From phosphatases to vanadium peroxidases: a similar architecture of theactive site. Proc. Natl. Acad. Sci. USA 94:2145–2149.

24. Hemrika, W., R. Renirie, S. Macedo-Ribeiro, A. Messerschmidt, and R.Wever. 1999. Heterologous expression of the vanadium-containing chlo-roperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae andsite-directed mutagenesis of the active site residues His496, Lys353, Arg360,and Arg490. J. Biol. Chem. 274:23820–23827.

25. Henderson, I. R., R. Cappello, and J. P. Nataro. 2000. Autotransporterproteins, evolution and redefining protein secretion. Trends Microbiol.8:529–532.

26. Henderson, I. R., and J. P. Nataro. 2001. Virulence functions of autotrans-porter proteins. Infect. Immun. 69:1231–1243.

27. Henderson, I. R., F. Navarro-Garcia, M. Desvaux, R. C. Fernandez, and D.Ala’Aldeen. 2004. Type V protein secretion pathway: the autotransporterstory. Microbiol. Mol. Biol. Rev. 68:692–744.

28. Horton, R. M., Z. Cai, S. N. Ho, and L. R. Pease. 1990. Gene splicing byoverlap extension: tailor-made genes using the polymerase chain reaction.BioTechniques 8:528–535.

29. Horton, R. M., H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease. 1989.Engineering hybrid genes without the use of restriction enzymes: gene splic-ing by overlap extension. Gene 77:61–68.

30. Hu, W. G., J. Chen, J. C. McMichael, and X. X. Gu. 2001. Functionalcharacteristics of a protective monoclonal antibody against serotype A and Clipooligosaccharides from Moraxella catarrhalis. Infect. Immun. 69:1358–1363.

31. Ishikawa, K., Y. Mihara, K. Gondoh, E. Suzuki, and Y. Asano. 2000. X-raystructures of a novel acid phosphatase from Escherichia blattae and its com-plex with the transition-state analog molybdate. EMBO J. 19:2412–2423.

32. Joardar, V., M. Lindeberg, R. W. Jackson, J. Selengut, R. Dodson, L. M.Brinkac, S. C. Daugherty, R. DeBoy, A. S. Durkin, M. G. Giglio, R. Madupu,W. C. Nelson, M. J. Rosovitz, S. Sullivan, J. Crabtree, T. Creasy, T. David-sen, D. H. Haft, N. Zafar, L. Zhou, R. Halpin, T. Holley, H. Khouri, T.Feldblyum, O. White, C. M. Fraser, A. K. Chatterjee, S. Cartinhour, D. J.Schneider, J. Mansfield, A. Collmer, and C. R. Buell. 2005. Whole-genomesequence analysis of Pseudomonas syringae pv. phaseolicola 1448A revealsdivergence among pathovars in genes involved in virulence and transposi-tion. J. Bacteriol. 187:6488–6498.

33. Jones, D. T. 1999. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292:195–202.

34. Kemmer, G., T. J. Reilly, J. Schmidt-Brauns, G. W. Zlotnik, B. A. Green,M. J. Fiske, M. Herbert, A. Kraiss, S. Schlor, A. Smith, and J. Reidl. 2001.NadN and e (P4) are essential for utilization of NAD and nicotinamidemononucleotide but not nicotinamide riboside in Haemophilus influenzae. J.Bacteriol. 183:3974–3981.

35. Kim, D. S., Y. Chao, and M. H. Saier, Jr. 2006. Protein-translocating trimericautotransporters of gram-negative bacteria. J. Bacteriol. 188:5655–5667.

36. Kirk, R. E. 1995. Experimental design: procedures for the behavioral sci-ences. Brooks/Cole Publishing Company, Pacific Grove, CA.

37. Lafontaine, E. R., N. J. Wagner, and E. J. Hansen. 2001. Expression of theMoraxella catarrhalis UspA1 protein undergoes phase variation and is regu-lated at the transcriptional level. J. Bacteriol. 183:1540–1551.

38. Linke, D., T. Riess, I. B. Autenrieth, A. Lupas, and V. A. Kempf. 2006.Trimeric autotransporter adhesins: variable structure, common function.Trends Microbiol. 14:264–270.

39. Lipski, S. L., C. Akimana, J. M. Timpe, R. M. Wooten, and E. R. Lafontaine.2007. The Moraxella catarrhalis autotransporter McaP is a conserved surfaceprotein that mediates adherence to human epithelial cells through its N-terminal passenger domain. Infect. Immun. 75:314–324.

40. Luke, N. R., and A. A. Campagnari. 1999. Construction and characterizationof Moraxella catarrhalis mutants defective in expression of transferrin recep-tors. Infect. Immun. 67:5815–5819.

41. Marchler-Bauer, A., J. B. Anderson, P. F. Cherukuri, C. Weese-Scott, L. Y.Geer, M. Gwadz, S. He, D. I. Hurwitz, J. D. Jackson, Z. Ke, C. J. Lanczycki,C. A. Liebert, C. Liu, F. Lu, G. H. Marchler, M. Mullokandov, B. A. Shoe-maker, V. Simonyan, J. S. Song, P. A. Thiessen, R. A. Yamashita, J. J. Yin,D. Zhang, and S. H. Bryant. 2005. CDD: a conserved domain database forprotein classification. Nucleic Acids Res. 33:D192–D196.

42. Marchler-Bauer, A., and S. H. Bryant. 2004. CD-Search: protein domainannotations on the fly. Nucleic Acids Res. 32:W327–W331.

43. Meng, G., N. K. Surana, J. W. St. Geme III, and G. Waksman. 2006.Structure of the outer membrane translocator domain of the Haemophilusinfluenzae Hia trimeric autotransporter. EMBO J. 25:2297–2304.

44. Mougous, J. D., M. E. Cuff, S. Raunser, A. Shen, M. Zhou, C. A. Gifford,A. L. Goodman, G. Joachimiak, C. L. Ordonez, S. Lory, T. Walz, A.Joachimiak, and J. J. Mekalanos. 2006. A virulence locus of Pseudomonasaeruginosa encodes a protein secretion apparatus. Science 312:1526–1530.

45. Murphy, T. F. 1996. Branhamella catarrhalis: epidemiology, surface antigenicstructure, and immune response. Microbiol. Rev. 60:267–279.

46. Murphy, T. F., A. L. Brauer, B. J. Grant, and S. Sethi. 2005. Moraxellacatarrhalis in chronic obstructive pulmonary disease: burden of disease andimmune response. Am. J. Respir. Crit. Care Med. 172:195–199.

47. Murphy, T. F., and M. R. Loeb. 1989. Isolation of the outer membrane ofBranhamella catarrhalis. Microb. Pathog. 6:159–174.

48. Oomen, C. J., P. van Ulsen, P. Van Gelder, M. Feijen, J. Tommassen, and P.Gros. 2004. Structure of the translocator domain of a bacterial autotrans-porter. EMBO J. 23:1257–1266.

49. Ou, Z., R. L. Felts, T. J. Reilly, J. C. Nix, and J. J. Tanner. 2006. Crystalli-zation of recombinant Haemophilus influenzae e (P4) acid phosphatase. ActaCrystallograph. Sect. F Struct. Biol. Cryst. Commun. 62:464–466.

50. Pan, C. J., K. J. Lei, B. Annabi, W. Hemrika, and J. Y. Chou. 1998. Trans-membrane topology of glucose-6-phosphatase. J. Biol. Chem. 273:6144–6148.

51. Pearson, M. M., E. R. Lafontaine, N. J. Wagner, J. W. St. Geme III, and E. J.Hansen. 2002. A hag mutant of Moraxella catarrhalis strain O35E is deficientin hemagglutination, autoagglutination, and immunoglobulin D-binding ac-tivities. Infect. Immun. 70:4523–4533.

52. Pearson, M. M., C. A. Laurence, S. E. Guinn, and E. J. Hansen. 2006.Biofilm formation by Moraxella catarrhalis in vitro: roles of the UspA1adhesin and the Hag hemagglutinin. Infect. Immun. 74:1588–1596.

53. Pei, J., and N. V. Grishin. 2007. PROMALS: towards accurate multiplesequence alignments of distantly related proteins. Bioinformatics 23:802–808.

54. Pohlner, J., R. Halter, K. Beyreuther, and T. F. Meyer. 1987. Gene structureand extracellular secretion of Neisseria gonorrhoeae IgA protease. Nature325:458–462.

55. Pohlner, J., R. Halter, and T. F. Meyer. 1987. Neisseria gonorrhoeae IgAprotease. Secretion and implications for pathogenesis. Antonie van Leeu-wenhoek 53:479–484.

56. Pukatzki, S., A. T. Ma, D. Sturtevant, B. Krastins, D. Sarracino, W. C.Nelson, J. F. Heidelberg, and J. J. Mekalanos. 2006. Identification of aconserved bacterial protein secretion system in Vibrio cholerae using theDictyostelium host model system. Proc. Natl. Acad. Sci. USA 103:1528–1533.

57. Reidl, J., S. Schlor, A. Kraiss, J. Schmidt-Brauns, G. Kemmer, and E.Soleva. 2000. NADP and NAD utilization in Haemophilus influenzae. Mol.Microbiol. 35:1573–1581.

58. Reilly, T. J., G. S. Baron, F. E. Nano, and M. S. Kuhlenschmidt. 1996.Characterization and sequencing of a respiratory burst-inhibiting acid phos-phatase from Francisella tularensis. J. Biol. Chem. 271:10973–10983.

59. Reilly, T. J., D. L. Chance, and A. L. Smith. 1999. Outer membrane lipopro-tein e (P4) of Haemophilus influenzae is a novel phosphomonoesterase. J.Bacteriol. 181:6797–6805.

60. Reilly, T. J., R. L. Felts, M. T. Henzl, M. J. Calcutt, and J. J. Tanner. 2006.Characterization of recombinant Francisella tularensis acid phosphatase A.Protein Expr. Purif. 45:132–141.

61. Reilly, T. J., B. A. Green, G. W. Zlotnick, and A. L. Smith. 2001. Contributionof the DDDD motif of H. influenzae e (P4) to phosphomonoesterase activityand heme transport. FEBS Lett. 494:19–23.

62. Riccio, M. L., G. M. Rossolini, G. Lombardi, A. Chiesurin, and G. Satta.1997. Expression cloning of different bacterial phosphatase-encoding genesby histochemical screening of genomic libraries onto an indicator mediumcontaining phenolphthalein diphosphate and methyl green. J. Appl. Micro-biol. 82:177–185.

63. Rossolini, G. M., S. Schippa, M. L. Riccio, F. Berlutti, L. E. Macaskie, andM. C. Thaller. 1998. Bacterial nonspecific acid phosphohydrolases: physiol-ogy, evolution and use as tools in microbial biotechnology. Cell. Mol. LifeSci. 54:833–850.

64. Saha, A. K., J. N. Dowling, A. W. Pasculle, and R. H. Glew. 1988. Legionellamicdadei phosphatase catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate in human neutrophils. Arch. Biochem. Biophys. 265:94–104.

65. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a


at UN

IV T

EX

AS

SW

ME

D C

TR


jb.asm.org

Dow

nloaded from

http://jb.asm.org

laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY.

66. Sethi, S., and T. F. Murphy. 2001. Bacterial infection in chronic obstructivepulmonary disease in 2000: a state-of-the-art review. Clin. Microbiol. Rev.14:336–363.

67. Thaller, M. C., S. Schippa, and G. M. Rossolini. 1998. Conserved sequencemotifs among bacterial, eukaryotic, and archaeal phosphatases that define anew phosphohydrolase superfamily. Protein Sci. 7:1647–1652.

68. Timpe, J. M., M. M. Holm, S. L. Vanlerberg, V. Basrur, and E. R. Lafon-taine. 2003. Identification of a Moraxella catarrhalis outer membrane proteinexhibiting both adhesin and lipolytic activities. Infect. Immun. 71:4341–4350.

69. Tsai, C.-M., and C. E. Frasch. 1982. A sensitive silver stain for detectinglipopolysaccharide in polyacrylamide gels. Anal. Biochem. 119:115–119.

70. Vanet, A., J. A. Plumbridge, and J. H. Alix. 1993. Cotranscription of two

genes necessary for ribosomal protein L11 methylation (prmA) and panto-thenate transport (panF) in Escherichia coli K-12. J. Bacteriol. 175:7178–7188.

71. Vincent, J. B., M. W. Crowder, and B. A. Averill. 1991. Evidence for aphosphoryl-enzyme intermediate in phosphate ester hydrolysis by purpleacid phosphatase from bovine spleen. J. Biol. Chem. 266:17737–17740.

72. Wang, W., and E. J. Hansen. 2006. Plasmid pWW115, a cloning vector foruse with Moraxella catarrhalis. Plasmid 56:133–137.

73. Ward, C. K., S. R. Lumbley, J. L. Latimer, L. D. Cope, and E. J. Hansen.1998. Haemophilus ducreyi secretes a filamentous hemagglutinin-like protein.J. Bacteriol. 180:6013–6022.

74. Whitby, P. W., D. J. Morton, and T. L. Stull. 1998. Construction of antibioticresistance cassettes with multiple paired restriction sites for insertional mu-tagenesis of Haemophilus influenzae. FEMS Microbiol. Lett. 158:57–60.


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