JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Aug. 1999, p. 5090–5093 Vol. 181, No. 16

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Moraxella (Branhamella) catarrhalis BRO b-Lactamase:a Lipoprotein of Gram-Positive Origin?

HESTER J. BOOTSMA,1* PIET C. AERTS,1 GEORGE POSTHUMA,2 THEO HARMSEN,1

JAN VERHOEF,1 HANS VAN DIJK,1 AND FRITS R. MOOI1,3

Eijkman-Winkler Institute for Microbiology, Infectious Diseases, and Inflammation, University Hospital Utrecht,1

and Department of Cell Biology and the Graduate School of Biomembranes, Utrecht University,2 Utrecht,and Research Laboratory for Infectious Diseases, National Institute of Public Health and the

Environment (RIVM), Bilthoven,3 The Netherlands

Received 22 February 1999/Accepted 2 June 1999

In the past 20 years, BRO b-lactamase-producing Moraxella catarrhalis strains have emerged. We show thatBRO is expressed as a 33-kDa lipoprotein associated with the inner leaflet of the outer membrane. To ourknowledge, this is the first description of a lipidated b-lactamase in a gram-negative species.

In recent years, Moraxella (Branhamella) catarrhalis hasemerged as a significant cause of upper respiratory tract infec-tions in children and lower respiratory tract infections in pa-tients with underlying chest disease (2, 4, 14). In addition tobeing directly pathogenic, M. catarrhalis may be indirectlypathogenic, because it produces penicillin-degrading enzymes(b-lactamases), which may protect concomitantly infecting,more virulent bacteria such as Streptococcus pneumoniae orHaemophilus influenzae from antibiotic therapy (10, 23). Whileampicillin-resistant M. catarrhalis strains were first describedonly 20 years ago (13, 17), over 90% of the M. catarrhalisstrains isolated currently worldwide are b-lactamase positive(6, 9). The b-lactamases produced by M. catarrhalis—theBRO-1 type and the BRO-2 type—are distinguishable by theirisoelectric-focusing patterns (22). BRO-1-producing strainsare more resistant to b-lactam antibiotics than BRO-2 produc-ers, most likely due to higher levels of BRO-1 (1, 9, 22).

In an earlier study, we reported the cloning and sequencingof the genes (bro) coding for M. catarrhalis BRO b-lactamases(1). The bro genes appeared to be located on the chromosomeand coded for polypeptides that differed in only one aminoacid. BRO production appeared to be constitutive, not affectedby the presence of antibiotics (21). Interestingly, we observedthat the G1C content of bro (31%) was quite different fromthat of the rest of the M. catarrhalis genome (41%), suggestingthat BRO was derived from another, as-yet-unknown species.In support of this notion, bro was found to encode a signalsequence characteristic for lipoproteins (LTGC), a feature un-characteristic of b-lactamases occurring in gram-negative bac-teria. Here we determine whether BRO is indeed a lipopro-tein.

Lipoproteins are synthesized as precursors with N-terminalsignal peptides that are modified by lipidation (3). The firststep in the lipid modification of such proteins is the addition ofa diglyceride to a cysteine residue. In the second step, lipopro-tein-specific signal peptidase II cleaves the signal peptide at theamino-terminal side of the modified cysteine, whereupon fur-ther fatty acylation of the cysteine residue takes place, and the

resultant fully modified lipoprotein is assembled into the mem-brane (3).

Lipid-modified, membrane-bound b-lactamases have beendescribed for a number of gram-positive organisms. The firstmembrane-bound enzyme was found in Bacillus cereus (18),and similar b-lactamases have been observed in Bacillus li-cheniformis (25) and Staphylococcus aureus (15). The mem-brane-bound forms of gram-positive b-lactamases have beenproposed to be the precursors of extracellular enzymes, gen-erated by proteolytic cleavage of the lipid anchor (16, 25).Several studies have indicated that BRO b-lactamases are, atleast in part, produced as membrane-bound proteins (7, 20).We have shown that in both M. catarrhalis and Escherichia coliharboring bro, 10 and 45% of b-lactamase activity, respectively,was found in the membrane compartment (1), suggestive of alipid anchor.

Identification of the bro product in M. catarrhalis and E. coli.To obtain specific antibodies, BRO-1 was produced in excessas a maltose-binding protein (MBP) fusion protein by using thepMAL-cRI vector (New England Biolabs, Beverly, Mass.).Five monoclonal antibodies (MAbs) directed against BROwere produced after immunization with 20 mg of MBP-BROaccording to standard protocols. Enzyme-linked immunosor-bent assay identified MAbs PA1 to PA5 as immunoglobulin Gtype 1 (IgG1) antibodies specific for BRO and not MBP. Amixture of PA1 to PA5 was used in immunoblotting to identifythe bro product in cell lysates of both M. catarrhalis and re-combinant E. coli. In M. catarrhalis, BRO b-lactamase wasidentified as a protein with an apparent molecular mass of 33kDa by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE; Fig. 1A, lanes 3 and 4), in good agree-ment with the calculated molecular size of 32 kDa (1). Nob-lactamase could be detected in M. catarrhalis lacking the brogene (Fig. 1A, lane 5). The expected higher expression level ofBRO-1, based on enzymatic assays, was confirmed by immu-noblots, which revealed a more intense band in BRO-1 strainsthan in BRO-2 strains (Fig. 1A, lanes 3 and 4, respectively).Two protein bands were detected on immunoblots of E. coliharboring bro (Fig. 1A, lane 1) that were absent in E. colistrains lacking bro (Fig. 1A, lane 2). The most intense band hadthe same mobility as the 33-kDa band detected in M. catarrha-lis and was associated with the membrane fraction of E. coli(Fig. 1B, lane 2), indicating that this protein band representedthe mature, lipid-modified BRO. The second, minor band may

* Corresponding author. Mailing address: Eijkman-Winkler Insti-tute for Microbiology, Infectious Diseases, and Inflammation, Univer-sity Hospital Utrecht, Room G04.614, P.O. Box 85,500, 3508 GAUtrecht, The Netherlands. Phone: (31) (30) 2506525. Fax: (31) (30)2541770. E-mail: H.J.Bootsma@lab.azu.nl.

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represent a nonlipidated precursor protein including a signalpeptide.

Lipid modification of M. catarrhalis BRO b-lactamase. Boththe membrane association and the signal peptide sequence ofbro suggested that BRO could be a lipoprotein. Lipid modifi-cation of M. catarrhalis b-lactamase was initially investigated inE. coli. Logarithmic cultures of E. coli harboring different(sub)-clones of bro were labeled with 50 mCi of [3H]palmiticacid per ml for 1 h. Labeling was stopped by the addition oftrichloroacetic acid (TCA) to a final concentration of 10%.Precipitates were then collected by centrifugation, washed withacetone and chloroform-methanol (2:1 [vol/vol]), dried, anddissolved in 50 mM Tris–1% SDS (pH 6.8) by heating at 100°Cfor 2 min. Subsequently, 20-ml aliquots were analyzed by SDS-PAGE and fluorography (Fig. 2). Since bro was present on a4-kb HindIII insert in E. coli that contained two other potentialopen reading frames (orf1 and orf3), a set of subclones wasused in which orf1 or orf3 was deleted to confirm that thelipid-modified band was indeed encoded by bro. Whenever anintact bro gene was present in E. coli, [3H]palmitic acid wasfound to be incorporated into a 33-kDa protein (Fig. 2, lanes 2,3, and 5). This labeled protein band disappeared when the brogene was disrupted (Fig. 2, lanes 1, 4, and 6). We could not,however, detect lipid-modified BRO in M. catarrhalis directly,most probably because it was masked by other lipidated pro-teins of a similar size. Definite proof that lipids were alsoincorporated in BRO in M. catarrhalis was obtained by immu-noprecipitation. TCA precipitates were suspended in 200 ml of25 mM Tris-HCl (pH 8.0) supplemented with 50 mM glucoseand 10 mM EDTA, after which b-lactamase was isolated byusing polyclonal antiserum raised against MBP-BRO, coupledto protein G-Sepharose beads (Pharmacia, Uppsala, Sweden)at a concentration of 30 mg of protein G/ml. By this approach,the 33-kDa protein was isolated from both E. coli and M.catarrhalis producing BRO-1 (Fig. 3). Although the lipoproteinsignal sequence of the BRO-2 enzyme is identical to that ofBRO-1 (1), we did not succeed in isolating [3H]palmitate-labeled BRO-2. Considering the lower level of BRO-2 produc-

tion, this lack of success was probably due to the limited sen-sitivity of the detection method.

Subcellular localization of M. catarrhalis BRO b-lactamase.Whether lipoproteins are targeted to the inner or outer mem-

FIG. 1. Immunoblot analysis of BRO b-lactamase expressed by M. catarrhalis and recombinant E. coli strains. (A) Whole-cell lysates of E. coli (1 3 108/lane) andM. catarrhalis (5 3 108/lane). Lane 1, E. coli pMC100 (bro1); lane 2, E. coli pUK21 (vector); lane 3, M. catarrhalis 43618 (BRO-1); lane 4, M. catarrhalis 43617 (BRO-2);lane 5, M. catarrhalis 3.21S (BRO negative). (B) Subcellular localization of BRO in E. coli pMC100. Lane 1, whole-cell fraction; lane 2, outer- plus inner-membranefractions. The outer- and inner-membrane protein fractions of E. coli harboring bro were obtained from cell-free extracts by centrifugation at 150,000 3 g for 60 minat 4°C. All samples were separated on an SDS–12.5% PAGE gel, blotted, and incubated with a mixture of 5 BRO-specific MAbs. Positions of molecular size markersare shown on the left.

FIG. 2. Incorporation of [3H]palmitic acid into BRO b-lactamase. Cells har-boring different (sub)clones of bro were labeled with [3H]palmitate for 1 h,collected, and analyzed by SDS-PAGE and fluorography. Lane 1, E. coli pUK21(vector); lane 2, E. coli pMC100 (orf11 bro1 orf31); lane 3, E. coli pMC105 (bro1

orf31); lane 4, E. coli pMC106 (orf31); lane 5, E. coli pMC097 (orf11 bro1); lane6, E. coli pMC096 (orf11 orf3). Positions of molecular size markers are indicatedon the left.

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brane is determined mainly by the residue next to the modifiedcysteine residue (24). Lipoproteins possessing aspartic acid atthis position are destined for the inner membrane, whereas anamino acid other than aspartic acid results in outer-membranelocalization. In the BRO-1 signal sequence, a lysine is presentat the position following the cysteine (1), thus predicting outer-membrane localization of the enzyme. We used immunogoldelectron microscopy to investigate the subcellular localizationof M. catarrhalis b-lactamase in E. coli. To separate the cyto-plasmic membrane from the outer membrane, we subjectedthe bacteria to plasmolysis by incubating them in 0.35 M su-crose (5). Then, the bacteria were pelleted at 1,000 3 g andsuspended in 2% paraformaldehyde (PFA) and 0.2% glutar-aldehyde in 0.1 M phosphate buffer (PB) (pH 7.4). After 2 h,the fixative was removed, and pelleted bacteria were stored in2% PFA in PB for 18 h. The bacteria were then washed threetimes with phosphate-buffered saline containing 0.15 M glycineand finally embedded in 10% gelatin in PB. Small cubes werecut at 4°C and infused with 2.3 M sucrose in PB at 4°C for atleast 2 h, after which they were frozen in liquid nitrogen.Ultrathin cryosections were prepared at 2120°C on an Ul-tracut S (Leica, Vienna, Austria) by using a diamond knife(Drukker International, Cuick, The Netherlands) according toLiou et al. (11). The sections were immunolabeled as describedpreviously (19) with a mixture of PA1 to PA5 (20 mg of each/mlin PBS-bovine serum albumin [1%]), followed by incubationwith rabbit anti-mouse IgG (DAKO, Glostrup, Denmark), and10-nm protein A-gold (Department of Cell Biology, UtrechtUniversity, Utrecht, The Netherlands). No specific labelingwas detected in M. catarrhalis, presumably due to the low levelof expression of BRO b-lactamase. However, specific labelingwas obtained in E. coli harboring the bro gene (Fig. 4). Over atotal of 200 bacteria, the percentages of gold particles associ-ated with the inner and outer membranes were 5 and 60%,respectively. This is highly significantly different from an equaldistribution of label over the two membranes (chi-square by

Mantel-Haenszel procedure, P , 0.0001). Thus, BRO b-lacta-mase was found to be largely associated with the outer mem-brane rather than the cytoplasmic membrane in E. coli. More-over, the enzyme appeared to be located on the inside of theouter membrane, facing the periplasm. The relatively highlabeling of the cytoplasm (35%) may be due to the presence of(uncompleted) polypeptide chains still associated with ribo-somes. No labeling was observed in E. coli without bro (notshown).

Concluding remarks. To our knowledge, BRO b-lactamaseis the first-described lipidated and, therefore, membrane-asso-ciated b-lactamase found in gram-negative bacteria. Two othergram-negative bacteria, Pseudomonas pseudomallei and Cap-nocytophaga sp., have been reported to produce membrane-bound b-lactamases, but no molecular proof was provided thatthese enzymes were lipoproteins (8, 12). It does not seem likelythat a gram-negative bacterium such as M. catarrhalis wouldbenefit much from a membrane-bound b-lactamase. For gram-

FIG. 3. Immunoprecipitation of lipid-modified b-lactamase. BRO b-lacta-mase was isolated by using polyclonal anti-BRO antiserum and analyzed bySDS-PAGE and fluorography. Lane 1, E. coli pMC100 (orf11 bro1 orf31); lane2, E. coli pMC105 (bro1 orf31); lane 3, E. coli pMC106 (orf31); lane 4, M.catarrhalis 43618 (BRO-1); lane 5, M. catarrhalis 43617 (BRO-2). Positions ofmolecular size markers are shown on the left.

FIG. 4. Electron micrograph of immunogold-labeled E. coli harboring theBRO-1 b-lactamase gene. Cells were subjected to sucrose shock and labeled witha mixture of MAbs recognizing BRO-1 b-lactamase. Arrows indicate outer (O)and cytoplasmic (C) membranes. Bar, 0.2 mm. The image is representative.

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positive organisms, lipoprotein modification of b-lactamasesefficiently retains a portion of the secreted enzyme close to thecell. Gram-negative bacteria do not depend on such a mecha-nism, since their enzymes are retained in a periplasmic com-partment. As we observed by electron microscopy, the mem-brane-bound enzyme appeared to be facing the periplasmicspace. Therefore, it may function as a periplasmic enzyme,similar to other b-lactamases produced by gram-negative bac-teria. The distinct G1C content of bro compared to those ofother M. catarrhalis genes is strong evidence for a relativelyrecent acquisition. Taken together, the present data suggestthat BRO b-lactamase originates from a gram-positive bacte-rium and that its lipidation is a remnant of its origin.

We are grateful to Joen Luirink for advice on labeling of lipopro-teins.

This work was supported by grant no. 32.96.67 from the DutchAsthma Foundation.

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