Cloning and Expression of Genes Responsible for Altered Penicillin- Binding Proteins 3a and 3b in Haemophilus influenzae

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1987, p /87/ $02.00/0 Copyright 1987, American Society for Microbiology Vol. 31, No. 2 Cloning and Expression of Genes Responsible for Altered Penicillin- Binding Proteins 3a and 3b in Haemophilus influenzae F. MALOUIN,* A. B. SCHRYVERS, AND L. E. BRYAN Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta T2N 4NJ, Canada Received 2 June 1986/Accepted 11 November 1986 A Haemophilus influenzae strain (T-1,3) possessing clinical,b-lactam resistance due to altered peniciflinbinding protein 3 was used to construct a recombinant cosmid gene bank in Escherichia coli. Three of the recombinant cosmids were capable of transforming a susceptible H. influenzae strain (Rd") simultaneously to moxalactam resistance and altered the binding of penicillin-binding proteins 3a and 3b to [35S]peniciUin G. Restriction endonuclease mapping of one of the recombinant cosmids, plb100, was performed to facilitate subsequent subcloning of the gene(s) responsible for the altered penicillin-binding protein 3 (a and b) binding phenotype. Subcloning of individual fragments derived from plb100 indicated that two adjacent fragments of DNA were both capable of transforming a susceptible Haemophilus strain to moxalactam resistance and altered penicillin-binding protein 3 binding. Expression of plasmid-coded proteins in minicells indicated that one fragment coded for a major 55,000-molecular-weight polypeptide and that the second contained a C-terminal coding region that expressed a 28,000-molecular-weight polypeptide when fused to the N-terminal region of the tetracycline resistance gene. Initial attempts at labeling the plasmid-coded proteins expressed in minicells with [35S]penicillin G were unsuccessful. Ampicillin and chloramphenicol are recommended for the treatment. of Haemophilus influenzae infections. However, the emergence of ampicillin- and chloramphenicol-resistant strains over the past few years (9, 10) and concern over the toxicity of chloramphenicol (3) have prompted a search for alternative antibacterial agents. Ampicillin resistance in H. influenzae is principally due to the presence of a TEM-type,-lactamase (19). Nevertheless, there are several reports on' non-p-lactamase-mediated ampicillin resistance in H. influenzae (2, 15, 17, 18, 25). Alteration of,-lactam target proteins in bacterial cells may lead to significant resistance. The importance of the role of such penicillin-binding proteins (PBPs) in non-p-lactamasemediated P-lactam resistance was recently reviewed (13). In our laboratory, the broad-spectrum P-lactam resistance of a P-lactamase-negative clinical isolate of H. influenzae type b was previously investigated (18); alteration in the binding capacity of PBPs 3a and 3b correlated with the,-lactam resistance of this strain. To understand how such resistance develops, determination of the molecular basis of this resistance is necessary. We report here the cloning and expression of the genes responsible for altered PBP 3a and 3b expression in H. influenzae. MATERIALS AND METHODS Bactgrial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Media. The medium used for the growth of H. influenzae was supplemented brain heart infusion agar or broth (Gibco Diagnostics, Madison, Wis.), as previously described (18). Agar plates were incubated in an atmosphere containing 5% CO2. Mueller-Hinton broth (Gibco), also supplemented (18), was used in the determination of,b-lactam MICs. LB me- * Corresponding author. 286 dium, agar or broth (14), was used for the growth of Escherichia coli. Methionine assay medium (Difco Laboratories, Detroit, Mich.) and M9 minimal salts medium (14) were used for the identification of plasmid-encoded products in minicells. Drugs and radiolabeled compounds. The following antibiotics were used in this work: ampicillin and penicilin G from Ayerst Laboratories, Montreal, Quebec, Canada; moxalactam from Eli Lilly & Co., Indianapolis, Ind., piperacillin and tetracycline from Lederle Laboratories, Pearl River, N.Y.; imipenem and cefoxitin from Merck Sharp & Dohme, Rahway, N.J.; chloramphenicol, kanamycin, and spectinomycin from Sigma Chemical Co., St. Louis, Mo. [35S]benzylpenicillin (4.9 Ci/mol) was purchased from New England Nuclear Corp., Lachine, Quebec, Canada: and L-[355]methionine (1, 170 Ci/mmol) was from Amersham Corp., Oakville, Ontario, Canada. Cosmid gene bank preparation. The cells from a 100-ml culture of H. influenzae T-1,3 grown overnight at 37 C were collected by centrifugation, and the chromosomal DNA was extracted by the method of Silhavy et al. (21). One milligram of chromosomal DNA was partially digested with 20 U of Sau3A at 23 C for 1 h and extracted by successive phenolchloroform and chloroform extractions before ethanol precipitation. The digested DNA was then fractionated on a 38-ml linear sucrose gradient (10 to 40%) as described by Maniatis et al. (14). The fractions containing DNA fragments from 25 to 50 kilobases (kb) in size were pooled (600,ug), dialyzed, butanol extracted, and then ethanol precipitated. Mixtures containing 30,g of chromosomal DNA and 15,ug of BamHI-cleaved and alkaline phosphatase-dephosphorylated phc79 vector in a total volume of 200,ul were ligated overnight at 4 C with 1 U of T4 DNA ligase. The ligated DNA was packaged with an in vitro packing mixture as described by Hohn and Collins (8), and the

2 VOL. 31, 1987 H. INFLUENZAE ALTERED PBP 3 EXPRESSION 287 TABLE 1. Bacterial strains and plasmids Strain or plasmid Relevant properties Source (reference) Haemophilus influenzae RDPOv Novobiocin resistant nonencapsulated: non-3-18 lactamase producer T-1, 3 Novobiocin resistant nonencapsulated non-1- Transformant of Rd""' with DNA from H. influenzae lactamase producer broad-spectrum [B-lac- type b clinical strain UCHI-2 (18) tam resistance phenotype, altered PBPs 3a and 3b HT100 HT200 HT300 Same as T-1,3 Transformants of Rd"O" with cosmids plb100, plb200, and plb300, respsectively (this paper) HT120 HT140 HT160 Same as T-1,3 Transformants of Rd"""'' with plasmids plb120, plb140, and plb160, respectively (this paper) Escherichia coli DH1 Genotype: K-12 gyra96 reca relal (?) endal American Type Culture Collection: ATCC (14) thi-i hsdrj7 sup44 A-; used for transduction and transformation experiments BHB2688 Genotype: N205 reca (X imm434 citsb2 red3 ATCC (14) Eam4 Sam7IA); used for in vitro packaging of cosmid DNA BHB2690 Genotype: N205 reca (X imm434 citsb2 red3 ATCC (14) DamlS Sam7/A); used for in vitro packaging of cosmid DNA SA-2742 Genotype: mina minb his thr; used as the Kenneth E. Sanderson, University of Calgary minicell-producing strain Cosmid phc79 Apr Tcr, "cos" region of lambda DNA Boehringer Mannheim Canada, Dorval, Quebec Plasmids pbr325 Apr Cmr Tcr Bethesda Research Laboratories, Inc., Gaithersburg, Md. puc-4k Apr Kmr Pharmacia Canada Inc., Dorval, Quebec recombinant cosmids were transduced in E. coli DH1 and selected on LB plates containing 40,ug of ampicillin per ml. Screening for the altered P1P 3 expression gene. Recombinant phc79 cosmids were extracted from ampicillinresistant, transduced E. coli DH1 isolates by a rapid isolation method (23). Each recombinant cosmid isolated from a 5-ml culture was used to transform competent H. influenzae Rd"'V cells (0.5 ml). Competence was developed in M-IV medium by the procedure of Herriott et al. (7). The cells were incubated for 30 mim at 37 C with slow agitation, and 9.5 ml of supplemented brain heart infusion broth was added before an additional 2 h of incubation. To screen for altered PBP 3 expression in RDnov, 1 ml of transformation mixture was added to 2 ml of top agar, mixed, and poured onto supplemented brain heart infusion agar plates containing 0.2 jig of moxalactam per ml. Resistance to moxalactam has been shown to correlate with altered PBP 3 expression in H. influenzae T-1,3 (18). Subcloning of the altered PBP 3 expression gene. Largescale isolation of recombinant cosmids conferring moxalactam resistance in RDno, was performed by the procedure of Maniatis et al. (14) with chloramphenicol amplification, lysis by sodium dodecyl sulfate, and purification by centrifugation to equilibrium in cesium chloride (1.55 g/ml)-ethidium bromide (600,ug/ml) gradients. A purified recombinant cosmid (plb100) was digested with BamHI or with BamHI and HindlIl successively. BamHI restriction fragments were isolated and purified with a 38 ml-linear sucrose gradient (14). Fractions containing fragments were then dialyzed and ethanol precipitated overnight. Ligation of each BamHI-purified fragment to BamHIcleaved and alkaline phosphatase-dephosphorylated pbr325 vector was performed as recommended by the T4 DNA ligase supplier at 18 C. Pooled, double-digested BamHI-HindIII DNA fragments were submitted to blind ligation to the corresponding dephosphorylated pbr325 vector, which was purified by sucrose gradient centrifugation. All ligation mixtures were used to transform E. coli DH1 by the calcium chloride procedure (14), and recombinant pbr325 plasmids were extracted from ampicillin-resistant, tetracycline-susceptible (20,ug/ml) strains by a rapid isolation method (23). H. influenzae Rdnov was then transformed with each recombinant plasmid to screen for altered PBP 3 expression as described above. Altered PBP expression in H. influenzae RD"nV. (i) Binding of penicillin G. The procedure for binding radiolabeled penicillin to whole cells was previously described (18). Cells were labeled for 45 min with a penicillin concentration of 0.22 jig/ml (4 p.ci/ml) and loaded for electrophoresis on 10% discontinuous sodium dodecyl sulfate-polyacrylamide gels (11) Gels were stained and destained (18) before being soaked for 20 min in Amplify (Amersham Corp.) before 10 days of fluorography on prefogged X-Omat AR film (Eastman Kodak Co., Rochester, N.Y.). (ii) Susceptibility testing. MICs of P-lactams were determined by a broth dilution method as previously described (18). (iii) Detection of Il-lactamase. 3-Lactamase activity was detected by using a slide test technique with nitrocefin (Oxoid Chemicals Ltd., Nepean, Ontario, Canada) (16). Expression of plasmid-encoded proteins in minicells. (i) Transformation of E. coli SA All recombinant plasmids giving the altered PBP 3 expression in H. influenzae Rdn"v were used to transform the minicell-producing strain E. coli SA-2742 by the calcium chloride procedure (14). (ii) Isolation of minicells. The method followed for isolation of minicells was essentially that of Reeve (20). Purified

3 288 MALOUIN ET AL. minicells from 750-ml cultures were finally suspended in M9 medium containing 30% (vol/vol) glycerol. Samples of 1 ml (2 x 1010 minicells) were frozen by immersion in liquid nitrogen and stored at -70 C until used. (iii) Labeling of plasmid-encoded polypeptides in nminicells. With the method described by Reeve (20), the minicells from frozen samples were pelleted by centrifugation and suspended in 1 ml of M9 medium containing 0.4% (wt/vol) glucose. Samples of 100,ul were incubated for 10 min at 37 C before the addition of 5,ul of L-[35S]methionine at a final concentration of 100,uCi/ml in methionine assay medium. Minicells were then incubated for 1 h at 37 C, pelleted by centrifugation, washed in M9 medium-glucose, and suspended in 50,ul of electrophoretic sample buffer consisting of 2% sodium dodecyl sulfate, 4% 2-mercaptoethanol, 10% glycerol, 1 M Tris (ph 6.8), and 0.002% bromophenol blue. The suspended minicells were heated to 100 C for 5 min, and 25,ul was loaded for electrophoresis in 12% discontinuous sodium dodecyl sulfate-polyacrylamide gels. Gels were submitted to 3 h of fluorography as described above. (iv) Binding of penicillin G. To study the binding of radiolabeled penicillin to minicell proteins, the construction of suitable recombinant plasmids was necessary. Inactivation of the ampicillin resistance marker in the recombinant pbr325 plasmids was performed by inserting the kanamycin gene block from the puc-4k vector into the PstI site. The minicells obtained from a 750-ml br,th culture as described above were collected by centrifugation, and the pellet was passed four times through a French pressure cell (Fred S. Carver, Inc., Summit, N.J.) at 15,000 lb/in2. Phenylmethylsulfonyl fluoride and DNase 1 (Sigma) were added to final concentrations of 1 mm and 30,ug/ml, respectively, and the cell lysate was centrifuged at 1,500 x g for 10 min to remove unbroken cells. The supernatant was collected and spun at 46,000 rpm in a 50 Ti Beckman rotor for 1 h. The cell membrane pellet was suspended in 100,ul of 100 mm Tris (ph 8.0)-i mm phenylmethylsulfonyl fluoride and frozen in working samples until used. The supematant was concentrated 10 times with an Amicon ultrafiltration unit no with PM10 Diaflo ultrafilters (Amicon Canada Ltd., Oakville, Ontario, Canada) and was frozen in working samples as the cytosol fraction. The procedure for binding radiolabeled penicillin to minicell fractions was modified from the method of Spratt (22) and used by Godfrey et al. (4). Fractions (-100 p,g of protein) were labeled for 45 min with a [35S]penicillin concentration of 0.22,ug/ml (4 p.ci/ml) and submitted to electrophoresis on a 8% discontinuous sodium dodecyl sulfate-polyacrylamide gel. Restriction endonuclease cleavage. The recombinant cosmid plb100 was too large to allow unambiguous assignment of restriction endonuclease recognition sites by conventional single and sequential restriction enzyme digestions (14). Therefore the mapping procedure of Legerski et al. (12) with the BAL 31 double-strand exonuclease was used. Approximately 10 p.g of pbl100 DNA was linearized with SstI, ethanol precipitated, and suspended in 160 jil of the BAL 31 reaction buffer recommended by the supplier (international Biotechnologies, Inc., New Haven, Conn.). At different times after the addition of 4 U of BAL 31 (mixed fast and slow species) to the mixture, samples of approximately 1,ug were taken, and the BAL 31 exonuclease activity was stopped by the addition of 10 mm ethylene glycol-bis (,-aminoethyl ether)-n,n,n',n'-tetraacetic acid followed by heat inactivation before ethanol precipitation. Samples were then digested with either BglII, BamHI or Sst BamB HI I U Sal l PuBgl 3 PVU1 plb F ANTIMICROB. AGENTS CHEMOTHER kb i.e 1Kb phc79 m I5349h NK$4\,I8I a coordinates /\ \ Tcs Apr TcS.. H. inliuenzae T-1,3 /t TcS /\ Apr Tct DNA m 2 2 1a 2 Hin id lil ; ihi + iind III FIG. 1. Restriction endonuclease map of plb100. Boxes represent individual fragments obtained by digestion with the indicated enzyme. Fragments are numbered according to decreasing size as observed on agarose gel electrophoresis. DNA fragments that retained the ability to transform H. influenzae Rdnov to moxalactam resistance are indicated by cross-hatching. Resistance markers: Apr, ampicillin; Tcs, inactivated tetracycline. HindIII and submitted to 1% agarose gel electrophoresis in a buffer containing 0.04 M Tris-acetate (ph 8.0) M EDTA-0.5 p,g of ethidium bromide per ml. RESULTS Screening for the altered PBP 3 expression gene. DNA fragments 25 to 50 kb in size obtained from a partial Sau3A digest of H. influenzae T-1,3 have been cloned into the BamHI site of cosmid vector phc79 by selecting for ampicillin-resistant E. coli transductants. The recombinant cosmid DNA isolated from each of 300 ampicillin-resistant transduced E. coli DH1 cells were then used to transform H. influenzae Rdn"v. The transformants were screened for altered PBP 3 expression by selection for moxalactam resistance. Three recombinant cosmids (plb100, plb200, and plb300) giving altered PBP 3 expression in Rdnov were isolated from E. coli; one of (plb100) was used for the subcloning of smaller DNA fragments. Subcloning of the altered PBP 3 expression gene. Only two of the three purified fragments from a BamHI digest of plb100 (42.2 kb) were subcloned into pbr325, because the largest fragment (29.1 kb) was too big for efficient subcloning into this plasmid vector. The smallest fragment (2.9 kb) subcloned in pbr325 (plb120) gave altered PBP 3 expression in H. influenzae Rdnov after transformation. A variety of recombinant plasmids were obtained after ligation of pbr325 to the BamHI-HindIII double-digested, pooled fragments of plb100. Two of the recoinbinant plasmids derived from BamHI-HindIII fragments, plb3140 and plb160 gave altered PBP 3 expression in Rdnov in addition to plb120. These three recombinant plasmids were used twice in transformation experiments and showed high specificity in their capability to give altered PBP 3 expression in Rdno0 compared with control vectors (without inserts) and other recombinant plasmids. Reisolation of these recombinant plasmids from Rdnov after transformation was unsuccessful, indicating recombination events in H. influenzae. Restriction endonuclease cleavage map of plb100. The cleavage sites on the recombinant cosmid plb100 (42.2 kb) were determined for several restriction endonucleases (Fig. 1). plb100 possessed two pieces of phc79 cosmid DNA and two pieces of H. influenzae T-1,3 DNA (26 and 4 kb). 1.~~~~~

4 VOL. 31, 1987 plb18o plb12o (plb121) H B P H. pbr325 (p LB001) plb14b plbl60 :... _ FIG. 2. Diagrammatical representation of the recombinant pbr325 plasmids. H. influenzae T-1,3 DNA inserts are indicated by cross-hatching with fragment numbers corresponding to the plb100 restriction map (Fig. 1) for BgII (t). BamHI (t), and BamHI- HindIlI (*). Plasmids in parentheses had the ampicillin marker inactivated by insertion of the kanamycin marker into the PstI site as in plb180. Resistance markers: amp, ampicillin; cam, chloramphenicol; kan, kanamycin; tet, tetracycline. Other abbreviations: ori, origin of replication; B, BamHI; H, Hindlll; P, Pstl. Cleavage sites are indicated by dotted lines. The three fragments of DNA that retained the ability to transform H. influenzae Rd"'9" to moxalactam resistance upon subcloning are indicated by cross-hatching in Fig. 1. The 2.0-kb BamHI-HindIII fragment no. 7b was present in plb140 and included in plb120, the 2.9-kb BamHI fragment no. 3 was present in plb120, and the 4.3-kb BamHI- HindlIl fragment no. 4 was present in plb160 (Fig. 2). These results indicated that the H. influenzae T-1,3 DNA region responsible for altered PBP 3 expression was largely covered by the 7.3-kb plb100 BglII fragment no. 3 (Fig. 1), a DNA fragment that, once subcloned into the BamHI site of pbr325, was subsequently used in minicell experiments. Altered PBP 3 expression in H. influenzae. Fluorographs of penicillin-labeled cells showed a remarkable difference in PBP profiles for H. influenzae Rdnov and the,-lactamresistant T-1,3 strain (Fig. 3). PBPs 3a and 3b bound radiolabeled penicillin with higher affinity in the susceptible Rd""1 strain than did the equivalent PBPs in the T-1,3 strain as k.n H. INFLUENZAE ALTERED PBP 3 EXPRESSION 289 TABLE 2. MICs of some 3-lactams against some H. influenzae strains and transformantsa Strain MICb (,g/ml) Moxalactam Piperacillin Penicillin G Rdnov 0.02 (1)c 0.01 (1) 0.19 (1) T (32) 0.05 (8) 1.54 (8) HT (16) 0.02 (4) 0.38 (2) HT (16) 0.02 (4) 0.38 (2) HT (16) 0.02 (4) 0.77 (4) HT (16) 0.05 (8) 0.77 (4) HT (16) 0.02 (4) 0.38 (2) a None of the strains produced f3-lactamase, as determined by a nitrocefin slide test. b MICs of drugs were determined by tube dilution with supplemented Mueller-Hinton broth. Strains were incubated for 20 h at 37C. ' Numbers within parentheses indicate ratios of MIC for indicated strain to MIC for strain Rdn," based on the initial doubling dilution series. previously resported (18). The Rdnov strain transformed with recombinant cosmids plb100, plb200, or plb300 or with recombinant plasmids plb120, plb140, or plb160 showed PBP profiles indentical to that of the resistant T-1,3 donor strain (Fig. 3). The MICs of moxalactam, piperacillin, and penicillin G against the resistant T-1,3 strain were similar to MICs obtained against all of these H. influenzae transformants (Table 2). As expected, similar MICs of cefoxitin and imipenem (data not shown) were obtained against all H. influenzae strains (including the susceptible strain Rdnov), since it has been reported that those r-lactams have primary targets other than PBPs 3a and 3b (18). Altered PBP 3 expression in minicells. The proteins synthetized in minicells were labeled with [35S]methionine to identify plasmid-encoded gene products (Fig. 4). A major 55-kilodalton (kda) polypeptide was synthetized from plb160 and plb180. The BglII fragment no. 3 in plb180 and the BamHI-HindIII fragment no. 4 in plb160 (Fig. 2) bore overlapping inserts as seen in the plb100 restriction maps (Fig. 1). In addition to the major 55-kDA polypeptide, the plb160 vector coded for two products (45 and 15 kda) which were apparently produced from the nonoverlapping region of the H. influenzae DNA insert, a region which did not transform strain Rdn"v for resistance as seen when the no. Rd"04 T-1,3 HTIOC HT200 HT300 1T120 HT14O 1T160 I I I 04 2A la- II lb a- 83 3b- I ~1# 7w~2~ ~ ;0 BLA 28- ~'"fw q CAT ~~~I KAPH CAT FIG. 3. Fluorographs of H. influenzae strains and transformants. Whole cells were labeled with [35S]penicillin G and electrophoresed in a Laemmli 10% sodium dodecyl sulfate-polyacrylamide gel system. Major PBP numbers and their apparent molecular weights (103) are indicated on the left. A B C FIG. 4. Fluorograph of labeled polypeptides expressed in minicells bearing different recombinant pbr325 plasmids. E. coli minicells were labeled with L-[35Sjmethionine and electrophoresed in a Laemmli 12% sodium dodecyl sulfate-polyacrylamide gel system. Plasmids: A, pbr325, B, plb120; C, plb160; D, plb180; E, plb001. Numbers indicated the apparent molecular weights (103) of insert-specific encoded polypeptides. Abbreviations: BLA. P- lactamase (30 kda); CAT, chloramphenicol acetyl transferase (26 kda); KAPH, aminoglycoside (kanamycin) amino 3'-phosphotransferase (29 kda).

5 290 MALOUIN ET AL. subcloned BglII fragment no. 5 was used in a transformation experiment (Fig. 1). No polypeptides were synthetized from plb140. However, plb120 coded for a 28-kDa polypeptide that was not observed in minicells containing plb140 or plb180, although all three recombinant plasmids possessed an insert that contained the BamHI-HindIII fragment no. 7b. It is salient to mention that plb140 is lacking the tetracycline promoter region from pbr325, whereas the BamHI and BglII Haemophilus DNA inserts of plb120 and plb180, respectively, are subcloned into the BamHI site within the tetracycline resistance gene (Fig. 2). The binding of penicillin G to minicell proteins was also investigated. There were no additional PBPs corresponding to the plasmid-coded polypeptides in fluorographs of membrane fractions or cytosol fractions from minicells bearing the recombinant plasmid plb180 or plb121 (a recombinant plasmid derivative of plb120; Fig. 2). DISCUSSION Previous work done in our laboratory demonstrated that alteration in PBPs 3a and 3b correlated with the 1-lactam resistance of an H. influenzae strain, UCHI-2, isolated from an immunocompromised adult with pneumonia (18). This clinical isolate showed up to a 32-fold increase in in vitro MICs of a wide variety of,-lactamase, including ampicillin, moxalactam, and cefotaxime, although no 1-lactamase activity was detected even after attempted induction. Transformation of this broad-spectrum 1-lactam resistance into the susceptible H. influenzae Rdnov strain was uniquely associated with the alteration of PBPs 3a and 3b. We concluded that the primary mechanism of resistance in strain UCHI-2 and its transformant T-1,3 was the alteration of the P-lactam binding capacity of PBP 3 (a and b) and that the genetic basis of the resistance was chromosomally determined. Our present objective is to identify the molecular modification involved in the altered penicillin-binding capacity of H. influenzae T-1,3 PBPs 3a and 3b to understand how such a resistance mechanism develops and how it can be overcome by modification of future P-lactams. As a first step, the cloning of the altered PBP 3 expression gene(s) was undertaken and reported in the present study. We chose the cosmid vector phc79 for the initial phase of cloning, since the large (25- to 50-kb) Haemophilus DNA inserts have the advantage of limiting the number of clones that need to be screened and should improve transformation frequencies of H. influenzae Rdnov by viture of their size (1). The former consideration was particularly important since initial screening was a several-step process involving recombinant cosmid isolation from individual transformants and subsequent transformation of H. influenzae by the cloned DNA followed by screening for moxalactam resistance. Consequently, using the cosmid bank to transform H. inf u- enzae Rdn"v, we rapidly identified three recombinant cosmids able to give Rdnov altered PBP 3 expression (Fig. 3) and similar broad-spectrum P-lactam resistance to that of the donor strain T-1,3 (Table 2). The subcloning of different restriction fragments from one of the recombinant cosmids (plb100) showed that three DNA fragments were individually able to give the altered PBP 3 phenotype to the sensitive strain Rdnov. Analysis of these DNA fragments showed that there was actually a single H. influenzae T-1,3 DNA region involved in altered PBP 3 expression (Fig. 1). Two of the subcloned fragments (fragments BamHI no. 3 and BamHI-HindIII no. 7b) were ANTIMICROB. AGENTS CHEMOTHER. overlapping, and the other (fragment BamHI-HindIII no. 4) was adjacent to them. Repeated transformation of H. influenzae Rd"' showed great specificity of the recombinant plasmids plb120, plb140, and plb160 to give altered PBP 3 expression to the isogenic susceptible recipient strain, although low transformation frequencies were observed (4 x 10-9 to 10 x 10-9). These low frequencies were probably related to the insert size of Haemophilus DNA (1) as well as to the recombination events that were needed to obtain the altered PBP 3 expression in Rdnov. As expected, with specific E. coli cloning vectors our inability to reisolate recombinant plasmids from the resistant H. influenzae transformants suggested that the expression of the mutant phenotype was dependent upon recombinational events with the host chromosomal DNA. Considering these recombinational events, our data suggested that at least two mutations were involved in the resistance phenotype, since two distinct adjacent T-1,3 DNA regions were able to give that phenotype to Rd"'. However, it was not clear whether these two mutations affected different genes, the same gene, or repeated identical gene sequences. Analysis of plasmid-encoded polypeptides in minicells revealed additional interesting information. The expression of the plb100 BgIII fragment no. 3 or BamHI-HindIII fragment no. 4 in minicells resulted in the production of a 55-kDa polypeptide coded by that altered T-1,3 DNA region (Fig. 4). This polypeptide did not bind radiolabeled penicillin under our experimental conditions, and its apparent molecular mass was different from that of the PBPs 3a and 3b expressed in H. influenzae T-1,3 (65 and 63 kda, respectively). The lack of penicillin binding does not preclude the possibility that the 55-kDa protein is coded by the mutant PBP 3 gene from H. influenzae T-1,3, since PBPs 3a and 3b are very poorly labeled in that strain (Fig. 3). The difference in electrophoretic mobility between PBPs 3a and 3b expressed in H. influenzae and the 55-kDa plasmid-encoded polypeptide could be due to difference in processing and modification in the E. coli host strains or may indicate that the latter protein is not a structural PBP 3 protein but is involved in posttranslational modifications of a PBP 3 polypeptide that affects binding properties. At present, we have no definitive evidence to exclude either of these possibilities. The second region of T-1,3 DNA that transforms the resistance and altered PBP 3 binding phenotype into H. influenzae is contained within BglII fragment no. 3, BamHI fragment no. 3, and BamHI-HindIII fragment no. 7b (Fig. 1). There is no polypeptide observed in minicells from the subcloned BamHI-HindIII fragment 7b, nor is any additional polypeptide expressed from the subcloned BglII fragment no. 3 covering this region. However, a 28-kDa polypeptide is expressed in minicells containing the subcloned BamHI fragment no. 3 (Fig. 4). This 28-kDa polypeptide is probably a fusion product between the N-terminal region of the tetracycline resistance gene of pbr325 and the C-terminal region of an H. influenzae polypeptide (Fig. 2). The reason that a similar product is not seen with the cloned BglII fragment is because it contains the C-terminal region of the tetracycline resistance gene from phc79 (Fig. 1) immediately adjacent to the C-terminal region of the presumed H. influenzae gene facing in opposite directions. The BamHI- HindlIl fragment no. 7b is cloned into a pbr325 vector lacking the tetracycline promoter region (excised with the small BamHI-HindIII fragments), and thus expression of a fusion polypeptide is impossible (Fig. 2). Unfortunately, plb100 does not contain H. influenzae T-1,3 DNA that

6 VOL. 31, 1987 would contain the promoter and N-terminal regions of this presumptive Haemophilus polypeptide, so information on the molecular weight and penicillin-binding properties of this polypeptide is not readily available. We are currently proceeding with elucidation of the role of the polypeptides involved in altered PBP 3 expression in H. influenzae. This work is a major undertaking since (i) the cloned Haemophilus DNA did not result in moxalactam resistance in E. coli, (ii) the available genetic backgrounds in H. influenzae are limited, and (iii) genetics systems for cloning and manipulating DNA in H. influenzae are also limited. However, the availability of cloned and characterized DNA should facilitate subsequent detailed analysis either on the structural PBP 3 polypeptide or on the components involved in processing and modification. This paper is the first report on cloning of gene(s) involved in naturally occuring altered PBP expression. Our data showed that the low penicillin-binding capacity of H. influenzae T-1,3 PBP and the T-1,3,B-lactam resistance profile may be attributed to at least two distinct chromosomal mutations. These results are in agreement with the recent work of Hedge and Spratt (5) which showed that four different amino acid substitutions needed to be introduced into PBP 3 to obtain laboratory-induced E. coli mutants with high levels of resistance to a variety of cephalosporins. These multiple modifications led to a marked reduction of the affinity of this PBP for,-lactams. The situation described in this paper differs, however, in that either of at least two distinct chromosomal mutations cloned separately reproduced the resistance phenotype and would resemble more the situation described by Hedge and Spratt (5) with lowerlevel resistant mutants. For instance, these authors reported a second-level mutant (two mutations) which showed a cephalexin MIC (60 p.g/ml) similar to that of a first-level mutant (one mutation, 50 p.g/ml). These investigators also showed (6) that a single mutation at different positions in the pbpb gene could give similar resistance phenotypes (for instance, their class 1 and class 2 mutants which had cephalexin MICs of 50,ug/ml). Therefore, it seems possible to have two different mutations that could individually give a resistance profile similar to that of a mutant parent strain, as in the case of H. influenzae T-1,3 and its transformants. With a two-gene model for the resistant strain H. influenzae T-1,3, a regulatory gene might be implicated. Tormo et al. (24) described recently that a mutation in the ftsa gene of E. coli, which codes for a 50-kDa FtsA product, influences penicillin binding to PBP3. In that regard, it seems possible that proteins with enzymatic, structural, and regulatory roles in septation interact with each other (24). ACKNOWLEDGMENTS This work was supported by Medical Research Council of Canada grant MT4350. F. M. is a recipient of a studentship from the Medical Research Council. A.B.S. is a recipient of a Medical Research Council fellowship. We thank S. Eikerman and K. Munro for assistance in preparation of this manuscript. LITERATURE CITED 1. Balganesh, M., and J. K. Setlow Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae. J. Bacteriol. 161: Bell, S. M., and D. Plowman Mechanism of ampicillin resistance in Haemophilus influenzae from respiratory tract. Lancet i: H. INFLUENZAE ALTERED PBP 3 EXPRESSION Bryan, L. E Bacterial resistance and susceptibility to chemotherapeutic agents, p. 53. Cambridge University Press, Cambridge. 4. Godfrey, A. J., L. E. Bryan, and H. R. Rabin P-Lactamresistant Pseudomonas aeruginosa with modified penicillinbinding proteins emerging during cystic fibrosis treatment. Antimicrob. Agents Chemother. 19: Hedge, P. J., and B. G. Spratt Resistance to P-lactam antibiotics by re-modelling the active site of an E. coli penicillinbinding protein. Nature (London) 318: Hedge, P. J., and B. G. Spratt Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin. Eur. J. Biochem. 151: Herriott, R. M., E. M. Meyer, and M. Vogt Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J. Bacteriol. 101: Hohn, B., and J. Collins A small cosmid for efficient cloning of large DNA fragments. Gene 11: Istre, G. R., J. S. Conner, M. P. Glode, and R. S. Hopkins Increasing ampicillin-resistance rates in Haemophilus influenzae meningitis. Am. J. Dis. Child. 138: Kenny, J. F., C. D. Isburg, and R. H. Michaels Meningitis due to Haemophilus influenzae type b resistant to both ampicillin and choramphenicol. Pediatrics 66: Laemmli, U. K., and F. Favre Maturation of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80: Legerski, R. J., J. L. Hodnett, and H. B. Gray, Jr Extracellular nucleases of Pseudomonas Bal 31. lii. Use of double-strand deoxyribonuclease activity as a basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acids Res. 5: Malouin, F., and L. E. Bryan Modification of penicillinbinding proteins as mechanisms of f-lactam resistance. Antimicrob. Agents Chemother. 30: Maniatis, T., E. F. Fritsch, and J. Sambrook Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Mendelman, P. M., D. 0. Chaffin, T. L. Stull, C. E. Rubens, K. D. Mack, and A. L. Smith Characterization of non-3- lactamase-mediated ampicillin resistance in Haernophilus influenzae. Antimicrob. Agents Chemother. 26: O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. H. Shingler A novel method for detection of P-lactamase by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1: Offit, P. A., J. M. Campos, and S. A. Plotkin Ampicillinresistant, 3-lactamase-negative Haemophilus influenzae type b. Pediatrics 69: Parr, T. R., Jr., and L. E. Bryan Mechanism of resistance of an ampicillin-resistant, P-lactamase-negative clinical isolate of Haemophilus influenzae type b to P-lactam antibiotics. Antimicrob. Agents Chemother. 25: Philpott-Howard, J Antibiotic resistance and Haemophilus influenzae. J. Antimicrob. Chemother. 13: Reeve, J. N Synthesis of bacteriophage and plasmidencoded polypeptides in minicells, p In A. Puhler and K. N. Timmis (ed.), Advanced molecular genetics. Springer- Verlag, New York. 21. Silhavy, T. J., M. L. Berman, and L. W. Enquist Experiments with gene fusions, p Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 22. Spratt, B. G Properties of the penicillin-binding proteins of Escherichia coli K-12. Eur. J. Biochem. 72: Takahashi, S., and Y. Nagano Rapid procedure for isolation of plasmid DNA and application to epidemiological analysis. J. Clin. Microbiol. 20: Tormo, A., J. A. Ayala, M. A. De Pedro, M. Aldea, and M. Vincente Interaction of FtsA and PBP3 proteins in the Escherichia coli septum. J. Bacteriol. 166: Westerman, E. L., J. Puls, and J. R. Medina Epiglottitis due to ampicillin-tolerant Haemophilus influenzae type b. South. Med. J. 77: