Cloning and Expression of Genes Responsible for Altered Penicillin-Binding Proteins 3a and 3b in Haemophilus influenzae
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1987, p. 286-291 Vol. 31, No. 2
0066-4804/87/020286-06$02.00/0
Copyright © 1987, American Society for Microbiology
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 peniciflin-
binding protein 3 was used to construct a recombinant cosmid gene bank in Escherichia coli. Three of the
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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 dium, agar or broth (14), was used for the growth of Esche-
treatment. of Haemophilus influenzae infections. However, richia coli.
the emergence of ampicillin- and chloramphenicol-resistant Methionine assay medium (Difco Laboratories, Detroit,
strains over the past few years (9, 10) and concern over the Mich.) and M9 minimal salts medium (14) were used for the
toxicity of chloramphenicol (3) have prompted a search for identification of plasmid-encoded products in minicells.
alternative antibacterial agents. Drugs and radiolabeled compounds. The following antibi-
Ampicillin resistance in H. influenzae is principally due to otics were used in this work: ampicillin and penicilin G from
the presence of a TEM-type ,-lactamase (19). Nevertheless, Ayerst Laboratories, Montreal, Quebec, Canada; moxalac-
there are several reports on' non-p-lactamase-mediated tam from Eli Lilly & Co., Indianapolis, Ind., piperacillin and
ampicillin resistance in H. influenzae (2, 15, 17, 18, 25). tetracycline from Lederle Laboratories, Pearl River, N.Y.;
Alteration of ,-lactam target proteins in bacterial cells may imipenem and cefoxitin from Merck Sharp & Dohme,
lead to significant resistance. The importance of the role of Rahway, N.J.; chloramphenicol, kanamycin, and spectino-
such penicillin-binding proteins (PBPs) in non-p-lactamase- mycin from Sigma Chemical Co., St. Louis, Mo.
mediated P-lactam resistance was recently reviewed (13).
In our laboratory, the broad-spectrum P-lactam resistance [35S]benzylpenicillin (4.9 Ci/mol) was purchased from New
of a P-lactamase-negative clinical isolate of H. influenzae England Nuclear Corp., Lachine, Quebec, Canada: and
type b was previously investigated (18); alteration in the L-[355]methionine (1, 170 Ci/mmol) was from Amersham
binding capacity of PBPs 3a and 3b correlated with the Corp., Oakville, Ontario, Canada.
,-lactam resistance of this strain. To understand how such Cosmid gene bank preparation. The cells from a 100-ml
resistance develops, determination of the molecular basis of culture of H. influenzae T-1,3 grown overnight at 37°C were
this resistance is necessary. We report here the cloning and collected by centrifugation, and the chromosomal DNA was
expression of the genes responsible for altered PBP 3a and extracted by the method of Silhavy et al. (21). One milligram
3b expression in H. influenzae. of chromosomal DNA was partially digested with 20 U of
Sau3A at 23°C for 1 h and extracted by successive phenol-
MATERIALS AND METHODS chloroform and chloroform extractions before ethanol pre-
cipitation. The digested DNA was then fractionated on a
Bactgrial strains and plasmids. The bacterial strains and 38-ml linear sucrose gradient (10 to 40%) as described by
plasmids used in this study are listed in Table 1. Maniatis et al. (14). The fractions containing DNA fragments
Media. The medium used for the growth of H. influenzae from 25 to 50 kilobases (kb) in size were pooled (600 ,ug),
was supplemented brain heart infusion agar or broth (Gibco dialyzed, butanol extracted, and then ethanol precipitated.
Diagnostics, Madison, Wis.), as previously described (18). Mixtures containing 30 ,g of chromosomal DNA and 15 ,ug
Agar plates were incubated in an atmosphere containing 5% of BamHI-cleaved and alkaline phosphatase-dephosphory-
CO2. Mueller-Hinton broth (Gibco), also supplemented (18), lated pHC79 vector in a total volume of 200 ,ul were ligated
was used in the determination of ,B-lactam MICs. LB me-
overnight at 4°C with 1 U of T4 DNA ligase.
The ligated DNA was packaged with an in vitro packing
* Corresponding author. mixture as described by Hohn and Collins (8), and the
286VOL . 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 33849 (14)
thi-I hsdRJ7 sup44 A-; used for transduc-
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tion and transformation experiments
BHB2688 Genotype: N205 recA (X imm434 cItsb2 red3 ATCC 35131 (14)
Eam4 Sam7IA); used for in vitro packaging
of cosmid DNA
BHB2690 Genotype: N205 recA (X imm434 cItsb2 red3 ATCC 35132 (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 Pooled, double-digested BamHI-HindIII DNA fragments
selected on LB plates containing 40 ,ug of ampicillin per ml. were submitted to blind ligation to the corresponding
Screening for the altered P1P 3 expression gene. Recombi- dephosphorylated pBR325 vector, which was purified by
nant pHC79 cosmids were extracted from ampicillin- sucrose gradient centrifugation. All ligation mixtures were
resistant, transduced E. coli DH1 isolates by a rapid isola- used to transform E. coli DH1 by the calcium chloride
tion method (23). Each recombinant cosmid isolated from a procedure (14), and recombinant pBR325 plasmids were
5-ml culture was used to transform competent H. influenzae extracted from ampicillin-resistant, tetracycline-susceptible
Rd"'V cells (0.5 ml). Competence was developed in M-IV (20 ,ug/ml) strains by a rapid isolation method (23). H.
medium by the procedure of Herriott et al. (7). The cells influenzae Rdnov was then transformed with each recombi-
were incubated for 30 mim at 37°C with slow agitation, and nant plasmid to screen for altered PBP 3 expression as
9.5 ml of supplemented brain heart infusion broth was added described above.
before an additional 2 h of incubation. To screen for altered Altered PBP expression in H. influenzae RD"nV. (i) Binding
PBP 3 expression in RDnov, 1 ml of transformation mixture of penicillin G. The procedure for binding radiolabeled
was added to 2 ml of top agar, mixed, and poured onto penicillin to whole cells was previously described (18). Cells
supplemented brain heart infusion agar plates containing 0.2 were labeled for 45 min with a penicillin concentration of
jig of moxalactam per ml. Resistance to moxalactam has 0.22 jig/ml (4 p.Ci/ml) and loaded for electrophoresis on 10%
been shown to correlate with altered PBP 3 expression in H. discontinuous sodium dodecyl sulfate-polyacrylamide gels
influenzae T-1,3 (18). (11) Gels were stained and destained (18) before being
Subcloning of the altered PBP 3 expression gene. Large- soaked for 20 min in Amplify (Amersham Corp.) before 10
scale isolation of recombinant cosmids conferring moxalac- days of fluorography on prefogged X-Omat AR film (East-
tam resistance in RDno, was performed by the procedure of man Kodak Co., Rochester, N.Y.).
Maniatis et al. (14) with chloramphenicol amplification, lysis (ii) Susceptibility testing. MICs of P-lactams were deter-
by sodium dodecyl sulfate, and purification by centrifugation mined by a broth dilution method as previously described
to equilibrium in cesium chloride (1.55 g/ml)-ethidium bro- (18).
mide (600 ,ug/ml) gradients. (iii) Detection of Il-lactamase. 3-Lactamase activity was
A purified recombinant cosmid (pLB100) was digested detected by using a slide test technique with nitrocefin
with BamHI or with BamHI and HindlIl successively. (Oxoid Chemicals Ltd., Nepean, Ontario, Canada) (16).
BamHI restriction fragments were isolated and purified with Expression of plasmid-encoded proteins in minicells. (i)
a 38 ml-linear sucrose gradient (14). Fractions containing Transformation of E. coli SA-2742. All recombinant plasmids
fragments were then dialyzed and ethanol precipitated over- giving the altered PBP 3 expression in H. influenzae Rdn"v
night. Ligation of each BamHI-purified fragment to BamHI- were used to transform the minicell-producing strain E. coli
cleaved and alkaline phosphatase-dephosphorylated pBR325 SA-2742 by the calcium chloride procedure (14).
vector was performed as recommended by the T4 DNA (ii) Isolation of minicells. The method followed for isolation
ligase supplier at 18°C. of minicells was essentially that of Reeve (20). Purified288 MALOUIN ET AL. ANTIMICROB. AGENTS CHEMOTHER.
minicells from 750-ml cultures were finally suspended in M9 pHC79 coordinates
medium containing 30% (vol/vol) glycerol. Samples of 1 ml
(2 x 1010 minicells) were frozen by immersion in liquid
Sst
BamB HI U
I
pLB100 42.2 kb
1
m m
nitrogen and stored at -70°C until used. Sal l .~~~~~
2
(iii) Labeling of plasmid-encoded polypeptides in nminicells. PuBgl 3 15 1 4 F 1 2
With the method described by Reeve (20), the minicells from PVU1 1a| 2
frozen samples were pelleted by centrifugation and sus- 3 | 89 1 1 16 1
6 2 Hin id lil ;
pended in 1 ml of M9 medium containing 0.4% (wt/vol) 1Kb
i.e
glucose. Samples of 100 ,ul were incubated for 10 min at 37°C iHI +
before the addition of 5 ,ul of L-[35S]methionine at a final I5349h NK$4\,I8I10 1 17a 1 6 1 2 114 iind III
concentration of 100 ,uCi/ml in methionine assay medium.
Minicells were then incubated for 1 h at 37°C, pelleted by /\ \ /t /\
centrifugation, washed in M9 medium-glucose, and sus- Tcs Apr TcS .. H. inliuenzae T-1,3 TcS Apr Tct
pended in 50 ,ul of electrophoretic sample buffer consisting of DNA
2% sodium dodecyl sulfate, 4% 2-mercaptoethanol, 10%
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FIG. 1. Restriction endonuclease map of pLB100. Boxes repre-
glycerol, 1 M Tris (pH 6.8), and 0.002% bromophenol blue. sent individual fragments obtained by digestion with the indicated
The suspended minicells were heated to 100°C for 5 min, and enzyme. Fragments are numbered according to decreasing size as
25 ,ul was loaded for electrophoresis in 12% discontinuous observed on agarose gel electrophoresis. DNA fragments that
sodium dodecyl sulfate-polyacrylamide gels. Gels were sub- retained the ability to transform H. influenzae Rdnov to moxalactam
mitted to 3 h of fluorography as described above. resistance are indicated by cross-hatching. Resistance markers: Apr,
(iv) Binding of penicillin G. To study the binding of ampicillin; Tcs, inactivated tetracycline.
radiolabeled penicillin to minicell proteins, the construction
of suitable recombinant plasmids was necessary. Inactiva- HindIII and submitted to 1% agarose gel electrophoresis in a
tion of the ampicillin resistance marker in the recombinant buffer containing 0.04 M Tris-acetate (pH 8.0)-0.002 M
pBR325 plasmids was performed by inserting the kanamycin EDTA-0.5 p,g of ethidium bromide per ml.
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 RESULTS
pellet was passed four times through a French pressure cell
(Fred S. Carver, Inc., Summit, N.J.) at 15,000 lb/in2. Screening for the altered PBP 3 expression gene. DNA
Phenylmethylsulfonyl fluoride and DNase 1 (Sigma) were fragments 25 to 50 kb in size obtained from a partial Sau3A
added to final concentrations of 1 mM and 30 ,ug/ml, respec- digest of H. influenzae T-1,3 have been cloned into the
tively, and the cell lysate was centrifuged at 1,500 x g for 10 BamHI site of cosmid vector pHC79 by selecting for ampicil-
min to remove unbroken cells. The supernatant was col- lin-resistant E. coli transductants. The recombinant cosmid
lected and spun at 46,000 rpm in a 50 Ti Beckman rotor for DNA isolated from each of 300 ampicillin-resistant
1 h. The cell membrane pellet was suspended in 100 ,ul of 100 transduced E. coli DH1 cells were then used to transform H.
mM Tris (pH 8.0)-i mM phenylmethylsulfonyl fluoride and influenzae Rdn"v. The transformants were screened for al-
frozen in working samples until used. The supematant was tered PBP 3 expression by selection for moxalactam resist-
concentrated 10 times with an Amicon ultrafiltration unit no. ance. Three recombinant cosmids (pLB100, pLB200, and
8010 with PM10 Diaflo ultrafilters (Amicon Canada Ltd., pLB300) giving altered PBP 3 expression in Rdnov were
Oakville, Ontario, Canada) and was frozen in working isolated from E. coli; one of (pLB100) was used for the
samples as the cytosol fraction. subcloning of smaller DNA fragments.
The procedure for binding radiolabeled penicillin to Subcloning of the altered PBP 3 expression gene. Only two
minicell fractions was modified from the method of Spratt of the three purified fragments from a BamHI digest of
(22) and used by Godfrey et al. (4). Fractions (-100 p,g of pLB100 (42.2 kb) were subcloned into pBR325, because the
protein) were labeled for 45 min with a [35S]penicillin con- largest fragment (29.1 kb) was too big for efficient subcloning
centration of 0.22 ,ug/ml (4 p.Ci/ml) and submitted to electro- into this plasmid vector. The smallest fragment (2.9 kb)
phoresis on a 8% discontinuous sodium dodecyl sul- subcloned in pBR325 (pLB120) gave altered PBP 3 expres-
fate-polyacrylamide gel. sion in H. influenzae Rdnov after transformation.
Restriction endonuclease cleavage. The recombinant A variety of recombinant plasmids were obtained after
cosmid pLB100 was too large to allow unambiguous assign- ligation of pBR325 to the BamHI-HindIII double-digested,
ment of restriction endonuclease recognition sites by con- pooled fragments of pLB100. Two of the recoinbinant plas-
ventional single and sequential restriction enzyme digestions mids derived from BamHI-HindIII fragments, pLB3140 and
(14). Therefore the mapping procedure of Legerski et al. (12) pLB160 gave altered PBP 3 expression in Rdnov in addition to
with the BAL 31 double-strand exonuclease was used. pLB120. These three recombinant plasmids were used twice
Approximately 10 p.g of pBL100 DNA was linearized with in transformation experiments and showed high specificity in
SstI, ethanol precipitated, and suspended in 160 jil of the their capability to give altered PBP 3 expression in Rdno0
BAL 31 reaction buffer recommended by the supplier (inter- compared with control vectors (without inserts) and other
national Biotechnologies, Inc., New Haven, Conn.). At recombinant plasmids. Reisolation of these recombinant
different times after the addition of 4 U of BAL 31 (mixed plasmids from Rdnov after transformation was unsuccessful,
fast and slow species) to the mixture, samples of approxi- indicating recombination events in H. influenzae.
mately 1 ,ug were taken, and the BAL 31 exonuclease Restriction endonuclease cleavage map of pLB100. The
activity was stopped by the addition of 10 mM ethylene cleavage sites on the recombinant cosmid pLB100 (42.2 kb)
glycol-bis (,-aminoethyl ether)-N,N,N',N'-tetraacetic acid were determined for several restriction endonucleases (Fig.
followed by heat inactivation before ethanol precipitation. 1). pLB100 possessed two pieces of pHC79 cosmid DNA
Samples were then digested with either BglII, BamHI or and two pieces of H. influenzae T-1,3 DNA (26 and 4 kb).VOL. 31, 1987
pLB18O
pLB12O
(pLB121)
pBR325
(p LB001)
pLB14B
H B
.......
_
P
k.n
H.
.................
H. INFLUENZAE ALTERED PBP 3 EXPRESSION
TABLE 2. MICs of some 3-lactams against some H. influenzae
Strain
Rdnov
T-1.3
HT100
HT300
HT120
strains and transformantsa
Moxalactam
0.02
0.77
0.38
0.38
0.38
(1)c
(32)
(16)
(16)
(16)
MICb (,g/ml)
Piperacillin
0.01 (1)
0.05 (8)
0.02 (4)
0.02 (4)
0.02 (4)
1.54 (8)
0.38 (2)
0.38 (2)
0.77 (4)
289
Penicillin G
0.19 (1)
: ........... HT140 0.38 (16) 0.05 (8) 0.77 (4)
pLBl60
HT160 0.38 (16) 0.02 (4) 0.38 (2)
a None of the strains produced f3-lactamase, as determined by a nitrocefin
FIG. 2. Diagrammatical representation of the recombinant slide test.
b MICs of drugs were determined by tube dilution with supplemented
pBR325 plasmids. H. influenzae T-1,3 DNA inserts are indicated by Mueller-Hinton broth. Strains were incubated for 20 h at 37C.
cross-hatching with fragment numbers corresponding to the pLB100
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' Numbers within parentheses indicate ratios of MIC for indicated strain to
restriction map (Fig. 1) for BgII (t). BamHI (t), and BamHI- MIC for strain Rdn," based on the initial doubling dilution series.
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, chloram- previously resported (18). The Rdnov strain transformed with
phenicol; kan, kanamycin; tet, tetracycline. Other abbreviations: recombinant cosmids pLB100, pLB200, or pLB300 or with
ori, origin of replication; B, BamHI; H, Hindlll; P, Pstl. Cleavage recombinant plasmids pLB120, pLB140, or pLB160 showed
sites are indicated by dotted lines. 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
The three fragments of DNA that retained the ability to MICs obtained against all of these H. influenzae transform-
transform H. influenzae Rd"'9" to moxalactam resistance ants (Table 2). As expected, similar MICs of cefoxitin and
upon subcloning are indicated by cross-hatching in Fig. 1. imipenem (data not shown) were obtained against all H.
The 2.0-kb BamHI-HindIII fragment no. 7b was present in influenzae strains (including the susceptible strain Rdnov),
pLB140 and included in pLB120, the 2.9-kb BamHI frag- since it has been reported that those r-lactams have primary
ment no. 3 was present in pLB120, and the 4.3-kb BamHI- targets other than PBPs 3a and 3b (18).
HindlIl fragment no. 4 was present in pLB160 (Fig. 2). Altered PBP 3 expression in minicells. The proteins synthe-
These results indicated that the H. influenzae T-1,3 DNA tized in minicells were labeled with [35S]methionine to
region responsible for altered PBP 3 expression was largely identify plasmid-encoded gene products (Fig. 4). A major
covered by the 7.3-kb pLB100 BglII fragment no. 3 (Fig. 1), 55-kilodalton (kDa) polypeptide was synthetized from
a DNA fragment that, once subcloned into the BamHI site of pLB160 and pLB180. The BglII fragment no. 3 in pLB180
pBR325, was subsequently used in minicell experiments. and the BamHI-HindIII fragment no. 4 in pLB160 (Fig. 2)
Altered PBP 3 expression in H. influenzae. Fluorographs of bore overlapping inserts as seen in the pLB100 restriction
penicillin-labeled cells showed a remarkable difference in maps (Fig. 1). In addition to the major 55-kDA polypeptide,
PBP profiles for H. influenzae Rdnov and the ,-lactam- the pLB160 vector coded for two products (45 and 15 kDa)
resistant T-1,3 strain (Fig. 3). PBPs 3a and 3b bound radio- which were apparently produced from the nonoverlapping
labeled penicillin with higher affinity in the susceptible Rd""1 region of the H. influenzae DNA insert, a region which did
strain than did the equivalent PBPs in the T-1,3 strain as 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 1-55-- _
2A
04 la-
I ~1# 7w~2~ ~ ;0
BLA _45
II lb- -KAPH
28 - ~'"fw q CAT
72 2-
65 3a- CAT ~~
~I
83 3b-
-15
42 -
A B C
FIG. 4. Fluorograph of labeled polypeptides expressed in
minicells bearing different recombinant pBR325 plasmids. E. coli
30 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;
FIG. 3. Fluorographs of H. influenzae strains and transformants. E, pLB001. Numbers indicated the apparent molecular weights (103)
Whole cells were labeled with [35S]penicillin G and electrophoresed of insert-specific encoded polypeptides. Abbreviations: BLA. P-
in a Laemmli 10% sodium dodecyl sulfate-polyacrylamide gel lactamase (30 kDa); CAT, chloramphenicol acetyl transferase (26
system. Major PBP numbers and their apparent molecular weights kDa); KAPH, aminoglycoside (kanamycin) amino 3'-phosphotrans-
(103) are indicated on the left. ferase (29 kDa).290 MALOUIN ET AL. ANTIMICROB. AGENTS CHEMOTHER.
subcloned BglII fragment no. 5 was used in a transformation overlapping, and the other (fragment BamHI-HindIII no. 4)
experiment (Fig. 1). No polypeptides were synthetized from was adjacent to them.
pLB140. However, pLB120 coded for a 28-kDa polypeptide Repeated transformation of H. influenzae Rd"' showed
that was not observed in minicells containing pLB140 or great specificity of the recombinant plasmids pLB120,
pLB180, although all three recombinant plasmids possessed pLB140, and pLB160 to give altered PBP 3 expression to the
an insert that contained the BamHI-HindIII fragment no. 7b. isogenic susceptible recipient strain, although low transfor-
It is salient to mention that pLB140 is lacking the tetracy- mation frequencies were observed (4 x 10-9 to 10 x 10-9).
cline promoter region from pBR325, whereas the BamHI and These low frequencies were probably related to the insert
BglII Haemophilus DNA inserts of pLB120 and pLB180, size of Haemophilus DNA (1) as well as to the recombina-
respectively, are subcloned into the BamHI site within the tion events that were needed to obtain the altered PBP 3
tetracycline resistance gene (Fig. 2). expression in Rdnov. As expected, with specific E. coli
The binding of penicillin G to minicell proteins was also cloning vectors our inability to reisolate recombinant plas-
investigated. There were no additional PBPs corresponding mids from the resistant H. influenzae transformants sug-
to the plasmid-coded polypeptides in fluorographs of mem- gested that the expression of the mutant phenotype was
brane fractions or cytosol fractions from minicells bearing dependent upon recombinational events with the host chro-
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the recombinant plasmid pLB180 or pLB121 (a recombinant mosomal DNA. Considering these recombinational events,
plasmid derivative of pLB120; Fig. 2). our data suggested that at least two mutations were involved
in the resistance phenotype, since two distinct adjacent
DISCUSSION T-1,3 DNA regions were able to give that phenotype to
Rd"'. However, it was not clear whether these two muta-
Previous work done in our laboratory demonstrated that tions affected different genes, the same gene, or repeated
alteration in PBPs 3a and 3b correlated with the 1-lactam identical gene sequences.
resistance of an H. influenzae strain, UCHI-2, isolated from Analysis of plasmid-encoded polypeptides in minicells
an immunocompromised adult with pneumonia (18). This revealed additional interesting information. The expression
clinical isolate showed up to a 32-fold increase in in vitro of the pLB100 BgIII fragment no. 3 or BamHI-HindIII
MICs of a wide variety of ,-lactamase, including ampicillin, fragment no. 4 in minicells resulted in the production of a
moxalactam, and cefotaxime, although no 1-lactamase ac- 55-kDa polypeptide coded by that altered T-1,3 DNA region
tivity was detected even after attempted induction. Trans- (Fig. 4). This polypeptide did not bind radiolabeled penicillin
formation of this broad-spectrum 1-lactam resistance into under our experimental conditions, and its apparent molec-
the susceptible H. influenzae Rdnov strain was uniquely ular mass was different from that of the PBPs 3a and 3b
associated with the alteration of PBPs 3a and 3b. We expressed in H. influenzae T-1,3 (65 and 63 kDa, respec-
concluded that the primary mechanism of resistance in strain tively). The lack of penicillin binding does not preclude the
UCHI-2 and its transformant T-1,3 was the alteration of the possibility that the 55-kDa protein is coded by the mutant
P-lactam binding capacity of PBP 3 (a and b) and that the PBP 3 gene from H. influenzae T-1,3, since PBPs 3a and 3b
genetic basis of the resistance was chromosomally deter- are very poorly labeled in that strain (Fig. 3). The difference
mined. in electrophoretic mobility between PBPs 3a and 3b ex-
Our present objective is to identify the molecular modifi- pressed in H. influenzae and the 55-kDa plasmid-encoded
cation involved in the altered penicillin-binding capacity of polypeptide could be due to difference in processing and
H. influenzae T-1,3 PBPs 3a and 3b to understand how such modification in the E. coli host strains or may indicate that
a resistance mechanism develops and how it can be over- the latter protein is not a structural PBP 3 protein but is
come by modification of future P-lactams. As a first step, the involved in posttranslational modifications of a PBP 3 poly-
cloning of the altered PBP 3 expression gene(s) was under- peptide that affects binding properties. At present, we have
taken and reported in the present study. no definitive evidence to exclude either of these possibilities.
We chose the cosmid vector pHC79 for the initial phase of The second region of T-1,3 DNA that transforms the
cloning, since the large (25- to 50-kb) Haemophilus DNA resistance and altered PBP 3 binding phenotype into H.
inserts have the advantage of limiting the number of clones influenzae is contained within BglII fragment no. 3, BamHI
that need to be screened and should improve transformation fragment no. 3, and BamHI-HindIII fragment no. 7b (Fig. 1).
frequencies of H. influenzae Rdnov by viture of their size (1). There is no polypeptide observed in minicells from the
The former consideration was particularly important since subcloned BamHI-HindIII fragment 7b, nor is any additional
initial screening was a several-step process involving recom- polypeptide expressed from the subcloned BglII fragment
binant cosmid isolation from individual transformants and no. 3 covering this region. However, a 28-kDa polypeptide is
subsequent transformation of H. influenzae by the cloned expressed in minicells containing the subcloned BamHI
DNA followed by screening for moxalactam resistance. fragment no. 3 (Fig. 4). This 28-kDa polypeptide is probably
Consequently, using the cosmid bank to transform H. inf u- a fusion product between the N-terminal region of the
enzae Rdn"v, we rapidly identified three recombinant tetracycline resistance gene of pBR325 and the C-terminal
cosmids able to give Rdnov altered PBP 3 expression (Fig. 3) region of an H. influenzae polypeptide (Fig. 2). The reason
and similar broad-spectrum P-lactam resistance to that of the that a similar product is not seen with the cloned BglII
donor strain T-1,3 (Table 2). fragment is because it contains the C-terminal region of the
The subcloning of different restriction fragments from one tetracycline resistance gene from pHC79 (Fig. 1) immedi-
of the recombinant cosmids (pLB100) showed that three ately adjacent to the C-terminal region of the presumed H.
DNA fragments were individually able to give the altered influenzae gene facing in opposite directions. The BamHI-
PBP 3 phenotype to the sensitive strain Rdnov. Analysis of HindlIl fragment no. 7b is cloned into a pBR325 vector
these DNA fragments showed that there was actually a lacking the tetracycline promoter region (excised with the
single H. influenzae T-1,3 DNA region involved in altered small BamHI-HindIII fragments), and thus expression of a
PBP 3 expression (Fig. 1). Two of the subcloned fragments fusion polypeptide is impossible (Fig. 2). Unfortunately,
(fragments BamHI no. 3 and BamHI-HindIII no. 7b) were pLB100 does not contain H. influenzae T-1,3 DNA thatVOL. 31, 1987 H. INFLUENZAE ALTERED PBP 3 EXPRESSION 291
would contain the promoter and N-terminal regions of this 3. Bryan, L. E. 1982. Bacterial resistance and susceptibility to
presumptive Haemophilus polypeptide, so information on chemotherapeutic agents, p. 53. Cambridge University Press,
the molecular weight and penicillin-binding properties of this Cambridge.
polypeptide is not readily available. 4. Godfrey, A. J., L. E. Bryan, and H. R. Rabin. 1981. P-Lactam-
We are currently proceeding with elucidation of the role of resistant Pseudomonas aeruginosa with modified penicillin-
the polypeptides involved in altered PBP 3 expression in H. binding proteins emerging during cystic fibrosis treatment. An-
timicrob. Agents Chemother. 19:705-711.
influenzae. This work is a major undertaking since (i) the 5. Hedge, P. J., and B. G. Spratt. 1985. Resistance to P-lactam
cloned Haemophilus DNA did not result in moxalactam antibiotics by re-modelling the active site of an E. coli penicillin-
resistance in E. coli, (ii) the available genetic backgrounds in binding protein. Nature (London) 318:478-480.
H. influenzae are limited, and (iii) genetics systems for 6. Hedge, P. J., and B. G. Spratt. 1985. Amino acid substitutions
cloning and manipulating DNA in H. influenzae are also that reduce the affinity of penicillin-binding protein 3 of Esche-
limited. However, the availability of cloned and character- richia coli for cephalexin. Eur. J. Biochem. 151:111-121.
ized DNA should facilitate subsequent detailed analysis 7. Herriott, R. M., E. M. Meyer, and M. Vogt. 1970. Defined
either on the structural PBP 3 polypeptide or on the compo- nongrowth media for stage II development of competence in
nents involved in processing and modification. Haemophilus influenzae. J. Bacteriol. 101:517-524.
8. Hohn, B., and J. Collins. 1980. A small cosmid for efficient
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This paper is the first report on cloning of gene(s) involved cloning of large DNA fragments. Gene 11:291-298.
in naturally occuring altered PBP expression. Our data 9. Istre, G. R., J. S. Conner, M. P. Glode, and R. S. Hopkins. 1984.
showed that the low penicillin-binding capacity of H. influ- Increasing ampicillin-resistance rates in Haemophilus influ-
enzae T-1,3 PBP and the T-1,3 ,B-lactam resistance profile enzae meningitis. Am. J. Dis. Child. 138:366-369.
may be attributed to at least two distinct chromosomal 10. Kenny, J. F., C. D. Isburg, and R. H. Michaels. 1980. Meningitis
mutations. These results are in agreement with the recent due to Haemophilus influenzae type b resistant to both ampicil-
work of Hedge and Spratt (5) which showed that four lin and choramphenicol. Pediatrics 66:14-16.
different amino acid substitutions needed to be introduced 11. Laemmli, U. K., and F. Favre. 1973. Maturation of bacterio-
phage T4. I. DNA packaging events. J. Mol. Biol. 80:575-599.
into PBP 3 to obtain laboratory-induced E. coli mutants with 12. Legerski, R. J., J. L. Hodnett, and H. B. Gray, Jr. 1978.
high levels of resistance to a variety of cephalosporins. Extracellular nucleases of Pseudomonas Bal 31. lII. Use of
These multiple modifications led to a marked reduction of double-strand deoxyribonuclease activity as a basis of a conve-
the affinity of this PBP for ,-lactams. The situation described nient method for the mapping of fragments of DNA produced by
in this paper differs, however, in that either of at least two cleavage with restriction enzymes. Nucleic Acids Res.
distinct chromosomal mutations cloned separately repro- 5:1445-1464.
duced the resistance phenotype and would resemble more 13. Malouin, F., and L. E. Bryan. 1986. Modification of penicillin-
the situation described by Hedge and Spratt (5) with lower- binding proteins as mechanisms of f-lactam resistance. Antimi-
level resistant mutants. For instance, these authors reported crob. Agents Chemother. 30:1-5.
14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
a second-level mutant (two mutations) which showed a cloning: a laboratory manual. Cold Spring Harbor Laboratory,
cephalexin MIC (60 p.g/ml) similar to that of a first-level Cold Spring Harbor, N.Y.
mutant (one mutation, 50 p.g/ml). These investigators also 15. Mendelman, P. M., D. 0. Chaffin, T. L. Stull, C. E. Rubens,
showed (6) that a single mutation at different positions in the K. D. Mack, and A. L. Smith. 1984. Characterization of non-3-
pbpB gene could give similar resistance phenotypes (for lactamase-mediated ampicillin resistance in Haernophilus influ-
instance, their class 1 and class 2 mutants which had enzae. Antimicrob. Agents Chemother. 26:235-244.
cephalexin MICs of 50 ,ug/ml). Therefore, it seems possible 16. O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. H. Shingler.
to have two different mutations that could individually give a 1972. A novel method for detection of P-lactamase by using a
resistance profile similar to that of a mutant parent strain, as chromogenic cephalosporin substrate. Antimicrob. Agents Che-
in the case of H. influenzae T-1,3 and its transformants. mother. 1:283-288.
17. Offit, P. A., J. M. Campos, and S. A. Plotkin. 1982. Ampicillin-
With a two-gene model for the resistant strain H. influ- resistant, 3-lactamase-negative Haemophilus influenzae type b.
enzae T-1,3, a regulatory gene might be implicated. Tormo et Pediatrics 69:230-232.
al. (24) described recently that a mutation in the ftsA gene of 18. Parr, T. R., Jr., and L. E. Bryan. 1984. Mechanism of resistance
E. coli, which codes for a 50-kDa FtsA product, influences of an ampicillin-resistant, P-lactamase-negative clinical isolate
penicillin binding to PBP3. In that regard, it seems possible of Haemophilus influenzae type b to P-lactam antibiotics. An-
that proteins with enzymatic, structural, and regulatory roles timicrob. Agents Chemother. 25:747-753.
in septation interact with each other (24). 19. Philpott-Howard, J. 1984. Antibiotic resistance and Haemo-
philus influenzae. J. Antimicrob. Chemother. 13:199-208.
20. Reeve, J. N. 1984. Synthesis of bacteriophage and plasmid-
ACKNOWLEDGMENTS encoded polypeptides in minicells, p. 212-223. In A. Puhler and
K. N. Timmis (ed.), Advanced molecular genetics. Springer-
This work was supported by Medical Research Council of Canada Verlag, New York.
grant MT4350. F. M. is a recipient of a studentship from the Medical 21. Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984.
Research Council. A.B.S. is a recipient of a Medical Research Experiments with gene fusions, p. 137-139. Cold Spring Harbor
Council fellowship. Laboratory, Cold Spring Harbor, N.Y.
We thank S. Eikerman and K. Munro for assistance in preparation 22. Spratt, B. G. 1977. Properties of the penicillin-binding proteins
of this manuscript. of Escherichia coli K-12. Eur. J. Biochem. 72:341-352.
23. Takahashi, S., and Y. Nagano. 1984. Rapid procedure for
LITERATURE CITED isolation of plasmid DNA and application to epidemiological
1. Balganesh, M., and J. K. Setlow. 1985. Differential behavior of analysis. J. Clin. Microbiol. 20:608-613.
plasmids containing chromosomal DNA insertions of various 24. Tormo, A., J. A. Ayala, M. A. De Pedro, M. Aldea, and M.
sizes during transformation and conjugation in Haemophilus Vincente. 1986. Interaction of FtsA and PBP3 proteins in the
influenzae. J. Bacteriol. 161:141-146. Escherichia coli septum. J. Bacteriol. 166:985-992.
2. Bell, S. M., and D. Plowman. 1980. Mechanism of ampicillin 25. Westerman, E. L., J. Puls, and J. R. Medina. 1984. Epiglottitis
resistance in Haemophilus influenzae from respiratory tract. due to ampicillin-tolerant Haemophilus influenzae type b.
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