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Vol. 175, No. 13JOURNAL OF BACTERIOLOGY, JUly 1993, p.
4008-40150021-9193/93/134008-08$02.00/0Copyright © 1993, American
Society for Microbiology
Pseudomonas aeruginosa lasBi Mutants Produce an
Elastase,Substituted at Active-Site His-223, That Is Defective
in
Activity, Processing, and SecretionKEVIN S. MCIVER,1 JOAN C.
OLSON,2 AND DENNIS E. OHMANl*
Department ofMicrobiology and Immunology, University of
Tennessee, and Veterans Affairs Medical Center,Memphis, Tennessee
381631 and Department ofPathology and Laboratory Medicine, Medical
University of
South Carolina, Charleston, South Carolina 294252
Received 5 January 1993/Accepted 25 April 1993
Pseudomonas aeruginosa secretes elastase in a multistep process
which begins with the synthesis of apreproelastase (53.6 kDa)
encoded by lasB, is followed by processing to proelastase (51 kDa),
and concludes withthe rapid accumulation of mature elastase (33
kDa) in the extracellular environment. In this study, mutants ofP.
aeruginosa were constructed by gene replacement which expressed
lasBI, an allele altered in vitro at anactive-site His-223-encoding
codon. The lasBI allele was exchanged for chromosomal lasB
sequences in two strainbackgrounds, FRD2 and PAO1, through a
selectable-cassette strategy which placed a downstream Tn501
markernext to lasB1 and provided the selection for homologous
recombination with the chromosome. Two lasB1mutants, FRD720 and
PDO220, were characterized, and their culture supernatants
contained greatly reducedproteolytic (9-fold) and elastolytic (14-
to 20-fold) activities compared with their respective parental
lasB+ strains.This was primarily due to the effect of His-223
substitution on substrate binding by elastase and thus
itsproteolytic activity. However, the concentration of supernatant
elastase antigen was also reduced (five- tosevenfold) in the mutant
strains compared with the parental strains. An immunoblot analysis
of cell extractsshowed a large accumulation of51-kDa proelastase
within lasBi mutant cells which was not seen in wild-type
cellextracts. A time course study showed that production of
extracellular elastase was inefficient in the lasB1 mutantscompared
with that ofparental strains. This showed that expression ofan
enzymatically defective elastase inhibitsproper processing of
proelastase and provides further evidence for autoproteolytic
processing of proelastase in P.aeruginosa. Unlike the parental
strains, culture supernatants of the lasBi mutants contained two
prominentelastase species that were 33 and 36 kDa in size.
Extracellular 51-kDa proelastase was barely detectable, eventhough
it accumulated to high concentrations within the lasBi mutant
cells. These data suggest that productionof an enzymatically
defective elastase affects proper secretion because autoproteolytic
processing of proelastase isnecessary for efficient localization to
the extracellular milieu. The appearance ofreduced amounts of
extracellularelastase and their sizes of 33 and 36 kDa suggest that
lasB1-encoded elastase was processed by alternate,less-efficient
processing mechanisms. Thus, proelastase must be processed by
removal of nearly all of the 18-kDapropeptide before elastase is a
protein competent for extracellular secretion.
Pseudomonas aeruginosa is an opportunistic pathogenwhich causes
a variety of disease manifestations in compro-mised hosts. The
ability of P. aeruginosa to secrete severaltoxic and degradative
enzymes into the environment is amajor contributor to the
pathogenesis of the organism.Elastase is one of several
extracellular proteases secreted byP. aeruginosa and is considered
a major virulence factor.This is supported by its ability to
degrade a number ofbiologically important proteins, including
elastin (18), somecollagens (8), immunoglobulins G (2) and A (7),
serumal-proteinase inhibitor (19), and complement components(23),
and it releases iron bound to transferrin (3).
Elastase is a neutral metalloprotease requiring one zincion per
molecule that is essential for its activity and acalcium ion for
stability (18). Elastase production and pro-cessing are facilitated
by a growth medium containing bothzinc and calcium ions (22). On
the basis of the inferred aminoacid sequence (1, 5, 24) and
crystallographic structure (25),elastase shares a high degree of
sequence and functionalhomology with the zinc metalloprotease
thermolysin ofBacillus thermoproteolyticus. These similarities to
thermol-
* Corresponding author.
ysin have allowed the prediction of specific residues in-volved
in elastase enzymatic activity and substrate binding,as well as
zinc and calcium binding.
Kessler and Safrin (9, 10) proposed a model for
elastasesecretion, now refined by DNA sequence information,
whichinvolves two proteolytic processing steps. Elastase, encodedby
lasB, is initially synthesized as a preproelastase with amolecular
mass of 53.6 kDa. During translocation throughthe inner membrane, a
2.6-kDa signal sequence is removedto form a 51-kDa proelastase
(12). The proelastase is rapidlyprocessed to a 33-kDa mature form
by cleavage of an 18-kDaN-terminal propeptide. The model proposes
that the propep-tide remains noncovalently associated with a
33-kDaperiplasmic elastase until further processing or
dissociationof the complex occurs, which is followed by secretion
of themature enzyme through the outer membrane.We have recently
shown that overexpression of lasB in
Escherichia coli results in the intracellular accumulation
ofprocessed and enzymatically active 33-kDa elastase; how-ever,
little 51-kDa proelastase is seen (16). When the codonin lasB
encoding His-223, an active-site residue, is changedto encode
Asp-223 (lasBl) or Tyr-223 (lasB2), overexpres-sion of these mutant
alleles in E. coli results in both loss ofenzymatic activity and
accumulation of the unprocessed
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P. AERUGINOSA lasBi MUTANTS 4009
TABLE 1. Bacterial strains and plasmids
Strain or plasmid Genotype or phenotypea Source or reference
E. coliHB101 proA2 leuB6 thi-1 lacYl hsdR hsdM rec413 supE44
rpsL20 This laboratoryJM109 end4l recA1 gyrA96 thi hsdR17(rk mk+)
relA1 supE44 Alac-proAB (F' traD36 proAB Promega
lacIq jM15)
P. aeruginosaPAO1 Prototrophic; lasB+ D. HaasFRD2 Prototrophic;
lasB+ 6FRD706 lasB+ TnS01-6 This studyFRD720 lasBi TnS01-6 This
studyPDO220 lasBi TnS01-6 This study
PlasmidspBluescript KS- High-copy-number cloning vector; Apr
StratagenepKK223-3 Inducible tac promoter expression vector; Apr
PharmaciapEMR2 pBR322::TnS cos oriT Apr Kmr 4pEMRZ3 pEMR2 with
lacZa multiple cloning DNA at BamHI site; Apr Kmr H.
SchweizerRSF1010::TnSOl IncQ Hgr 21pRK2013 ColE1-Tra(RK2)+ Kmr
6pKSM5 pKK223-3 with 2.6-kb P. aeruginosa DNA containing lasBi; Apr
16pKSM15 pLAFR3 (IncP1) with 8-kb EcoRI P. aeruginosa DNA
containing lasB; Tcr This studypKSM20 pEMRZ3 with 4.8-kb P.
aeruginosa DNA containing lasBi and TnS01-6; Apr Kmr Hgr This
studyp720-BAM pEMR2 with 19.2-kb BamHI FRD720 DNA containing lasBi
and TnS01-6; Apr Kmr Hgr This study
a Abbreviations for phenotypes: Tcr, tetracycline resistance;
Hgr, mercury resistance; Apr, ampicillin resistance; Kmr, kanamycin
resistance; Cbr, carbenicillinresistance; Tra, transfer by
conjugation.
51-kDa proelastase (16). These results suggest that the
rapidprocessing of proelastase to mature elastase is
autocatalytic.
In the present study, we developed a gene replacementstrategy to
construct defined mutants of P. aeruginosa usinga cloned gene
modified in vitro by a single base pair change(e.g., lasB1). To
study processing and secretion of anenzymatically defective
elastase in the native host, weconstructed mutants of P. aeruginosa
with a chromosomallyencoded lasBI allele expressed under its native
promoter.These studies showed that modifying the
substrate-bindingresidue, His-223, affected not only enzyme
activity but alsoproelastase processing and extracellular secretion
of elastasein P. aeruginosa. These results indicate that the
pathway ofelastase secretion in P. aeruginosa includes
autoprocessing.
MATERIALS AND METHODSBacterial strains and plasmids and media.
The bacterial
strains and plasmids used in this study are listed in Table
1.Bacteria were cultured in L broth (1% tryptone, 0.5%
yeastextract, 0.5% NaCl [pH 7.5]) or a minimal medium (27).Media
were solidified with 1.5% Bacto agar (Difco). Unlessotherwise
specified, antibiotics were used at the followingconcentrations
(per milliliter): ampicillin, 100 ,ug for E. coli;carbenicillin,
300 ,ug for P. aeruginosa; kanamycin, 30 ,ug forE. coli or 500 ,ug
for P. aeruginosa; tetracycline, 15 ,ug for E.coli or 100 ,ug for
P. aeruginosa; and mercuric chloride, 18,ug for both E. coli and P.
aeruginosa. Casein-agar platescontained 1.5% skim milk (Difco) and
0.8% nutrient broth(Difco). Elastin-agar plates contained 0.5%
elastin (Sigma)and 0.8% nutrient broth (Difco).DNA manipulations.
Routine DNA manipulations and
plasmid extractions were performed as described elsewhere(14).
Triparental matings were used to mobilize recombinantplasmids from
E. coli to P. aeruginosa as previously de-scribed (6). DNA
sequences were determined by the chaintermination technique with
Sequenase (U.S. Biochemical) at42°C by using 5'-[a-32P]dCTP
(>6,000 Ci/mmol, 10 mCi/ml;
Amersham) and 7-deaza-dGTP. Oligonucleotides used forsequencing
primers were synthesized on an Applied Biosys-tems 380B DNA
synthesizer in the Molecular ResourcesCenter of the University of
Tennessee, Memphis.SDS-PAGE and immunoblotting. Protein samples
were
suspended in sodium dodecyl sulfate (SDS) sample buffer (60mM
Tris-HCI, 2% SDS, 10% glycerol, 0.1 mg of bromophe-nol blue per ml,
5% 2-mercaptoethanol [pH 6.8]), and loadedonto a 12.5%
polyacrylamide gel for polyacrylamide gelelectrophoresis (PAGE)
(13). Proteins in polyacrylamidegels were electrotransferred to
nitrocellulose in a Trans-Blotapparatus (Bio-Rad) for 2 h at 160 mA
and 4°C. Immuno-blotting was performed as previously described (16)
withrabbit anti-elastase immunoglobulin G (a gift of E. Kessler)as
the primary antibody and then with a goat anti-rabbithorseradish
peroxidase conjugate (Sigma).Gene replacement in P. aeruginosa FRD2
with selectable
cassettes. An adjacent Tn5Ol (encoding mercury resistance)was
used as a selectable marker to recombine the lasB1mutant allele
into the chromosome of P. aeruginosa. Adja-cent to lasB are a
2.2-kb PstI fragment and a 4.3-kb KpnIfragment (Fig. 1). Because
TnS01 contains no PstI or KpnIsites, such restriction fragments
containing TnS01 can beused as selectable cassettes that can be
ligated next to aDNA fragment containing lasBi. pKSM15 (Fig. 1)
andplasmids containing fragments of pKSM15 were subjected toTn501
mutagenesis as previously described (21). The relativepositions of
insertions, mapped by restriction analysis, areshown on the map of
pKSM15 (Fig. 1). The DNA fragmentswith TnS01 insertions were
exchanged for chromosomalsequences in FRD2 by a transduction method
and withphage F116L as previously described (21), and the
strainsconstructed were examined for any defects in
proteaseproduction that might occur as a result of the insertion.
The2.2-kb PstI and 4.2-kb KpnI fragments containing TnS01-6(8.3 kb)
were cloned from pKSM15::TnSOl-6 into pBlue-script KS- to provide a
source of these selectable cassettes
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4010 McIVER ET AL.
PLASMID(vector)
pKSM15(pLAFR1)
pBlue/P/Tn6(pBluescript KS-)
pBlue/KiTn6(pBluescript KS-)
pBlue/lasBl(pBluescript KS-)
pBlue/lasBl/iTn6(pBluescript KS-)
pZ3/lasBl(pEMRZ3)
pKSM20(pEMRZ3)
Chromosomal A
R lasB+ X
0 2
p KC f f pI
4 6
PK P2.2 kb+Tn5Ol
K
R lasBI- p
R lasBl
R lasBlcII
R lasBl _b
R A lasB+ I>.v I I
K R
88kb
K.j 4.2 kb+Tn5Ol
2.6kb
P K
P KI 1 2.9 kb
P KI
p
XK-J 7.1 kb+Tn501
R'V
Recombination in FRD2FIG. 1. Restriction maps of P. aeruginosa
DNA in plasmids used in this study to generate lasB1 mutants. The
lasB1 allele, previously
described (16), contains a single base pair alteration changing
the codon for His-223 (CAC) to Asp-223 (GAC). Circles represent
Tn501insertions in DNA downstream of lasB. DNA fragments containing
lasB1 and the TnS01-6 insertion were ligated together in the
genereplacement vector pEMRZ3 to form pKSM20. The bottom of figure
illustrates homologous recombination between pKSM20 and
thechromosome of FRD2 by selection for mercury resistance (TnS01-6)
and coinheritance of lasBi. R, EcoRI; P. PstI; K, KpnI.
(pBlue/P/Tn6 and pBlue/K/Tn6, respectively) (Fig. 1). A2.6-kb
EcoRI-PstI fragment containing the lasBi allele wascut from pKSM5
(16) and cloned into pBluescript KS-(pBlue/lasBl). The PstI-TnSOl
cassette was cloned in cor-rect orientation into the single PstI
site in pBlue/lasB1(pBlue/lasBl/Tn6), from which a 2.9-kb
EcoRI-KpnI frag-ment containing lasBi was cloned into the gene
replacementvector pEMRZ3 (pZ3/lasBl). The KpnI-TnSOl cassette
wascloned in correct orientation into the 1inl site of pZ3/lasBlto
form pKSM20 (Fig. 1) for use in the gene replacementprocedures
described below.
Wild-type lasB was replaced with the lasBi allele on the
P.aeruginosa FRD2 chromosome by the excision marker res-cue method
previously described (4), with the followingmodifications. pKSM20
was conjugated into FRD2 by tripa-rental mating, and merodiploid
colonies were selected forgrowth on minimal agar containing
carbenicillin. Colonieswere pooled, inoculated into 10 ml of L
broth containing alow level (3 pg/ml) of HgCl2 to promote
expression ofTnS01-encoded mercury resistance, and grown with
shakingovernight at 370C to allow for excision of the vector
anddiploid sequences by homologous recombination. Dilutionsof the
culture were plated onto L agar containing selectablelevels (18
pg/ml) of HgCl2 and incubated at 370C. Colonieswere screened for
loss of vector-encoded resistances tokanamycin and carbenicillin. A
Southern blot analysis ofdigested genomic DNA obtained from
potential mutants,with a 2.6-kb EcoRI-PstI lasB-containing probe,
was used toverify single-copy gene replacement (data not shown).
Pro-teolytic and elastolytic activities of potential lasBi
mutantswere screened on casein and elastin agar plates,
respec-tively, and then characterized by more sensitive
azocaseinand elastin Congo red assays (described below).
Gene replacement in P. aeruginosa PAO1. A lasBi mutantwas
constructed in the PAO strain background as follows.FRD720 (lasBi
TnS01-6) genomic DNA was digested withBamHI, ligated into the BamHI
site of the gene replacementvector pEMR2, packaged in vitro into X
particles (GigapackII X packaging kit; Stratagene), and transduced
into E. coliHB101 with selection on L agar containing HgCl2.
TnSOJdoes not have a BamHI site. Plasmids from mercury-resis-tant
colonies were screened by restriction analysis, and aclone
(p720-Bam) was identified containing a single BamHIfragment which
included TnS01-6 (8.3 kb) and 19.6 kb of P.aeruginosa DNA. Sequence
analysis was used to verify thatthe lasBi mutation and the
downstream TnS01-6 werepresent in this clone (data not shown). The
p720-Bamconstruct was used to introduce the lasBi mutation into
theP. aeruginosa PA01 chromosome by the excision markerrescue
method as described above. Gene replacement wasverified through a
Southern blot analysis (data not shown).Growth curves and sampling
for elastase production. Log-
arithmic-phase (optical density at 600 nm [OD6.] of 0.6)cultures
of P. aeruginosa strains were used to inoculate(1:100) L broth (250
ml, 1-liter flask), and then they wereincubated at 370C with
maximum aeration. Sample sizeswithdrawn each hour were 1 ml for the
first 5 h and 10 ml forthe next 13 h. OD600 was used to approximate
the celldensity. Samples were prepared for immunoblot analysisand
enzyme assays as follows: a 0.5-ml aliquot was centri-fuged (14,000
x g for 2 min at room temperature), and thepellet was resuspended
in SDS sample buffer and incubatedat 100'C for 5 min (cell extract
fraction). The remainder ofthe sample was centrifuged (8,000 x g
for 10 min at 40C), and2 ml of the resulting supernatant was stored
at -70'C untilused in assays for enzyme activity and elastase
antigen
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P. AERUGINOSA lasBi MUTANTS 4011
(described below). To prevent further nonspecific proteoly-sis,
phenylmethylsulfonyl fluoride (1 mM) and EDTA (5mM) were added to
the supernatant remaining, which wasthen concentrated 25-fold in a
Minicon B15 unit (Amicon).SDS sample buffer (25 ,ul) was added to
each, and sampleswere incubated for 6 min at 950C (supernatant
fraction). Cellextract and supernatant samples from each strain
wereimmunoblotted with antielastase as described above.
Assays of proteolytic and elastolytic activities.
Standardizedcultures of P. aeruginosa strains were used for assays
ofenzyme activities. L broth was inoculated (1:100) withovernight
cultures, grown to an OD6. of 0.6, and thecultures were used to
inoculate (1:100) 10 ml of Luria broth,which was incubated at 370C
with aeration. Maximal elastaseaccumulated in the extracellular
medium by 18 h. Dilutionsof samples were assayed for proteolytic
and elastolyticactivities to establish the linear range of the
reactions.Proteolytic activity was determined as previously
described(11, 15). Elastolytic activity was determined as
previouslydescribed (15, 20).
Quantitation of elastase by ELISA. Relative concentrationsof
elastase antigen in culture supernatants were measured bya modified
direct-binding enzyme-linked immunosorbent as-say (ELISA) as
previously described (22). Briefly, samplesof culture supernatants
(stored at -70'C) were diluted 1:50in ELISA coating buffer (5 mM
sodium carbonate [pH 9.6]),and 100 pl of each diluted supernatant
was added to ELISAwells. The ELISA plate was placed in a 100°C
water bath for3 min and then placed at 4°C overnight. Under these
condi-tions, the proteolytic activity of elastase on
immunoglobu-lins was inhibited; however, elastase maintained the
poten-tial to be specifically recognized by antibodies as
determinedby reproducible quantitation of various dilutions of
superna-tant aliquots (22). Elastase was quantified by adding 100
pu ofa 1:250 dilution of rabbit antielastase serum to each
well.Addition of a 1:1,000 dilution of peroxidase-conjugated
goatanti-rabbit (Cappel Research) and then of the
substrate4-chloro-1-naphthol (Sigma) allowed detection. All
assayswere performed in triplicate, with the mean ELISA
valuerecorded as OD450 per milliliter of culture supernatant.
RESULTS
Construction of a lasBI mutant of P. aeruginosa FRD2utilizing an
adjacent selectable marker. To obtain a selectablemarker next to
the lasB gene, DNA downstream of lasB wassubjected to TnS01
mutagenesis, and four distinct sites ofinsertion were mapped (Fig.
1). Since these insertions are inunknown loci that could
potentially affect elastase produc-tion, each was exchanged for
chromosomal sequences bygene replacement in FRD2. None of the
transposon inser-tions shown in Fig. 1 had any apparent effect on
expressionof lasB or extracellular proteolytic activity (data not
shown).Although our preliminary studies (15) suggested that
recom-binants with chromosomal insertions at TnSOJ-1 were
ad-versely affected in elastase production, further study
showedthat this phenotype was related to a spontaneous
mutation(lasC) in the strain's background that was not
directlyassociated with the insertions. The nature of this mutation
isunder investigation.We previously described the lasBI allele
which contains a
single base pair mutation that changed the codon for His-223(a
substrate-binding residue) to encode Asp-223, a mutationwhich
adversely affects proteolytic activity when expressedin E. coli
(16). By a selectable cassette strategy, DNAcontaining the lasBI
allele was cloned next to DNA contain-
ing Tn501-6 in the gene replacement vector pEMRZ3 (seeMaterials
and Methods). The plasmid formed, pKSM20 (Fig.1), was used in a
gene replacement procedure to exchangethe chromosomal wild-type
allele in FRD2 for the mutantlasB1 allele with the adjacent TnSOl
for selection. Amongthe colonies tested, about 4% had undergone
gene replace-ment as evidenced by the presence of TnS01
(mercuryresistance) and sensitivity to vector-encoded markers
(car-benicillin and kanamycin). All eight of the potential
lasB1mutants obtained exhibited a reduction in extracellular
pro-teolytic activity and had barely detectable elastolytic
activityon plate assays. One of these mutant strains,
designatedFRD720, was chosen for further characterization.To verify
that FRD720 contained the lasB1 allele, a
BamHI fragment was cloned from FRD720 genomic DNAwhich conferred
mercury resistance in E. coli. p720-Bamwas a 19-kb BamHI fragment
of P. aeruginosa DNA fromthe FRD720 chromosome in pEMR2 which
containedTnS01-6 and the presumptive lasB1 allele. The
mutationcausing the His-223 substitution disrupts a restriction
siterecognized by SnoI and ApaLI within the lasB gene
codingsequence (16). Digestion of p720-Bam with SnoI (or
ApaLI)demonstrated the loss of this restriction site and
suggestedthe presence of the lasB1 mutant allele (data not
shown).This was confirmed by sequence analysis of DNA thatincluded
codon 223 on p720-Bam, which showed the pres-ence of the lasB1
mutation (data not shown).
Construction of a lasBi mutant of P. aeruginosa PAO1 byusing the
cloned mutant allele from FRD720. A lasB1 mutantwas also made in
the P. aeruginosa PA01 background forcomparison with FRD720 and to
control for any strain-dependent phenomena. Gene replacement with
pKSM20(lasB1 TnS01-6) in FRD2 was not a frequent event, andlimited
attempts to construct lasB1 mutants in PA01 withpKSM20 were
unsuccessful. However, p720-Bam (lasB1TnS01-6), described above,
was a clone similar to pKSM20and contained another 11 kb of P.
aemginosa DNA. Follow-ing conjugation of p720-Bam into PAO1,
mercury-resistant(TnS01-6) colonies showed loss of vector-encoded
antibioticresistance markers at high frequency, approaching 80%.
All80 colonies examined that showed loss of the vector se-quences
exhibited reduction in proteolytic activity andbarely detectable
elastolytic activity in agar plate assays.One lasB1 mutant of PAO1,
designated PDO220, was used inthe subsequent studies.
Proteolytic and elastolytic activities in supernatants
oflasB1mutant strains. To determine the consequence of the
lasB1mutation on extracellular proteolytic activity in P.
aerugi-nosa, supernatants from 18-h standardized cultures
wereobtained from the lasB1 mutants (FRD720 and PDO220) andtheir
respective wild-type strains (FRD2 and PAO1).FRD706, which contains
a chromosomal TnS01-6 insertionand wild-type lasB allele, was also
included in this analysisto control for any effects due to the
transposon. The hydro-lysis of two substrates was examined:
azocasein, for quan-titation of general proteolytic activity, and
elastin Congored, for quantitation of elastolytic activity. All
three strains(FRD2, FRD706, and PAO1) expressing the wild-type
lasBallele exhibited high levels of both proteolytic and
elastolyticactivities (data not shown). In contrast, the levels of
proteo-lytic activity in the culture supernatants of the two
lasB1mutant strains (FRD720 and PDO220) were eight- to
ninefoldlower than those observed with their respective
wild-typestrains. Compared with the activity of parent strains,
elas-tolytic activity was reduced in the lasBI mutants by 14-
and20-fold (FRD720 and PDO220, respectively) (data not
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Time (hours)10
Time (hours)FIG. 2. Time course study comparing growth with
supernatant elastase concentrations for wild-type and lasBi mutant
strains of P.
aeruginosa FRD and PAO. Strains were grown in L broth at 370C
with aeration under standardized conditions. Growth (open symbols)
isexpressed as culture turbidity determined by A6w. Supernatant
elastase concentrations (closed symbols) were determined by ELISA
andexpressed as micrograms of elastase per milliliter of
supernatant. Time points were taken at hourly intervals from 1 to
18 h for growth and6 to 18 h for supernatant elastase
concentrations. (A) Wild-type FRD706 and FRD706 lasBi growth and
elastase concentrations; (B) wild-typePA01 and PDO220 lasBi growth
and elastase concentrations.
shown). These data supported earlier lasBi expression stud-ies
in E. coli which established that the His-223 residue wasimportant
for the enzymatic activity of P. aeruginosa elas-tase.Reduced
extracellular elastase antigen in lasBi mutant
cultures. The reduction in supernatant proteolytic and
elas-tolytic activities observed above with lasBi mutants was
notattributable to reduced growth rates. Measurements of cul-ture
turbidity, taken at 1-h intervals over an 18-h period ofgrowth,
showed no difference between FRD720 (lasBiTnS01-6) and FRD706
(lasB+ TnS01-6) (Fig. 2A [open sym-bols]). Likewise, PDO220 (1asBi
TnS01-6) and PAO1 (lasB+)showed the same growth pattern over time
(Fig. 2B [opensymbols]) and demonstrated that the TnS01-6 insertion
didnot affect growth under these conditions. The aliquots re-moved
during the growth analysis were also examined forthe concentration
of elastase protein by ELISA. The twowild-type strains, FRD706
(Fig. 2A) and PA01 (Fig. 2B),exhibited a biphasic expression of
elastase antigen, with aninitial burst between 6 and 9 h and then a
plateau inproduction between 10 and 12 h, which was followed by
arapid rise in elastase concentration through the final timepoint
at 18 h. In contrast, cultures of the lasBi mutants(FRD720 and
PDO220) demonstrated a much slower rise inelastase concentration,
and 18-h supernatants containedapproximately five- and sevenfold
less elastase antigen,respectively, compared with the amounts of
their wild-typestrains (Fig. 2).
Effect of lasBI mutation on proelastase processing andsecretion.
Expression of wild-type lasB in E. coli resulted intranslocation of
some mature (33-kDa), enzymatically activeelastase to the
periplasm, although this heterologous host (E.coli) was unable to
secrete elastase to the extracellularmedium (16). The product of
lasB1 in E. coli was defectivenot only in activity but also in
processing and translocation
to the periplasm (16). Here, we examined the potential for
asimilar lasBl-mediated defect in P. aeruginosa which af-fected
both processing and translocation. A defect in trans-location, in
this case, to the extracellular medium wouldexplain the reduced
extracellular elastase antigen in culturesof lasBI mutants
described above. To test this, cell extractsand supernatant
fractions were taken during the time coursestudy described above
and analyzed for elastase antigen byimmunoblot analysis. Cell
extracts of the lasB+ strains,FRD706 (Fig. 3A) and PAO1 (Fig. 4A),
showed mature-size(33-kDa) elastase both within the cell and
localized to thesupernatants (Fig. 3B and 4B) at all time points.
Thecell-bound elastase species (33 kDa) from both wild-typestrains
appeared to form a doublet, which suggests thepossibility of two
intracellular species, a feature previouslynoted by Kessler and
Safrin (9, 10). In the lasBI mutants,proelastase (51 kDa) was the
dominant species in cell ex-tracts of FRD720 (Fig. 3C) and PDO220
(Fig. 4C), indicatinga defect in processing. Both lasBi mutants
accumulatedlarge amounts of 51-kDa proelastase within the cell as
earlyas 6 h. In these P. aeruginosa lasBi extracts, many sizes
ofproelastase breakdown products were observed (Fig. 3C and4C),
including a 33-kDa form, which was similar to that seenin our
previous E. coli expression studies (16) and whichsuggests that the
lasBi 51-kDa proelastase was susceptibleto general proteolytic
digestion. Interestingly, the elastaseappearing in the lasB1 mutant
culture supernatants was oftwo sizes, a 33-kDa mature-size species
and a novel 36-kDaelastase species.
DISCUSSION
The secretion of P. aeruginosa elastase is a multistepprocess
which begins with the synthesis of a 53.6-kDapreproelastase and
results in the rapid accumulation of
A.
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P. AERUGINOSA lasBI MUTANTS 4013VOL. 175, 1993
A. FRD706 Cell Extracts6 7 a 9 10 11
C. FRD720 Cell Extracts
51-kDa -
B. FRD706 Supernatants D. FRD720 Supematants
*< 33-kDaX->
FIG. 3. Immunoblots of samples taken in a time course study
showing elastase-related proteins in cell extracts and supernatants
ofwild-type and lasBi mutant strains of P. aenrginosa FRD. The
number above each lane corresponds to the time point (hours) at
which eachsample was taken and can be directly compared with Fig.
2. Positions corresponding to the 33-kDa mature elastase and the
51-kDa proelastaseare indicated. (A) FRD706 lasB+ cell extract
samples; (B) FRD706 lasB+ supernatant samples; (C) FRD720 lasBi
cell extract samples; (D)FRD720 lasBl supernatant samples.
mature 33-kDa elastase in the extracellular environment. Inan
effort to better understand this pathway, we altered theactive-site
His-223-encoding codon in the structural gene forelastase to form
the lasBI allele to study its plasmid-borneexpression in E. coli
(16). In the present study, we con-structed P. aeruginosa strains
which expressed the lasBIallele from the chromosome to examine the
effect of thisenzymatic defect on proelastase processing and
secretion inthe native organism. The lasBI allele was exchanged
forwild-type lasB sequences in two strain backgrounds, FRD(FRD720)
and PAO (PDO220). This was accomplishedthrough a
selectable-cassette strategy which placed a down-stream TnS01
marker next to lasBI and provided the selec-tion pressure needed
for homologous recombination with thechromosome. In general, the
strategy employed here al-lowed the introduction of a defined,
single-base pair alter-ation into the chromosome ofP. aeruginosa
and should havegeneral application to other genetic studies ofP.
aeruginosa.
Supernatants from the two lasBi mutant strains, FRD720and
PDO220, contained greatly reduced proteolytic (9-fold)and
elastolytic (14- to 20-fold) activities compared with thoseof their
respective wild-type parent strains. In part, thiscould be
attributed to the low concentration of supernatantelastase, as
detected by ELISA, which was reduced by five-to sevenfold in the
mutant strains (FRD720 and PDO220,respectively) compared with
parental strains grown underthe same conditions. However, reduction
in proteolytic andelastolytic activities in the supernatants of
lasBi mutantswas primarily due to the His-223 substitution, which
affectssubstrate binding by elastase and thus its proteolytic
activ-ity. This is in agreement with our previous studies with
lasBiexpression in E. coli, which resulted in almost total loss
of
both proteolytic and elastolytic activities (16). There areother
proteases in the supernatant of P. aeruginosa, includ-ing alkaline
protease and LasA, which have been reported toexhibit some
elastolytic activity (26, 28), and they mayaccount for the residual
elastolytic activity. Determinationof the specific proteolytic
activity of lasBi elastase, purifiedfrom mutant culture
supernatants, is in progress and willshow whether any residual
enzymatic activity remains fol-lowing a substitution at His-223.The
five- to sevenfold reduction in the level of elastase
produced by the lasBl mutants was not attributable to
anydetectable growth defect, since the two mutants demon-strated
growth patterns comparable to those of the wild-typestrains. This
was also not due to reduced rates of lasBtranscription in the
mutants; a lasB-cat operon fusion,constructed in a low-copy-number
plasmid, produced chlor-amphenicol acetyltransferase levels that
were almost identi-cal in all strains (17). However, the immunoblot
analysis ofcell extracts showed a large accumulation of the
51-kDaproelastase form in lasBI mutant cells which was not seen
inwild-type cell extracts. This is further evidence that
expres-sion of an enzymatically defective elastase inhibits
properprocessing of proelastase. We recently have shown
thatcultures of wild-type P. aeruginosa deprived of zinc andcalcium
ions, which are required for elastase enzymaticfunction, also
result in an accumulation of proelastase (22).It was of interest
that the accumulating proelastase in P.aeruginosa lasBI cell
extracts generally appeared to degradein a nonspecific fashion,
although much of the products wasfound in a stable 33-kDa form.
Even 51-kDa lasBiproelastase that was overproduced in E. coli,
which alsounderwent nonspecific proteolysis, formed significant
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4014 McIVER ET AL.
A. PA01 Cell Extracts C. PDO220 Cell Extracts6 7 8 9 10 11
51-kDa -*
*- 33-kDa -*
B. PA01 Supematants D. PDO220 Supernatants6 7 8 910 11 12 14 16
18 6789 01112141618
51-kDa->
if~~~~~~~< 33-kk--
FIG. 4. Immunoblots of samples taken in a time course study
showing elastase-related proteins in cell extracts and supernatants
ofwild-type and lasBi mutant strains of P. aeruginosa PAO. The
number above each lane corresponds to the time point (hours) at
which eachsample was taken and can be directly compared with Fig.
2. Positions corresponding to the 33-kDa mature elastase and the
51-kDa proelastaseare indicated. (A) PA01 lasB+ cell extract
samples; (B) PAO1 lasB+ supernatant samples; (C) PDO220 lasBi cell
extract samples; (D)PDO220 lasBi supernatant samples.
amounts of 33-kDa mature-size elastase (16). These
resultssuggest that the lasBi protease may have residual
autopro-cessing enzymatic activity or that the normal processing
sitein proelastase may be a preferred site for other
proteaseswithin the cell, thus generating the 33-kDa elastase.
Thesepossibilities are under investigation.
Extracellular secretion of elastase by the wild-type strainswas
efficient, with 33-kDa mature elastase appearing inrelatively large
amounts by 6 h and continuing to accumulatethrough the 18-h time
point. This was consistent with theELISA results, which showed a
rapid increase in elastasesupernatant concentration well into the
stationary phase(Fig. 2). On the other hand, the mutant strains
lagged behindin their ability to secrete elastase across the outer
mem-brane, with significant amounts not appearing until 9 h.
Inaddition, there were clearly two elastase species found inlasBi
mutant supernatant fractions, an approximately 36-kDa form as well
as the mature-size 33-kDa form. The33-kDa form was detectable
first, and the larger speciesbegan to appear soon afterward.
Interestingly, the superna-tant fractions of lasBi mutants
contained only barely detect-able amounts of 51-kDa proelastase,
even though it accumu-lated to significant amounts within the cell.
These datasuggest that production of an enzymatically defective
elas-tase perturbs secretion because proelastase must be pro-cessed
for efficient extracellular secretion to take place. Theappearance
of the extracellular 33- and 36-kDa speciessuggests that nearly all
of the propeptide must be removedbefore elastase becomes a protein
that is competent forsecretion. Thus, the propeptide may contain
sequenceswhich, under most circumstance, prevent exoproteins
fromtraversing the membrane. In vitro degradation experimentswere
performed with trypsin on lasBi proelastase produced
by E. coli, and a similar 36-kDa protein product was ob-served
(data not shown). Thus, there may be a generalprotease cleavage
site upstream of the normal maturationsite, at which other
proteases in the cell can cleaveproelastase. This may provide a
less-efficient secondarypathway for processing and secretion of an
inactiveproelastase. Both the smaller 33- and the 36-kDa
specieswere secreted across the outer membrane, suggesting
thatsecretion through the outer membrane can proceed onceprocessing
has occurred by either method.
ACKNOWLEDGMENTS
We thank Efrat Kessler for the kind gift of anti-elastase
immuno-globulin and helpful discussions. Excellent technical
assistance wasprovided by Iulia Kovari at the Molecular Resources
Center,University of Tennessee, Memphis, in oligonucleotide
synthesis.
This work was supported by Public Health Service grant
AI-26187from the National Institute of Allergy and Infectious
Diseases(D.E.O.) and Medical University of South Carolina
InstitutionalResearch Funds (J.C.O.).
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