From the Department of Microbiology, School of Medicine, University
of Washington, Seattle, Washington 98195
Subunits of type IV pili and a subset of proteins
of the type II extracellular protein secretion apparatus undergo two
consecutive post-translational modifications: leader peptide cleavage,
followed by methylation of the newly created N-terminal amino acid.
These two reactions are carried out by a single bifunctional enzyme encoded in Pseudomonas aeruginosa by the pilD
gene. Properties of PilD mutants at positions Gly95 and/or
Lys96 which were differentially affected in leader
peptidase and N-methyltransferase function were
characterized. None of the single amino acid substitutions showed a
significant alteration in their ability to cleave the prepilin leader
peptide; however, two double mutants did exhibit a modest reduction in
the efficiency of cleavage. In contrast, a significant decrease of
N-methyltransferase activity was detected in PilD having
substitutions at Gly95. Mutants with substitutions at
position Lys96 showed a variable effect on
N-methyltransferase activity with an apparent requirement
for any charged amino acid at this position. Absence of
N-methyltransferase activity did not appear to interfere with the ability of P. aeruginosa to assemble functional
pili. Moreover, pilin monomers isolated from P. aeruginosa
expressing PilD with Gly95 substitutions were not
methylated. Although complete methylation does not appear to be
absolutely required for pilus assembly in P. aeruginosa,
this modification may be important for pilus function in its natural
habitat.
 |
INTRODUCTION |
The respiratory distress and morbidity in individuals suffering
from cystic fibrosis is most often caused by the opportunistic pathogen
Pseudomonas aeruginosa (1). This organism has at its disposal a variety of adhesins (2), including type IV pili, to
establish an infection in a susceptible host (3). Upon attachment and
multiplication, P. aeruginosa can secrete a variety of
hydrolytic enzymes and exotoxins (4). Central to these two processes
(attachment and secretion) is PilD (also called XcpA), a bifunctional
enzyme that catalyzes post-translational modifications of several
proteins required for assembly of type IV pilin subunits into filaments and for assembly of a functional extracellular protein secretory apparatus (5, 6).
Type IV pili are made up of polymerized pilin monomers that are
synthesized as precursors with a basic, short leader peptide. Other
proteins required for pilus biogenesis (PilE, PilV, PilW, and PilX), as
well as some components of the type II general secretory apparatus
(XcpT, XcpU, XcpV, XcpW), are synthesized with a type IV pilin-like
leader sequence and share extensive sequence similarity with the
N-terminal hydrophobic portion of mature type IV pilin subunit (7, 8).
PilD proteolytically removes the leader sequence from each of these
substrate proteins and subsequently monomethylates the
-amino group
of the newly exposed N-terminal phenylalanine (9, 10). Although removal
of the leader peptide by PilD is essential for each of its substrates
to be functional in formation of pili or for extracellular protein
secretion, it has not been established whether N-methylation
is essential for function of these related proteins as well.
Structure and function analysis of PilD, an integral membrane protein,
has shown that 4 cysteines in the cytoplasmic domain are required for
both proteolysis and methylation (11). However, cleavage and
methylation can occur independently in vitro, and methylation can be inhibited without affecting cleavage, which indicates that these two functions are separable (10, 11). The initial
structure and function study was extended here to identify further
amino acid residues of PilD that constitute each of the active sites.
We report that the type IV leader peptidase and
N-methyltransferase active sites are indeed functionally
separable by introducing single amino acid substitutions at
predetermined positions. The cleavage activity of several different
site-specific PilD mutants was unaffected as determined by kinetic
analysis. In contrast, the N-methyltransferase activity of
these same PilD mutants varied from undetectable to wild-type levels.
Specifically, the glycine at position 95 of PilD, and to a lesser
extent lysine at position 96, appeared to be essential for wild-type
N-methyltransferase activity. With the exception of one
mutant, the leader peptidase and N-methyltransferase
activities of the PilD mutants were essentially identical when examined
in vitro and in vivo; however, the methylation defect observed in vivo was dependent on PilD levels.
Interestingly, P. aeruginosa expressing any one of the
different PilD mutants showed no defect in pilus biogenesis or
extracellular protein secretion. This indicates that PilD substrates
may not need to be fully N-methylated for these proteins to
be functional.
 |
MATERIALS AND METHODS |
Bacterial Strains, Phage, Plasmids, and Growth
Conditions--
All bacterial strains, phage, and plasmids used in
this study are listed in Table I. Escherichia coli
S17-1
pir (12) was used to conjugally transfer all plasmids to
P. aeruginosa. E. coli were grown in 2× YT medium when
single-stranded DNA template was to be prepared for mutagenesis or
sequencing. All other bacterial cultures were grown with aeration at
37 °C in Luria broth (13). The following antibiotic concentrations
(in µg/ml) were used when appropriate: for P. aeruginosa,
carbenicillin (150), neomycin (200); for E. coli, ampicillin
(150), chloramphenicol (20). IPTG1 was used at a final
concentration of 1 mM unless otherwise indicated.
Oligonucleotide-directed Site-specific Mutagenesis and DNA
Sequencing--
Mutations in the Gly95 and
Lys96 codons of pilD (14) were generated by
oligo-directed site-specific mutagenesis of the phagemid pILD1 using
the method of Kunkel et al. (15). Mutations were targeted
for the first two positions of each codon such that random changes
occurred in the Gly95 codon alone, in the Lys96
codon alone, or in both the Gly95 and Lys96
codons. The mutagenic oligonucleotide used was DGK-1
(5'-GCGCTGGGCNNCNNGTGCTCGTCCTGC-3'); the underlined codons
were originally GGC (Gly95) and AAG (Lys96).
Each mutated pilD gene from pILD1 was subcloned immediately downstream of the tac promoter on plasmid pMMB66EH (Table
I), which is a broad-host range plasmid that can replicate in P. aeruginosa.
Preparation of Enzymes and Substrates--
P.
aeruginosa PAK-2B18CC (pilD::Tn5)
harboring either the cloned wild-type or mutant pilD genes
was grown for 14-16 h in the presence of IPTG to overexpress PilD.
Total membranes containing PilD were prepared as described previously
(16), resuspended to approximately 30 mg/ml in 25 mM
triethanolamine HCl, pH 7.5, 10% glycerol and stored as 50-µl
aliquots at
20 °C. The amount of PilD present was determined by
comparison with known amounts of purified PilD on Western immunoblots
or by comparison to known amounts of carbonic anhydrase resolved by
SDS-PAGE and stained with Coomassie Blue. The concentration of
membrane-associated PilD recovered was on average 0.19 mg/ml, which
constituted approximately 0.55% of the total membrane protein.
Prepilin substrate used for leader peptidase and
N-methyltransferase assays was obtained by overexpressing
the pilA gene from the tac promoter on the
plasmid pMStac27PD in P. aeruginosa PAK-2B18CC. Total membranes containing prepilin, prepared as described above, were
either used as the source of substrate for the enzyme assays or the
prepilin was purified as described previously (17). The amount of
membrane-associated pilin was determined by comparison with known
amounts of purified pilin on Western immunoblots or by comparison to
known amounts of lysozyme resolved by SDS-PAGE and stained with
Coomassie Blue. Purified prepilin was resuspended in 25 mM
triethanolamine HCl, pH 7.5, 10% glycerol to 10 mg/ml and stored at
20 °C.
Unmethylated mature pilin was generated by cleaving purified or
membrane-associated prepilin with wild-type PilD in a 2-h leader
peptidase assay (see below). PilD was inactivated by heating to
80 °C for 20 min, and then the pilin product was concentrated to
approximately 10 mg/ml by acetone precipitation. Complete in vitro processing of prepilin to mature pilin was verified by
SDS-PAGE, which was then used as a substrate in an
N-methyltransferase assay (see below).
In vivo generated pilin substrate for
N-methyltransferase assays was isolated as follows. P. aeruginosa strains grown on Luria broth agar plates at 37 °C in
the presence or absence of IPTG were resuspended in 10 mM
MgCl2, and pili were sheared from the cell surface by
vortexing for 30 s, then isolated away from whole cells by
centrifugation and concentrated to approximately 10 mg/ml in a
speed-vac.
PilD Enzyme Assays--
Enzyme activity assays were performed
using total membrane extracts since previously it was shown that PilD
in this form catalyzed prepilin cleavage at a 50-fold higher rate than
did immunoaffinity purified enzyme (17). All assays were carried out in
triplicate for each enzyme preparation and dilution.
The leader peptidase reaction was performed as follows. A mixture of
prepilin, PilD-enriched membrane extracts, dithiothreitol (5 mM final), and cardiolipin (0.05% final) were incubated at 37 °C for 2 min. The cleavage reaction was then started by adding 5× assay buffer (125 mM triethanolamine HCl, pH 7.5, 2.5%
Triton X-100) to a final volume of 10 µl, and then allowed to proceed at 37 °C for 0-45 min, depending on the experiment. The
N-methyltransferase assay was essentially identical to the
peptidase assay except that 5 mCi of [3H]AdoMet (Amersham
Pharmacia Biotech) was added as the methyl donor, and the incubation at
37 °C was increased to 1 h. Enzyme assays were stopped with 1 volume of 2× sample buffer (100 mM Tris-HCl, pH 6.8, 4%
SDS, 20% glycerol, 200 mM dithiothreitol, 0.2% bromphenol
blue).
Kinetics of Leader Peptidase Activity--
Leader peptidase
reactions were carried out over several intervals with saturating
levels of prepilin and various concentrations of PilD. Cleavage
reactions containing 7.56 nM PilD and 30-180 µM (i.e. 0.1-1 Km)
prepilin, and proceeding for 15 min, were used for kinetic studies
since these reactions were linear as a function of time and enzyme
concentration. The velocity of each reaction was calculated by
determining what fraction of prepilin was converted to mature pilin as
measured by laser densitometry. Previously measured PilD catalysis was
found to follow normal Michaelis-Menten kinetics with an apparent
substrate affinity (Km) of 650 µM and
a turnover rate (kcat) of 180 min
1
(17); however, in this study it was found that PilD has a slightly higher affinity for prepilin (3-fold) and that the
kcat for PilD was 1300 min
1, which
means this enzyme is actually 7-fold more efficient at cleaving its
substrate than originally postulated.
All analyses were on measurements of initial reaction velocities with a
minimum of six different substrate concentrations and at least three
separate reactions per concentration. Values for Km
(µM) and Vmax (mmol/min/mmol of
enzyme) were derived from substrate concentrations and velocities by
using the program Enzyme Kinetics, version 1.0c (Hypercard Stack for
MacIntosh) (18) which calculates these values in a two-step procedure
of first direct linear estimates (19) followed by a maximum likelihood estimation method (20). By using these units for
Vmax directly gives the substrate turnover rate
per min (kcat).
SDS-PAGE and Western Immunoblot Analysis--
The leader
peptidase reaction mixtures were separated by 18% SDS-PAGE (21). After
staining the gels with Coomassie Brilliant Blue R250, the percent
conversion of prepilin to mature pilin was quantitatively assessed by
laser densitometry. To examine N-methylation of pilin, the
gels were prepared for fluorography after staining by washing in water
for 30 min, treating with 0.5 M salicylate, 1.5% glycerol
for 30 min, then drying them under vacuum at 52 °C, and exposing the
gels to x-ray film (Kodak X-Omat).
For measuring PilD levels, samples were separated by 12% SDS-PAGE and
then electrophoretically transferred to nitrocellulose. Nitrocellulose
membranes were incubated with rabbit polyclonal anti-PilD antibody at a
dilution of 1:500. Binding of the primary antibody was visualized by
incubating with goat anti-rabbit IgG conjugated to alkaline phosphatase
and then developing with 5-bromo-4-chloro-3-indolyl phosphate and nitro
blue tetrazolium as described (22). PilD detected by immunoblot
analysis was quantitated by densitometry with the Foto/Analyst image
analysis system (Fotodyne).
Bacteriophage PO4 Sensitivity, Exoprotein Secretion Assays, and
Adherence Assay--
For the determination of bacteriophage PO4
sensitivity, single colony forming units of P. aeruginosa
PAK-2B18CC harboring either the cloned wild-type or mutant
pilD genes were streaked in a single line on Luria
broth-carbenicillin agar plates with (5, 10, 25, and 50 µM and 1 mM) or without IPTG. The plates were incubated at 37 °C for 10-12 h after spotting 4 µl of
bacteriophage PO4 (approximately 1 × 1010
plaque-forming units/ml) in the center of the streak. P. aeruginosa that are assembling functional pili will be lysed by
bacteriophage PO4; therefore, phage sensitivity was scored as a zone of
clearing in the area of phage inoculation.
To quantitatively examine the extracellular secretion of PLC, P. aeruginosa PAK-2B18CC harboring either the cloned wild-type or
mutant pilD genes were grown in triplicate, with or without IPTG, to an A600 of 1.0. Note that P. aeruginosa PAK-2B18CC constitutively expresses PLC; therefore, the
cultures were grown in Luria broth and not a low-phosphate medium that
is needed to induce expression of chromosomally encoded PLC.
Extracellular, periplasmic, and cell-associated fractions were isolated
and colorimetrically assayed for PLC activity with the chromogenic
substrate p-nitrophenylphosphorylcholine as described (23).
The extracellular activity of PLC and elastase was qualitatively
monitored by growing the same strains, in the presence or absence of
IPTG, on Luria broth agar plates containing 0.8% sheep red blood cells
or 1% skim milk, respectively. A zone of clearing around individual
colony-forming units indicated extracellular secretion.
Adherence of P. aeruginosa PAK-2B18CC, harboring either the
cloned wild-type or mutant pilD genes, to A549 lung
epithelial cells was performed as described previously (2). Bacteria
were added to subconfluent monolayers at a multiplicity of infection of
approximately 100. Cell-associated bacteria were released with 1%
Triton X-100, and viable counts were determined by plating on Luria
broth agar plates. Typically, 3% of the original inoculum of wild-type
P. aeruginosa PAK, or P. aeruginosa PAK-2B18CC
expressing wild-type PilD, would adhere to the monolayer, whereas
100-fold less (0.02%) P. aeruginosa PAK-2B18CC with or
without pMMB66EH would adhere.
Electron Microscopy--
P. aeruginosa PAK-2B18
(pilD::Tn5) harboring either the cloned
wild-type or mutant pilD genes were grown on
LB-carbenicillin agar plates with (10 µM or 1 mM) or without IPTG for 14-16 h at 37 °C, gently
resuspended in 10 mM MgCl2, and adsorbed to
200-mesh Formvar, carbon-coated grids for 2 min. Samples were washed
with saline and water, negatively stained with either 1% uranyl
acetate (4 min) or 2% phosphotungstic acid, pH 7.2 (30 s), and
air-dried after excess stain was drained against filter paper. Samples
were examined with a JEOL 1200 transmission electron microscope
operating at 60 kV.
 |
RESULTS |
Site-directed Mutagenesis of Conserved Amino Acids within a
Potential N-Methyltransferase Box of PilD--
The post-translational
modifications carried out by the bifunctional enzyme PilD of P. aeruginosa are necessary for type IV pilus biogenesis and for the
assembly of a functional apparatus of the general secretory pathway.
PilD is a leader peptidase and an N-methyltransferase, and
both activities appear to involve enzymatic mechanisms that are
unrelated to other known enzymes. It has been clearly established that
the leader peptidase activity of PilD is essential for pilus assembly
and exoprotein secretion; however, it has not been determined whether
N-methylation by PilD is biologically essential (9-11).
Initial structure and function analysis of the 290-amino acid PilD
polypeptide showed that four cysteine residues (Cys72,
Cys75, Cys97, and Cys100) are
required for full peptidase and methyltransferase activities (11).
Limited homology to other thiol methyltransferases was found in a
six-amino acid region of PilD, which is located within the most highly
conserved domain of the family of PilD homologs (11). Interestingly,
included in the potential methyltransferase box is Cys97 of
PilD (Leu-Gly-Gly-Lys-Cys97-Ser) (11), and similarly, a
cysteine is found in the same position of the homologous regions from
the E. coli EcoRI (INGKCP) (24) and EcoRII
(INGKCS) (25) methyltransferases. Alignment of 14 PilD homologs shows
that two of the six amino acids within this putative methyltransferase
box are highly conserved (Fig. 1). The
glycine at position 95 (Gly95) is invariant, and the lysine
at position 96 (Lys96) was found in half of the homologs,
whereas the other half had a conserved change to another basic amino
acid, arginine. One notable exception is the E. coli homolog
BfpP, which has glutamic acid in the position equivalent to
Lys96 of PilD. Regardless, Gly95 and
Lys96 are the most highly conserved amino acids in the
putative methyltransferase box of PilD; therefore,
oligonucleotide-directed site-specific mutagenesis of Gly95
and Lys96 was carried out to determine whether these two
residues are part of the PilD N-methyltransferase active
site.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 1.
Alignment of a highly conserved region of
PilD homologs that contains a potential methyltransferase box. The
conserved cysteines (dots) are required for leader peptidase
and N-methyltransferase function (11). The box
delineates the region of homology to EcoRI and
EcoRII methyltransferases. Only four PilD homologs had been
identified when this study began, and within the methyltransferase box
it was found that the glycine at position 95 of P. aeruginosa PilD (PilD-Pa) was invariant and that the
lysine at position 96 was highly conserved; the Gly95 and
Lys96 residues were targeted for mutagenesis. The
Gly95 residue of PilD-Pa turns out to be invariant
(shaded) in all of the PilD homologs identified to date. The
different PilD homologs are from the following strains:
BfpP, enteropathogenic E. coli; ComC,
Bacillus subtilis; FimP, Dichelobacter nodosus;
OutO-Ecc, Erwinia carotovora; OutO-Ech, Erwinia
chrysanthemi; PilD-Ah, Aeromonas hydrophila;
PilD-Ng, Neisseria gonorrhoeae; PilD-Pp, Pseudomonas
putida; PilD-Syn, Synechocystis sp.; PulO,
Klebsiella oxytoca; TcpJ, Vibrio cholerae; VvpD,
Vibrio vulnificus; and XpsO, Xanthomonas campestris.
Two additional PilD family members not included here, GspO of E. coli and Hemophilus influenzae, are homologous in the
membrane-anchoring C terminus but lack homology to the domain that
contains the active sites of PilD.
|
|
A degenerate oligonucleotide was used to introduce single or double
mutations into the first two nucleotides of the Gly95
and/or Lys96 codons of pilD by the method of
Kunkel et al. (15). A total of 18 unique pilD
mutants was identified by DNA sequencing, including 12 single mutants
(3 at Gly95 and 9 at Lys96) and 6 double
mutants. In order to analyze the effect of the different mutations on
PilD function, 11 of the mutant pilD genes were subcloned
from M13mpRBS-L into pMMB66EH for further analysis (Table
I). The pMMB66EH plasmid was used since
the various mutant pilD genes could be cloned downstream of
the IPTG-inducible tac promoter, which allowed for
controlled expression, and because the plasmid could replicate in
P. aeruginosa.
Subsequently, the 10 plasmids capable of expressing comparable levels
of mutant PilD protein in E. coli were mobilized into the
P. aeruginosa pilD mutant PAK-2B18CC
(pilD::Tn5) by conjugation. It was then
necessary to determine whether any of the
Gly95/Lys96 mutations in PilD affected the
stability or proper localization of the enzyme to the inner membrane of
P. aeruginosa. The envelope fraction isolated from P. aeruginosa PAK-2B18CC harboring the recombinant plasmids and grown
in the presence of IPTG was analyzed by Western blot using anti-PilD
antibody. It was found that similar amounts of mutant and wild-type
PilD were detectable in the P. aeruginosa PAK-2B18CC
membrane preparations (Fig. 2). This
showed that the mutations had no detectable effect on the stability or membrane localization of PilD.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
Western immunoblot of total membrane extracts
containing mutant PilD. P. aeruginosa 2B18CC
(pilD::Tn5), harboring wild-type or
mutant pilD genes cloned into pMMB66EH under the control of
the tac promoter, were grown in the presence of 1 mM IPTG. Western blots of total membrane fractions,
containing approximately 0.1 µg of PilD per lane, were probed with
anti-PilD. Purified PilD (0.015 µg) was used as a positive control in
lane 1 and P. aeruginosa 2B18CC harboring the
vector pMMB66EH alone was used as a negative control in lane
2.
|
|
In Vitro Analysis of the Leader Peptidase and N-Methyltransferase
Activities of the Mutant PilD--
Membrane fractions from P. aeruginosa PAK-2B18CC expressing the
Gly95/Lys96 pilD mutants were
analyzed for leader peptidase and N-methyltransferase activities, employing assays previously developed in this laboratory (9, 10). Purified PilD was previously shown to cleave one of its
substrates (prepilin) in the presence of a non-ionic detergent and
acidic phospholipids (9); however, the most efficient reactions occurred when PilD and prepilin were part of total crude membrane preparations (17). In this study, membrane-associated
Gly95/Lys96 PilD mutants with either purified
or membrane-associated prepilin as substrate were used in the leader
peptidase assay as described under "Materials and Methods."
Cleavage was assessed by the increased mobility of the mature pilin by
SDS-PAGE, when compared with prepilin. All of the
Gly95/Lys96 PilD mutants were able to process
prepilin into mature pilin as efficiently as wild-type PilD (Fig.
3); therefore, these two amino acids are
not essential for leader peptide cleavage activity.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 3.
In vitro leader peptidase assay of the
Gly95/Lys96 PilD mutants. The leader
peptidase reactions were carried out for 20 min at 37 °C with 200 ng
of prepilin and 8.0 ng of membrane-associated PilD (molar ratio of
50:1, substrate to enzyme). The reaction products were separated by
18% SDS-PAGE and stained with Coomassie Blue. Purified prepilin (200 ng, lane 1) and in vitro generated pilin (200 ng,
lane 2) were included as reference size standards for
substrate and product, respectively. The negative control for peptidase
activity was total membrane fractions from P. aeruginosa
2B18CC harboring the vector pMMB66EH alone, which lacks the PilD
protein (lane 3).
|
|
The in vitro N-methylation assay is essentially identical to
the leader peptidase assay except that the radioactive methyl donor
[3H]AdoMet was added to the reaction, with incorporation
of radioactivity into mature pilin being determined by SDS-PAGE and
fluorography. There was no detectable methylation of pilin by any
of the three Gly95 PilD mutants (PilD-G95D, -G95S, and
-G95Y) (Fig. 4). In contrast, there was a
variable degree of pilin methylation by the four Lys96 PilD
mutants as compared with wild-type PilD, and some could be detected
only after longer exposure of the fluorographs (Fig. 4, lower
panel). PilD-K96R was capable of methylating substrate as well as
wild-type PilD, and the activity of PilD-K96E was estimated to be
~5% of the wild-type levels. Pilin methylation by PilD-K96L was very
poor, and it was estimated to correspond to approximately 1% of
wild-type activity, whereas N-methyltransferase activity was
detected in strains expressing PilD-K96G. It is interesting to note
that the two mutants with the most N-methyltransferase activity were PilD-K96R and PilD-K96E, which reflect changes that occur
naturally in other PilD homologs (Fig. 1). All of the PilD double
mutants (PilD-G95D, K96E; -G95S, K96G, and -G95SK96R) were not
detectably methylating pilin, presumably due to the Gly95
mutations being dominant. Thus, a single amino acid substitution was
sufficient to abolish the in vitro N-methyltransferase
activity of PilD without detectably affecting its ability to cleave
substrate.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 4.
Fluorograph of the pilin product from an
N-methyltransferase assay with the
Gly95/Lys96 PilD mutants. The
N-methyltransferase reaction was essentially the same as the
leader peptidase reaction, except the following changes were
introduced: [3H]AdoMet was provided as the methyl donor,
the amount of membrane-associated PilD used was increased to 36 ng, and
the reaction was allowed to proceed for 1 h. Total membrane
fractions containing wild-type PilD were used as a positive control for
activity and membranes lacking PilD (from P. aeruginosa
2B18CC harboring the vector pMMB66EH alone) were used as a negative
control. The upper panel is a short exposure (15 h) of the
lower panel (8 days).
|
|
The Gly95/Lys96 PilD mutants had no detectable
decrease in leader peptidase function, but their ability to methylate
substrate was quite variable, which might have been due to a decrease
in functional stability over the 20-min to 1-h time course of the in vitro cleavage and N-methyltransferase assays,
respectively. To determine whether the heat stability of the
Gly95/Lys96 PilD mutants had been altered, the
enzymatic activity of each mutant was assessed after preincubation for
20 min at various temperatures, ranging from 37 to 80 °C. The
pretreated PilD mutants were subsequently used in a prepilin cleavage
reaction that included an additional 20-min incubation at 37 °C, and
then the percent conversion of prepilin to mature pilin was quantitated
by densitometry. It was found that wild-type PilD remained fully active
even when pre-heated to 50 °C, it retained on average 45% activity
when heated to 65 °C, and it was finally rendered inactive when
heated to 80 °C (Fig. 5). Each of the
Gly95/Lys96 PilD mutants showed a level of heat
stability similar to wild-type PilD (data not shown), although at
65 °C the cleavage activity of the mutants was reduced 75-85% as
opposed to 55% for wild type, as shown for PilDG95D (Fig. 5). In
addition, the N-methyltransferase activity of wild-type PilD
and of the Gly95/Lys96 PilD mutants was not
affected by pre-heating the enzymes at 50 °C (data not shown). The
N-methyltransferase activity of enzymes heated at 65 °C
or higher was not analyzed since cleavage is required for methylation
to occur. Therefore, the only functionally significant difference
between the Gly95/Lys96 PilD mutants and
wild-type PilD appears to be in their ability to methylate substrate
in vitro.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
Heat stability of the
Gly95/Lys96 PilD mutants. Total membranes
containing each of the PilD mutants were incubated for 20 min at
various temperatures (37, 50, 65, and 80 °C as indicated) before
being used in a leader peptidase reaction. The reactions were carried
out for 20 min at 37 °C in a final volume of 10 µl that contained
200 ng of membrane-associated prepilin and 3.5 ng of pretreated PilD.
Prepilin was included (lane 1) as a reference size standard
for substrate, and untreated wild-type PilD was included as a positive
control for fully active enzyme. All of the
Gly95/Lys96 PilD mutants showed a similar heat
stability phenotype.
|
|
Kinetic Analysis of the Leader Peptidase Activity of the Mutant
PilD--
PilD must remove the leader peptide from its substrate(s) in
order to monomethylate the
-amino group of the newly exposed N-terminal phenylalanine; consequently, leader peptide cleavage by PilD
is a rate-limiting step for the N-methyltransferase activity of PilD. A kinetic analysis of the leader peptidase activity for the
Gly95/Lys96 PilD mutants was performed to
determine whether the reduced in vitro N-methyltransferase
activity was due to a decrease in catalytic efficiency of cleavage.
To initiate these studies, the reaction rate of substrate cleavage for
each of the Gly95/Lys96 PilD mutants was
determined first. The rate of prepilin processing for each
Gly95/Lys96 PilD mutant was similar to
wild-type PilD in that they were linear as a function of enzyme
concentration and time when the substrate concentration was at
saturating levels. The results from a typical experiment are shown in
Fig. 6, and in this particular experiment the molar ratio of substrate (prepilin) to enzyme (PilD) in each reaction was 4000:1. However, the maximum reaction rate for 7 of the 10 Gly95/Lys96 PilD mutants was reduced 25-65%
relative to wild-type PilD. Three exceptions showed a similar rate of
reaction to wild-type PilD: 1) PilD-G95S (Fig. 6A), which is
interesting because this mutant was unable to methylate pilin in
vitro (Fig. 4); 2) PilD-K96R (Fig. 6B), which
represents a substitution that is found naturally in many of the PilD
homologs (Fig. 1); and 3) PilD-G95S, K96R (Fig. 6C), which
includes the previous two mutations together. The above measured
initial reaction rates for several substrate concentrations were used
in a steady state kinetic analysis to quantitatively determine whether
substrate affinity and/or maximum rate of catalysis of the
Gly95/Lys96 PilD mutants were significantly
affected.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Steady state kinetics of leader peptidase
activity for the Gly95/Lys96 PilD mutants.
The cleavage reaction was carried out at 37 °C for 30 min in 10 µl. Membrane fractions from P. aeruginosa 2B18CC
(pilD::Tn5) overexpressing either
wild-type or mutant PilDs from a plasmid were used as the source of
enzyme, and purified prepilin was used as the substrate. Each reaction
contained 51.5 µM prepilin and 16.4 nM PilD,
which is equivalent to a molar ratio of 4000:1 (substrate:enzyme).
A, rate of reaction for the Gly95 PilD mutants.
B, rate of reaction for the Lys96 PilD mutants.
C, rate of reaction for the PilD double mutants at positions
Gly95 and Lys96.
|
|
It was previously shown that PilD-catalyzed cleavage of prepilin
follows normal Michaelis-Menten kinetics with the measured reaction
velocity being dependent on substrate concentration (17). The
kcat and Km values for each
of the Gly95/Lys96 PilD mutants are listed in
Table II. Only 2 of the 10 Gly95/Lys96 PilD mutants, PilD-G95D,K96E and
PilD-G95S,K96G, showed a significant decrease in catalytic efficiency
in comparison to wild-type PilD, as evidenced by the
kcat/Km ratio. These two
double mutants showed a slightly higher affinity for substrate relative
to wild-type PilD, but their turnover rates were reduced by more than
25-fold (Table II). The inability of double mutants PilD-G95D,K96E and PilD-G95S,K96G to methylate substrate in vitro may have been
due to an 18-fold reduction in the catalytic efficiency
(kcat/Km ratio) of cleavage
in comparison to wild-type PilD; however, in this case, the inability
to methylate pilin in vitro was probably not caused by a
reduction in cleavage efficiency because three of the four
substitutions introduced singly (G95D, G95S, and K96G) were essentially
as efficient as wild-type PilD in catalyzing cleavage of their
substrates (Table II), yet these mutants could not methylate pilin
(Fig. 4).
Previous work from this laboratory has shown that PilD methylates
prepilin and mature pilin with similar kinetics, although the former
reaction requires both cleavage and methylation, whereas the latter
reaction requires only methylation. Thus, the
N-methyltransferase activity of the
Gly95/Lys96 PilD mutants was assessed by using
mature pilin as substrate to bypass the rate-limiting cleavage
reaction. It was found that the altered methylation phenotype of the
Gly95/Lys96 PilD mutants was identical whether
the substrate was prepilin (Fig. 4) or mature pilin (data not shown),
which is consistent with the results from the kinetic analysis above.
Overall, these data indicate that the
Gly95/Lys96 PilD mutant deficiency in
N-methyltransferase activity is not due to an inability or
reduced ability to cleave the prepilin substrate.
Effect of Amino Acid Substitutions in PilD on Pilus Biogenesis and
Extracellular Protein Secretion--
The
Gly95/Lys96 mutant PilD enzymes were examined
for their ability to complement the type IV pilus biogenesis and type
II protein export defects of P. aeruginosa PAK-2B18CC.
Complementation of pilus assembly was probed with the pilus-specific
bacteriophage PO4 since piliated P. aeruginosa are
sensitive to killing by this phage, whereas non-piliated P. aeruginosa, such as strain PAK-2B18CC, are resistant to killing by
PO4. P. aeruginosa PAK-2B18CC expressing each of the 10 Gly95/Lys96 pilD mutant clones at a
high level (i.e. when induced by IPTG) or low level
(i.e. in the absence of IPTG) were sensitive to phage PO4
(data not shown). Furthermore, electron microscopic examination of the
recombinant strains showed that the pilD mutant P. aeruginosa PAK-2B18CC (Fig.
7A) can form pili when
expressing pilDG95S (Fig. 7C), and they are
indistinguishable from bacteria expressing wild-type pilD
(Fig. 7B) in average number per cell and number and in
morphology. Complementation of the P. aeruginosa PAK-2B18CC
protein export defect can be detected when this normally non-hemolytic
strain becomes hemolytic on blood agar plates due to the secretion of phospholipase C (PLC). As with pilus biogenesis, each of the
Gly95/Lys96 PilD mutants could complement the
PLC secretion defect of P. aeruginosa PAK-2B18CC (data not
shown). It appears that the substitutions at Gly95 or
Lys96 did not significantly affect PilD function; however,
it was still not known whether the Gly95/Lys96
mutant PilD retained their ability to N-methylate substrate
in vivo.

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 7.
Transmission electron micrographs of P. aeruginosa 2B18CC (pilD::Tn5)
harboring wild-type or mutant pilD genes cloned into
pMMB66EH under the control of the tac promoter and grown in
the presence (shown) or absence of 10 µM IPTG. The
morphology and abundance of pili produced under either condition was
indistinguishable. A, P. aeruginosa 2B18CC (pMMB66EH).
B, P. aeruginosa 2B18CC (pRBS-L) (wild-type
pilD). C, P. aeruginosa 2B18CC (pMDG95S) (mutant
pilDG95S). All strains examined produced polar flagella, and
each of the Gly95/Lys96 PilD mutants showed a
similar pilus biogenesis phenotype. Magnification is 10,000-fold.
|
|
Enzymatic Activity of the PilD Mutants in Vivo--
The above
results showed that most of the Gly95/Lys96
PilD mutants cleaved prepilin as efficiently as wild-type in
vitro and that P. aeruginosa PAK-2B18CC expressing
either wild-type or mutant PilD could assemble functionally similar
pili. Most of the Gly95/Lys96 PilD mutants had
a moderate to severe defect in their ability to N-methylate
pilin in vitro, but it was still unknown whether the mutated
enzymes had a similar defect when functioning in vivo.
The expression of the plasmid-encoded mutant pilD genes is
under the control of the IPTG-inducible tac promoter;
however, low levels of PilD are produced even in the absence of IPTG
(Fig. 8). The level of PilD being
produced in the absence or presence of IPTG was quantitated by Western
blot analysis combined with densitometry. As compared with the levels
of PilD produced by wild-type P. aeruginosa PAK, it was
determined that in P. aeruginosa PAK-2B18CC (a mutant with
an insertional disruption of the pilD gene), approximately
20-fold less (Fig. 8) and 12-fold more (data not shown) plasmid-encoded
PilD was produced in the absence and presence of 1 mM IPTG,
respectively. The leader peptidase activity of the wild-type and mutant
PilDs was unaltered when present at 20-fold below chromosomal levels
(
IPTG) since prepilin and preXcpT were fully processed (Fig.
9). Furthermore, P. aeruginosa
PAK-2B18CC, expressing mutant PilD in the absence of induction by IPTG
were sensitive to killing by bacteriophage PO4, and their export of PLC
and elastase was similar to wild-type as was their adherence to A549
lung epithelial cells (data not shown).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 8.
Western immunoblot of differentially
expressed PilD mutants. P. aeruginosa 2B18CC
(pilD::Tn5) harboring either
wild-type or mutant pilD genes, cloned into pMMB66EH under
the control of the tac promoter, were grown in the presence
of different amounts of IPTG as indicated. Western blots of whole cell
extracts were probed with anti-PilD antibody. In the absence of IPTG,
these strains expressed PilD at 20-fold below wild-type P. aeruginosa PAK chromosomal levels of PilD (although it is
difficult to see in the photograph, PilD was visible on the original
immunoblot). All of the Gly95/Lys96 PilD
mutants showed a similar pattern of expression.
|
|

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 9.
Western immunoblot of pilin and XcpT to
examine whether the PilD mutants were capable of cleaving substrate
in vivo when expressed below wild-type P. aeruginosa PAK chromosomal levels. P. aeruginosa
2B18CC (pilD::Tn5) harboring
either wild-type or mutant pilD genes, cloned into pMMB66EH
under the control of the tac promoter, were grown in the
presence (+) or absence ( ) of 1 mM IPTG. The same whole
cells used in Fig. 10 were probed with anti-pilin (upper
panel) and anti-XcpT (lower panel). All of the
Gly95/Lys96 PilD mutants showed a similar
in vivo cleavage phenotype.
|
|
An assay for the assessment of methylation in P. aeruginosa
(i.e. in vivo) was developed based on an earlier observation
of Strom et al. (10)) who showed that in vitro
PilD could not further methylate a previously methylated substrate.
This work also established that the N-methyl modification of
PilD substrates was very stable. In our assay, pili sheared from the
surface of P. aeruginosa were harvested, and then the
methylation state of the in vivo modified pilin subunits
was probed with wild-type PilD in an in vitro
N-methyltransferase assay. In this assay, wild-type PilD would not
be capable of methylating pilin subunits in vitro if they
were already methylated, but wild-type PilD would further methylate
in vivo generated pilin that was unmethylated or only
partially methylated.
By using this in vivo N-methyltransferase assay it was found
that the reduced level of wild-type PilD, 20-fold less than normal level, in the absence of IPTG induction results in 80-95% of
substrate being methylated (Fig. 10),
which shows that the in vivo N-methylation of pilin, as was
observed in vitro, was kinetically slower than cleavage of
the leader peptide (compare Figs. 9 and 10). In contrast, reduced
amounts of some Gly95/Lys96 PilD mutants, such
as PilD-G95S, resulted in pilin that was not detectably methylated
(Fig. 10). However, the methylation defect of the
Gly95/Lys96 PilD mutants could be mitigated
when these enzymes were overproduced by the addition of IPTG (Fig. 10).
Plasmid pMDK96R constitutively expresses PilD-K96R; therefore, P. aeruginosa PAK-2B18CC(pMDK96R) overproduces PilD-K96R in the
presence or absence of IPTG, which results in fully methylated pilin
under either condition (Fig. 10). The only
Gly95/Lys96 PilD mutant that showed a different
N-methyltransferase phenotype in vitro versus in
vivo was PilD-K96G, where in vitro it showed no
detectable N-methyltransferase activity and in
vivo it methylated pilin as efficiently as wild-type PilD (Fig.
10). Although several of the Gly95/Lys96 PilD
mutants had a defect in their N-methyltransferase activity in vitro, this defect did not appear to be as prominent
in vivo; however, the level of pilin methylation in
vivo did correlate with the level of mutant enzyme being
produced.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 10.
Fluorograph of pilin processed by mutant
PilD in vivo and used as a substrate for wild-type PilD in
an in vitro N-methyltransferase assay. P. aeruginosa 2B18CC
(pilD::Tn5) harboring either
wild-type or mutant pilD genes, cloned into pMMB66EH under
the control of the tac promoter, were grown in the presence
(+) or absence ( ) of 1 mM IPTG. Pili were sheared from
the bacterial cell surface by vigorous vortexing, concentrated, and
then quantitated. 200-300 ng of sheared pili were used as a substrate
(equivalent to 13-19 µM in the final reaction mix) in a
methylation reaction with 1.1 µM wild-type PilD. As a
positive control, an equivalent amount of either prepilin or in
vitro generated (i.e. unmethylated) mature pilin was
used as substrate. As a negative control, pili isolated from wild-type
P. aeruginosa PAK were used as substrate. Each of the
Gly95/Lys96 PilD mutants showed an
N-methyltransferase phenotype that paralleled their
corresponding in vitro phenotype (see Fig. 5).
|
|
In Vivo N-Methyltransferase Activity of the PilD Mutants Expressed
at Chromosomal Levels--
The induced level of plasmid-encoded PilD
was modulated by varying the IPTG concentration in growing cultures to
determine whether "native" levels of mutant PilD were sufficient to
fully N-methylate pilin in vivo. It was found
that 50 µM IPTG was enough to maximally induce
plasmid-encoded PilD expression, which was approximately 7-12-fold
above wild-type P. aeruginosa PAK chromosomal PilD levels
(Fig. 8). In addition, it was found that 10 and 25 µM
IPTG induced expression comparable to and 2-fold above chromosomally produced PilD levels, respectively (Fig. 8).
A range of IPTG concentrations (0, 10, 25, and 50 µM) was
used to induce mutant PilD expression in P. aeruginosa
PAK-2B18CC, and then assembled pili were sheared from the bacterial
cell surface, and their methylation state was probed with wild-type
PilD in an in vitro N-methyltransferase assay. The same
cultures used to harvest pili were also probed for PilD expression by
Western blot analysis using anti-PilD antibody to verify enzyme levels being produced under the growth conditions examined (Fig. 8). When
plasmid-encoded wild-type PilD was induced with as little as 5 µM, which yielded enzyme at 3-fold below chromosomal
levels, pilin subunits were always 100% methylated (i.e.
these pilin subunits would not be further methylated by wild-type PilD
in an in vitro N-methyltransferase assay). The
PilD-Lys96 mutants methylated 65-85% of substrate when
expressed 20-fold below chromosomal levels as compared with 80-95% by
wild-type PilD at similar levels (i.e. in the absence of
IPTG induction). This difference represents only a slight defect in the
N-methyltransferase activity of the mutant enzyme (Fig.
11). In order for the pilin subunits to
be fully methylated by the PilD-K96 mutants, enzyme expression needed
to be induced with 10 µM IPTG or greater (Fig. 11). In
contrast, pilin subunits that had been processed by
PilD-Gly95 mutants or
PilD-Gly95,Lys96 double mutants in
vivo remained unmethylated when the mutant enzyme was present
below chromosomal levels (Figs. 10 and 11), but chromosomal levels of
mutant enzyme methylated 50-60% of the pilin subunits, and mutant
enzyme present at 2-fold above chromosomal levels methylated 75-80%
of the pilin subunits (Fig. 11). In general then, a
Gly95/Lys96 PilD mutant that shows a
methylation defect in vitro also shows a similar defect
in vivo; therefore, there is a strong correlation between
the in vitro and in vivo N-methyltransferase
activities of these PilD mutants.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 11.
Fluorograph of pilin processed by
chromosomal levels of mutant PilD in vivo and used as a
substrate for wild-type PilD in an in vitro
N-methyltransferase assay. P. aeruginosa 2B18CC
(pilD::Tn5) harboring either
wild-type or mutant pilD genes, cloned into pMMB66EH under
the control of the tac promoter, were grown in the presence
of different amounts of IPTG as indicated. The same bacterial cells
examined for PilD levels in Fig. 8 were used here. Pili were sheared
from the bacterial cell surface by vigorous vortexing, concentrated,
and then quantitated. 200-300 ng of sheared pili were used as
substrate (equivalent to 13-19 µM in the final reaction
mix) with 1.13 µM final wild-type PilD in the
N-methyltransferase assay. Each of the
Gly95/Lys96 PilD mutants showed an
N-methyltransferase phenotype that paralleled their
corresponding in vitro phenotype.
|
|
 |
DISCUSSION |
P. aeruginosa and many other bacteria simultaneously
carry out two specialized localization processes, secretion of
extracellular enzymes and assembly of type IV pili. It has been
recognized in recent years that the basic mechanisms of extracellular
protein secretion via the general secretory pathway and assembly of
pilin subunits are related based on similarities of individual
components that comprise the machinery of protein secretion and pilus
assembly. Moreover, in P. aeruginosa, the two export
pathways require the activity of one common enzyme, the product of the
pilD gene, which specifies a bifunctional leader
peptidase/N-methyltransferase (10).
Comparison of in vitro cleavage and methylation reactions,
catalyzed by PilD, has shown that the rate of cleavage of prepilin substrates is significantly faster than N-methylation. Under
optimal conditions, complete cleavage was observed after 10 min, while complete N-methylation required 60 min (10). We have
extended the previous work to show that the difference in the two
reactions rates can also be observed in vivo by limiting
pilD expression, and by examining the extent of methylation
of pilin subunits that were assembled into pili. PilD produced at
20-fold below normal chromosomal levels in P. aeruginosa
could fully cleave prepilin, but as much as 20% of the pilin subunits
isolated from assembled pili were not N-methylated. Under
these conditions, PilD cleaved many more prepilin subunits than it was
capable of methylating, yet the number, morphology, and function of the
type IV pili produced by P. aeruginosa was unaffected. This
result indicates that not every subunit has to be methylated for
assembly of functional pili. In addition, this result showed that the
in vitro leader peptidase and N-methyltransferase
assays accurately reflect the relative reaction rates measured in
vivo.
We had also previously shown that the two post-translational
modifications carried out by PilD can occur independently in vitro and that both active sites for prepilin processing and for N-methylation are adjacent within a relatively large
(approximately 68 amino acid) cytoplasmic loop domain (10, 11). This
work also suggested that the two active sites are non-overlapping. The
use of sulfhydryl-reactive alkylating agents and site-directed mutagenesis also showed that both enzymatic activities of PilD were
dependent on four highly conserved cysteines (Cys72,
Cys75, Cys97, and Cys100) located
within the cytoplasmic loop (11). However, it was not clear if the
cysteines were directly involved in leader peptidase activity or if
they were required for the formation or the maintenance of the active
site conformation because there was only a slight stimulation of
cleavage by the addition of thiols to the reaction mixture (10).
The involvement of cysteines in the methylation reaction catalyzed by
PilD led to a comparison with other thiol methyltransferases and a
region of limited homology, restricted to a 6-amino acid region of PilD
that included one cysteine (Leu-Gly-Gly-Lys-Cys97-Ser), was
identified (11). Comparison of this region with other members of the
PilD family of homologs showed that Gly95 and
Lys96 were the most highly conserved amino acids of this
potential methyltransferase box, which were targeted for site-directed
mutagenesis in this study. In order to assess the role of
N-methylation in the assembly and/or function of the type II
secretion apparatus and type IV pili, a PilD mutant that lacks
N-methyltransferase activity and is unaffected for leader
peptidase activity is required. This is the exact functional phenotype
the Gly95/Lys96 PilD mutants showed, which for
the most part was reproducible under both in vitro and
in vivo conditions.
By using an in vitro functional analysis of 10 different
single and double Gly95/Lys96 PilD mutants, we
showed that the leader peptidase and N-methyltransferase functions were separable with a single amino acid change. Any substitution for Gly95, which is invariant in the 14 known
PilD homologs, abolished the N-methyltransferase activity of
PilD in vitro. In contrast, the Lys96 mutants
showed a variable degree of N-methyltransferase activity depending on the amino acid change. Two substitutions for
Lys96, arginine and glutamic acid, resulted in mutant PilD
that retained significant N-methyltransferase activity
in vitro, with the former being a conservative change that
is found in approximately half of the PilD homologs, whereas the latter
changes the charge and is found in only one PilD homolog, BfpP of
enteropathogenic E. coli (26). A substitution to glycine was
the only change in Lys96 that completely abolished
N-methyltransferase activity in vitro. Amino acid
Gly95 appears to be essential for the
N-methyltransferase activity of PilD, but, in contrast,
Lys96 can be substituted for other amino acids with mutant
PilD showing a range of N-methyltransferase activities.
Additionally, it appears that PilD retains more of its
N-methyltransferase activity when Lys96 is
substituted with a charged amino acid. These data clearly establish
that the leader peptidase and N-methyltransferase active sites are non-overlapping within the large cytoplasmic loop domain of
PilD.
PilD must function as a leader peptidase before it can
N-methylate its substrate; therefore, each PilD mutant was
examined for any alterations in leader peptidase activity as compared
with wild-type PilD. In contrast to the PilD mutants in the cysteine residues (11), the kinetic parameters of the leader peptidase activity
for most of the Gly95/Lys96 PilD mutants were
unaltered. By using membrane-associated PilD and prepilin, measured
catalysis was previously shown to follow normal Michaelis-Menten
kinetics with an apparent substrate affinity (Km) of
650 µM and a turnover rate (kcat)
of 180 min
1 (17). Here we have found that the
Km and kcat for wild-type
PilD to be 210 µM and 1300 min
1,
respectively. Hence, PilD has a slightly higher affinity for substrate
and is actually 7-fold more efficient at cleaving substrate than
originally postulated. All of the PilD mutants that had a single
mutation in either Gly95 or Lys96 were as
efficient at cleaving prepilin as wild-type PilD; however, two of the
three double Gly95/Lys96 PilD mutants showed a
significant reduction in leader peptidase activity. The inability of
the double mutants to methylate pilin in vitro was not
likely due to a reduction in cleavage efficiency since the four
substitutions introduced singly (G95D, G95S, K96E, and K96G)
essentially catalyzed cleavage as efficiently as wild-type PilD, yet
three of these mutants singly could not methylate pilin. When the
cleavage reaction is bypassed by providing mature pilin as substrate,
which is N-methylated as efficiently as prepilin by
wild-type PilD, each of the 10 PilD mutants showed a similar defective
N-methyltransferase phenotype. In vivo, each of
the Gly95/Lys96 PilD mutants was found to
cleave prepilin and preXcpT as efficiently as wild-type PilD, even when
mutant PilD expression was 20-fold below normal chromosomal levels.
In addition to PilD, two other bacterial leader peptidases, catalyzing
the cleavage of signal peptides from secreted proteins, have been
biochemically characterized. The typical signal sequence of proteins
entering the sec-dependent secretion system are cleaved by
either leader peptidase I or II (LPaseI or LPaseII). LPaseI acts on a
wide variety of substrates with a reasonable affinity (Km = 16.5 µM) and has a modest
turnover rate (kcat = 520 min
1)
(27). LPaseII specifically recognizes and cleaves lipoproteins; this
enzyme has a high affinity (Km = 6 µM)
for its substrates in addition to an efficient turnover rate
(kcat = 1800 min
1) (28). In
comparison, PilD has a lower affinity for its substrates than the
LPases that may reflect the variety of related but not identical
substrates that have to be processed by this enzyme, yet it efficiently
cleaves one class of highly abundant proteins (type IV pilin
precursors), with each pilus filament containing between 500 and 1000 subunits.
We also compared the functional phenotypes of the
Gly95/Lys96 PilD mutants in vitro.
Because assembled, fully methylated pilins cannot function as
substrates for further methylation (10) the extent of in
vivo methylation can be accurately assessed by examining the
ability of pilin subunits of pili, isolated from various PilD mutants,
to accept methyl groups in vitro. By using this knowledge, we developed an assay where wild-type PilD was used in an in
vitro N-methyltransferase assay to probe the methylation state of
pilin that had been processed and modified in vivo by
wild-type or mutant PilD. If pilin is N-methylated in
vivo, then PilD would not further methylate this substrate in the
in vitro assay. Conversely, if pilin is only partially
methylated, or not methylated at all, then PilD will
N-methylate the population of pilin subunits that was not
N-methylated in vivo, with the corresponding
signal produced in vitro being inversely proportional to the
amount of substrate that was N-methylated in
vivo.
By using this in vivo N-methyltransferase assay we found a
strong correlation between in vitro and in vivo
activities for the mutant PilD, but the in vivo activity
depends on the amount of mutant enzyme being produced. At the reduced
levels of wild-type PilD (approximately 5% of the levels made by
wild-type bacteria expressing a chromosomal pilD gene)
80-95% of the pilin subunits isolated from assembled pilus filaments
were methylated. At the same expression level, the Gly95
PilD mutants were not detectably N-methylating pilin in
P. aeruginosa, whereas the Lys96 PilD mutants
were able to methylate approximately 15-35% of the processed pilin
subunits. However, in contrast to the in vitro activities,
the Gly95 PilD mutants were able to fully
N-methylate pilin in vivo when expressed at
approximately 5-fold above chromosomal PilD levels, but when expressed
at chromosomal levels, only 50-60% of the pilus subunits were
N-methylated. Similarly, the Lys96 PilD mutants
could overcome their defect in N-methylating substrate when
expressed above chromosomal levels, but these mutants showed a
consistently moderate defect in N-methyltransferase activity in vivo as opposed to variable levels of activity in
vitro. These data indicate that the Gly95 PilD mutants
possess some residual N-methyltransferase activity that is
detectable in vivo, but very little, if any, methylation is
required for the assembly and function of type IV pili or the type II
secretion apparatus.
The currently available data suggest a model for a series of
interactions between PilD and its substrates, which takes place on the
cytoplasmic face of the inner (cytoplasmic) membrane. The positively
charged leader peptide and N-terminal domain of prepilin are necessary
and sufficient to promote translocation across the cytoplasmic
membrane, as shown previously. The basic leader in front of the
N-terminal hydrophobic domain may very likely function to orient and
anchor the prepilin in the membrane. Because of the predicted
orientation, the cleavage of the substrate takes place in the
cytoplasmic side of the membrane, where methylation of mature pilin
takes place as well. The site of the two modification reactions is
consistent with the topological location of the PilD domain containing
the putative active site residues (11) and the presence of the methyl
donor AdoMet. Although PilD is a bifunctional enzyme, the comparison of
kinetics of processing and methylation (10) suggests that the two
reactions are not necessarily coupled, and it is conceivable that
methylation is not carried out by the same PilD molecule that was
responsible for leader peptide cleavage.
P. aeruginosa produces several extracellular and
surface-associated proteins, and the ability to export these virulence
determinants is primarily dependent on PilD. Clearly the maturation of
prepilin and prepilin-like proteins is essential for extracellular
secretion, but it is not clear what consequences the two modification
steps have on the subsequent fate and function of the processed
proteins. Processing of signal peptide anchors by LPase I is
responsible for the release of secreted proteins from the cytoplasmic
membrane (29); however, it is unlikely that processed pilin monomers similarly dissociate from the membrane. Following cleavage of the
short, basic leader peptide, the hydrophobic N-terminal domain of pilin
would very likely prevent the release of the mature subunits into the
periplasm. It is, however, conceivable that the leader peptide cleavage
and methylation of pilin subunits may lead to conformational changes
necessary for polymerization or interactions with the assembly
machinery located in the cell envelope. The leader peptide may
therefore serve to prevent premature polymerization of the subunits
until they have been processed into some form of an assembly complex.
It is also conceivable that the sole function of
N-methylation is to protect subunits from degradation by
proteases during a particular stage of pilus filament biogenesis or
type II protein secretion machinery assembly. Such degradative enzymes may only be present in P. aeruginosa when the bacteria are
propagated under specific conditions reflective of their natural
habitat, including soil, water, or the tissues of infected patients.
Protection of pilin subunits by N-methylation may not be
necessary under conditions when bacteria are propagated in artificial
laboratory media, accounting for the lack of phenotype of strains
expressing PilD mutants with decreased or absent
N-methyltransferase activity.
We thank Leah Turner, Dave Simpson, Mark
Strom, Cyndy Pepe, Wendy Loomis, and Tim Motley for helpful
discussions, and Stephanie Lara and J. Cano Lara for help with the
electron microscopy.