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Journal of Bacteriology, February 1999, p. 973-980, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effect of wzx (rfbX)
Mutations on A-Band and B-Band Lipopolysaccharide Biosynthesis
in Pseudomonas aeruginosa O5
Lori L.
Burrows and
Joseph S.
Lam*
Department of Microbiology, University of
Guelph, Guelph, Ontario, Canada N1G 2W1
Received 15 July 1998/Accepted 17 November 1998
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ABSTRACT |
The wbp cluster of Pseudomonas aeruginosa
O5 encodes a number of proteins involved in biosynthesis of the
heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the
O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly wbpF), contiguous with wzy in
the wbp cluster, is predicted to encode a highly
hydrophobic protein with multiple membrane-spanning domains. This
secondary structure is consistent with that of Wzx (RfbX), the putative
O-antigen unit translocase or "flippase." Insertion of a
Gmr cassette at two separate sites within the putative
wzx gene led in both cases to the loss of B-band
lipopolysaccharide (LPS) O-antigen production. To our knowledge, this
is the first report of the successful generation of chromosomal
wzx gene replacement mutations. Surprisingly, inactivation
of wzx also led to a marked delay in production of the
ATP-binding cassette-transporter-dependent, D-rhamnose
homopolymer, A-band LPS. This effect on A-band LPS synthesis was
alleviated by supplying multiple copies of WbpL in trans.
WbpL, a WecA (Rfe) homologue, was shown recently to be essential for
the initiation of both A-band and B-band LPS synthesis in P. aeruginosa O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103-1119, 1998). These results suggest that the delay in A-band LPS production may arise
from insufficient access to WbpL when the completed B-band O unit is
not successfully translocated to the periplasm. Without adequate WbpL,
A-band LPS synthesis is delayed. A subset of wzx mutants
appeared to have accumulated second-site mutations which either
restored the normal expression of A-band LPS or abolished A-band
expression completely. Complementation studies showed that all of the
additional mutations affecting LPS synthesis that were characterized in
this study were located within the B-band LPS genes.
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INTRODUCTION |
Current models explaining the
biosynthesis and assembly of lipopolysaccharide (LPS) O antigens invoke
two separate pathways, termed the Wzy (Rfc)-dependent and the
Wzy-inde-pendent-ATP-binding cassette (ABC)-transporter-depen-dent pathways
(reviewed in reference 36). Wzy is the O-antigen
polymerase and is involved in the biosynthesis of heteropolymeric O
antigens. Two other proteins required in the Wzy-dependent pathway are
Wzx (RfbX), the putative O-antigen unit translocase or "flippase,"
and Wzz (Rol, Cld), the regulator of O-antigen chain length
(36). Individual O-antigen sugar units are synthesized on
the isoprenoid lipid carrier undecaprenol phosphate (C55P)
at the cytoplasmic face of the inner membrane. Following synthesis,
individual O-antigen units are thought to be translocated by an
integral membrane protein, Wzx, to the periplasmic face of the
cytoplasmic membrane, where they are polymerized by Wzy (25,
36). The chain length of the growing heteropolymer is controlled
by the Wzz protein (3, 4, 7, 28) via an unknown mechanism.
The heteropolymer is then covalently attached to the previously
synthesized core lipid A by the O-antigen ligase, encoded by
waaL (rfaL) in Escherichia coli and
Salmonella enterica serovar Typhimurium.
In contrast, homopolymeric O antigens appear to be synthesized
processively on C55P without an O-antigen polymerase
(36). Most homopolymeric O antigens are transported via a
two-component, ABC-type transporter, which moves the assembled
homopolymer across the cytoplasmic membrane (19, 29, 36).
Once the homopolymer has been translocated to the periplasmic face of
the cytoplasmic membrane, it can be ligated to core lipid A by the
O-antigen ligase. Recently, an alternative transport mechanism for the
O:54 homopolymer of Salmonella enterica serovar Borreze was
proposed, in which the O antigen is assembled and transported across
the cytoplasmic membrane in the absence of an ABC transporter
(18).
Pseudomonas aeruginosa simultaneously produces two forms of
LPS, called A-band and B-band LPS. A-band LPS contains a neutral homopolymer of 
-D-rhamnose (23). The A-band
biosynthetic cluster has been cloned and sequenced and has been shown
to contain genes encoding a typical two-component transporter system
(29). B-band LPS is the O-antigen-containing form and is a
heteropolymer of di- to pentasaccharides, containing uronic acids and
very rare sugars, such as pseudaminic acid (20). The
wbp cluster encoding the biosynthesis of the B-band O
antigen of serotype O5 has been cloned and sequenced (6, 7,
24). It resembles other gene clusters encoding the synthesis of
heteropolymeric O antigens in that it contains wzx,
wzy, and wzz homologues. The wzy and wzz genes from serotype O5 have previously been
characterized in our lab (7, 9). The start codon of the
putative wzx gene (formerly wbpF) overlaps the
stop codon of wzy (6). Analysis of the deduced
amino acid sequence of Wzx showed that it is a hydrophobic protein with
multiple membrane-spanning domains in its predicted secondary
structure, similar to that of other Wzx proteins. Homologues of
wzx have been identified in many bacterial species in both
LPS O-antigen and capsular biosynthetic clusters (37). While
the primary sequence homologies of Wzx from different bacteria can be
poor, the proteins share similar secondary structures. Wzx has not been
extensively studied, probably due to obstacles encountered during the
cloning of the gene in isolation and creation of null mutants (25,
26). Chromosomal mutations in wzx have been described
as deleterious and difficult to study (31). However, Liu and
coworkers (25) were able to use a plasmid-encoded
O-antigen cluster carrying a nonpolar transposon insertion in
wzx to demonstrate that wzx mutants appear to
accumulate O-antigen units on the cytoplasmic face of the inner membrane.
In this study, we examined the function of Wzx in P. aeruginosa O5 through the creation of chromosomal wzx
knockout mutants. As far as we are aware, this is the first report of
the successful generation of such mutants. Analysis of these knockout
strains confirmed the involvement of Wzx in B-band LPS biosynthesis and helped define the interrelationship between the Wzy-dependent and the
Wzy-independent pathways of O-antigen biosynthesis in P. aeruginosa O5.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are listed in Table
1. P. aeruginosa strains
were grown either on Pseudomonas isolation agar (Difco),
Luria broth and agar (Sigma), or Davis minimal medium (Fisher).
E. coli strains were grown on Luria broth and agar (Sigma).
Where necessary, antibiotics (all from Sigma) were added as described
previously (9).
DNA manipulations.
Chromosomal DNA was isolated from
P. aeruginosa using the method of Goldberg and Ohman
(13). Plasmid DNA was isolated by using the Qiagen midi-prep
or mini-plasmid kits (Qiagen Inc.) as directed by the manufacturer.
Restriction and modification enzymes were used as directed by the manufacturers.
Plasmids were introduced into
E. coli by CaCl
2
transformation (
17) and into
P. aeruginosa
by electroporation using a Bio-Rad
(Richmond, Calif.) Gene Pulser
apparatus following the manufacturer's
protocols. Electrocompetent
cells of
P. aeruginosa were prepared
by growing the
cells to mid-log phase in Luria broth and then
washing the cells twice
for 5 min each in sterile 10% room temperature
glycerol followed by
immediate resuspension in the same solution.
Cells were either used
immediately or frozen at
80°C for future
use. Plasmids were also
introduced into
P. aeruginosa by biparental
mating with
E. coli SM10 carrying mobilizable plasmids of interest
(
34).
Creation of isogenic chromosomal knockout mutants.
The gene
replacement strategy of Schweizer and Hoang (33) was used
for the creation of knockout mutations in wzx as described previously (8, 9). In the event that isolates were obtained in which only single crossover events had occurred, these merodiploids were plated overnight at 37°C on modified Luria medium containing 5%
sucrose and no NaCl (38). This treatment selects for cells which have lost the sacB-containing vector DNA following a
double crossover event that generates true recombinants. Correct gene replacement was ascertained through Southern blot analysis of chromosomal DNA isolated from gentamicin-resistant, sucrose-sensitive, carbenicillin-sensitive mutants.
A similar strategy was used to create a double knockout mutant. An
A
L (for A late)
wzx strain, S2, was used as the
background for the
introduction via electroporation of a copy of
wbpM (
6) inactivated
by a nonpolar carbenicillin
resistance cassette, and cloned into
the
sacB-containing suicide vector pEX18Tc (pFV169-18TcCb)
(
16).
Correct gene replacement was ascertained by Southern
blot analysis
of chromosomal DNA from gentamicin-resistant,
carbenicillin-resistant,
sucrose-sensitive, tetracycline-sensitive
mutants.
Southern blot analysis.
Restriction-enzyme-digested
chromosomal DNA was separated on 0.8% agarose gels, transferred to
Zetaprobe nylon membrane (Bio-Rad) by capillary transfer, and
crosslinked to the membrane using a Stratalinker (Stratagene, La
Jolla, Calif.). For detection of specific fragments, probe DNA was
labelled with digoxigenin-dUTP (Boehringer Mannheim, Laval, Quebec,
Canada), and hybridization and detection were performed according to
the manufacturer's directions.
SDS-PAGE and Western immunoblot analysis.
LPS from
P. aeruginosa was prepared by the method of Hitchcock
and Brown (15). The LPS preparations were separated on
standard discontinuous sodium dodecyl sulfate (SDS)-12%
polyacrylamide gels and visualized by silver staining using the method
of Dubray and Bezard (11). For immunoblotting, LPS separated
by SDS-polyacrylamide gel electrophoresis (PAGE) was transferred to
nitrocellulose (5). Nitrocellulose blots were blocked with
3% skim milk followed by overnight incubation with hybridoma culture
supernatants containing monoclonal antibody (MAb) MF15-4 (specific for
O5 B-band LPS) (21) or MAb N1F10 (specific for A-band LPS)
(22). A goat anti-mouse F(ab')2-alkaline
phosphatase second antibody conjugate (Jackson Immunoresearch, West
Grove, Pa.) was used to detect the first antibody. The blots were
developed using a substrate containing 0.3 mg of Nitro Blue
Tetrazolium/ml and 0.15 mg of BCIP (5-bromo-4-chloro-3-indolylphosphate toluidine)/ml in 0.1 M bicarbonate buffer (pH 9.8).
Time course experiments.
Growth curves of strains PAO1, S2,
and X10 showed that there were no significant differences in the rates
of growth of the parent and mutant strains (not shown). To demonstrate
the appearance of A-band LPS over time, the mutant and parent strains
were grown in 50 ml of Luria broth at 37°C with 200-rpm shaking.
Aliquots of 0.5 ml were removed beginning at 12 h after
inoculation (early stationary phase) and then at 6- to 12-h intervals
up to 60 h postinoculation, and the optical densities at 600 nm of
the samples were determined. The cells were harvested and used to
prepare LPS via the method of Hitchcock and Brown (15). The
LPS was analyzed by SDS-PAGE and Western immunoblotting as outlined above.
Nucleotide sequence accession number.
The corrected DNA
sequence of the wzx gene is available from GenBank under
accession no. U50396.
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RESULTS AND DISCUSSION |
Characterization of Wzx.
In a previous study (6),
we reported Wzx (formerly WbpF) to be 316 amino acids (aa) long, a
length inconsistent with those of other Wzx proteins, which range from
approximately 400 to 500 aa. Reanalysis of the DNA sequence in the
region containing wzx showed the actual size of the
wzx gene to be 1,236 bp. This open reading frame is
predicted to encode a protein of 411 aa, in agreement with the sizes of
other Wzx proteins.
Repeated attempts to express Wzx by both in vivo and in vitro methods
were not successful, although the HisH and HisF proteins
encoded
immediately downstream of
wzx (Fig.
1) and on the same
recombinant plasmid
(pFV162-26) were readily expressed (
6).
Our observations are
consistent with previous reports that the
high hydrophobicity and
presence of rare or modifying codons within
the coding regions for Wzx
and Wzy proteins make them extremely
difficult to express using
currently available methods (
9,
26,
27).
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FIG. 1.
Physical map of locations of plasmids used in this study
with respect to the B-band LPS gene cluster. The individual open
reading frames within the wbp cluster of serotype O5 are
shown as arrows at the top of the figure. The wzx gene
described in this study is shown in black, while the wbpL
and wbpM genes are shown in grey. The positions of the two
individual Gmr cassette insertions within the
wzx gene are shown as black triangles. Mutants with an
insertion at SstI are designated
wzxs, while mutants with an insertion at
XhoI are designated wzxx. B,
BamHI; B2, BglII; H,
HindIII; N, NruI; S, SalI; Ss,
SstI; X, XhoI; Xb, XbaI. For clarity,
only selected restriction sites are shown.
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Analysis of chromosomal wzx mutants.
In order to
demonstrate the function of Wzx in P. aeruginosa
O-antigen biosynthesis, a nonpolar Gmr cassette was used to
insertionally inactivate the wzx gene of serotype O5. The
first set of wzx mutants constructed had a gentamicin cassette inserted at a unique SstI site located 462 bp from
the 3' end of the gene (wzxs; Fig. 1). However,
there were concerns that insertion of the Gmr cassette near
the 3' end of the gene may have led to the generation of a truncated
but potentially functional peptide. This consideration prompted the
construction of a second set of wzx mutants. The second
group, wzxx, had the nonpolar Gmr
cassette inserted in a XhoI site 200 bp from the 5' end of
wzx (Fig. 1). In our hands, the yield of both types of
wzx mutants compared to that obtained for other
P. aeruginosa LPS genes using the same methodology was
very low (6, 7, 9, 29, 30). The poor yield is consistent
with the reported difficulties encountered by others during attempts to
isolate such mutants (25, 26). Correct insertion of the
gentamicin resistance cassette within wzx in both sets of
mutants was confirmed by Southern immunoblot analysis (Fig.
2).
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FIG. 2.
Southern blot analysis of selected wzx
mutants. Chromosomal DNA from representative AL (S2 and
X10) and A+ (S7 and X24) wzx mutants from the
wzxs and wzxx series and
a representative A (X14) wzxx
mutant was digested with HindIII and separated on a
0.8% agarose gel, transferred to a nylon membrane, and probed with a
dUTP-digoxigenin-labelled BamHI-BglII fragment
corresponding to the insert of pFV162-26. All mutants show an increase
in the size of the HindIII fragment of approximately 0.9 kb, corresponding to the size of the Gmr cassette. No gross
rearrangements that could be responsible for the varied A-band LPS
phenotypes of these mutants were found in this region.
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On silver-stained SDS-polyacrylamide gels, the gentamicin-resistant
wzxs mutants were devoid of B-band LPS (Fig.
3). Interestingly,
of 21
wzxs mutants generated, 20 also lacked the
ladder-like banding
pattern typical of A-band LPS on silver-stained
SDS-polyacrylamide
gels. This result was confirmed using MAb N1F10,
which is specific
for A-band LPS (Fig.
3). This is the first instance
in which mutation
of a
wzx gene has been shown to affect
synthesis of a Wzy-independent,
homopolymeric polysaccharide.
Similar results were obtained for
the
wzxx set
of mutants (not shown).
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FIG. 3.
Analysis of LPS from a representative AL
wzxs mutant, S2. LPS isolated from 12-h cultures
of the AL wzxs mutant S2 and from
the O5 parent strain was analyzed with silver-stained
SDS-polyacrylamide gels as well as by Western immunoblotting with
LPS-specific antibodies. The mutant produced no detectable A-band or
B-band LPS. Complementation with pFV162-26 (wzx,
hisHF; Fig. 1) restored both A- and B-band LPS
biosynthesis.
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Production of A-band LPS is delayed in wzx
mutants.
During analysis of the wzxs
mutants, we noted that LPS preparations made from fresh overnight
plates contained no detectable A-band LPS on silver-stained
SDS-polyacrylamide gels or Western immunoblots. In contrast,
preparations made from plates that were several days old appeared to
have substantial amounts of A-band LPS. This delay in A-band LPS
production was reproducible and occurred in cultures grown on solid
media (both Luria agar and Pseudomonas isolation agar) as
well as in broth. Strains which displayed this phenotype were
designated AL to distinguish them from those which were
truly A.
Analysis of LPS production by A
L
wzxs mutants over time compared with the parent
strain PAO1 was performed. Comparison of the
growth rates of the parent
and a representative A
L wzxs mutant
(S2) showed no significant differences (not shown).
While the parent
strain had substantial amounts of both A- and
B-band LPS after 12 h of growth, the S2 mutant produced only rough
LPS (core-lipid A) with
no detectable A band or B band (Fig.
4).
After 18 to 24 h, A-band LPS became detectable in the preparations
from the S2 culture (Fig.
4). In contrast, no B-band LPS could
be
detected at any time during the experiment.
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FIG. 4.
Analysis of the production of A-band LPS over time. (A)
LPS was harvested from the O5 parent strain and from the AL
mutant wzxs S2 as described in Materials and
Methods and analyzed on silver-stained SDS-polyacrylamide gels. While
the parent strain (O5) produced substantial amounts of both A- and
B-band LPS after 12 h of growth, the S2 mutant produced no
perceptible amounts of either LPS after 12 h of growth. After 24 to 36 h of growth, the mutant produced sufficient A-band LPS to be
detectable on silver-stained SDS-polyacrylamide gels. (B) Western
immunoblot analysis of LPS from the AL wzx
mutants S2 (wzxs) and X10
(wzxx) using LPS-specific MAbs. In comparison
with 12-h cultures of the parent strain (O5), which contain both A- and
B-band LPS, 12-h cultures of S2 and X10 contain no detectable A- or
B-band LPS. A-band LPS is detectable after 18 to 24 h of growth,
while no B-band LPS could be detected over the duration of the
experiment. The effect of both mutations in wzx appears to
be the same.
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As mentioned above, our concern that production of a truncated but
partially functional Wzx protein by
wzxs mutants
was somehow
responsible for the A
L phenotype was addressed
through generation and analysis of the
wzxx
series of mutants. Analysis of the LPS produced by
wzxx mutants
on silver-stained
SDS-polyacrylamide gels showed that of 39 mutants
obtained, none
produced B-band LPS, and the majority produced
little or no A-band LPS
after 12 h of growth. Results from a time
course experiment
similar to the one described above showed that,
again, the amount of
A-band LPS produced by a representative
wzxx mutant (X10) increased over time from undetectable to substantial
(Fig.
4).
Analysis of atypical A+ or A
wzx mutants.
In contrast to the AL
wzx mutants described above, a subset of wzx
mutants (1 of 21 wzxs and 4 of 39 wzxx mutants) produced substantial amounts
of A-band LPS during all phases of growth (representative mutants are
shown in Fig. 5). In addition, at least 3 of 39 wzxx mutants produced no A-band LPS at
all, even upon prolonged incubation (Fig. 5). These two types of
wzx mutants were designated A+ and
A, respectively. Despite the difference in A-band
phenotype among the wzx mutants, Southern blot analysis of
representative strains showed no gross rearrangements in ~5 kb of DNA
encompassing the site of the insertional mutation (Fig. 2). The
synthesis of A-band LPS by both the AL (after prolonged
growth) and the A+ wzx mutants confirmed that
Wzx is not directly necessary for A-band LPS synthesis. Therefore, the
mere lack of Wzx could not explain the deficiency in A-band LPS
biosynthesis in AL (during early growth phases) and
A wzx mutants.
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FIG. 5.
Analysis of LPS from atypical wzx mutants.
LPS from representative wzx mutants which either produced
A-band LPS without delay (wzxs mutant S7 and
wzxx mutant X24) or produced no A-band LPS even
after prolonged growth (wzxx mutants X6 and X14)
was analyzed on silver-stained SDS-polyacrylamide gels and Western
immunoblots using LPS-specific MAbs. The LPS from the O5 parent strain,
S7, and X24 was prepared from 12-h cultures, while the LPS from the X6
and X14 cultures was prepared from 36-h cultures. None of the mutants
made B-band LPS at any point during growth.
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The delay in A-band LPS expression in A
L strains was
thought to be due to the reduction in some component required for
production
of both types of LPS, stemming from the interruption in
B-band
LPS synthesis. A-band and B-band LPS have clearly been shown to
be synthesized by different pathways (
7,
9,
29). However,
there are components shared by both pathways. Recently, we showed
that
WbpL, which initiates synthesis of B-band LPS by transferring
N-acetyl-6-deoxygalactosamine-1-P (Fuc2NAc-1-P) to
C
55P (
6),
is also required to initiate the
synthesis of A-band LPS (
30).
WbpL is a homologue of
E. coli WecA (Rfe), an enzyme that initiates
the synthesis
of enterobacterial common antigen and a variety
of O antigens through
the addition of
N-acetylglucosamine-1-P
(GlcNAc-1-P) to
C
55P. In the case of homopolymeric O antigens,
including A-band LPS, the residue added by WecA-WbpL acts solely
as a
primer and does not become part of the O repeat unit (
36).
WbpL and WecA were both able to initiate the synthesis of A-band
LPS in a
wbpL::Gm
r mutant of
P. aeruginosa O5, likely through the addition of
GlcNAc-1-P
to C
55P (
30).
We postulated that the interruption of B-band biosynthesis after
formation of the O-antigen unit, but prior to its translocation,
might
in some way affect the availability of WbpL. A reduction
in the
availability of WbpL would hinder the initiation of A-band
polymer
synthesis, generating the A
L phenotype. Alternatively, the
introduction of errors or rearrangements
in the putative operon
wbpG-wbpL, following the recombination
events required to
generate the knockout mutant, could have polar
effects on the
expression of
wbpL. The latter prospect is less
likely,
since a number of individual
wzx mutants with an
A
L phenotype, each presumably arising from unique
recombination
events, were isolated. In addition, Southern
blot analyses of
the
wzy-wbpG region in which the
recombination events occurred
show that there are no gross
rearrangements (Fig.
2), suggesting
that polar effects are unlikely to
be the cause of the A
L phenotype.
The growth rates of A
L wzx mutants (which do not
appear to have acquired additional mutations affecting LPS
biosynthesis) do
not seem adversely affected (not shown), as one
might expect of
cells with insufficient free C
55P to
support normal peptidoglycan
synthesis. However, based on the
potentially deleterious nature
of
wzx mutations, it is
possible that those mutants eventually
isolated already contain
compensatory mutations that permit them
to grow in the presence of the
wzx mutation.
The delay in A-band LPS production is alleviated by supplying WbpL
in trans.
To test the hypothesis that the availability of
WbpL was limiting in AL cells, we transformed
AL wzxs mutant S2 with a
high-copy-number plasmid carrying the serotype O5 wbpL gene
under the control of the lac promoter (pFV110). If insufficient WbpL is available in AL cells, provision of
excess WbpL in trans should mitigate the delay in A-band LPS
expression. Analysis of LPS from S2 transformed with pFV110
(wbpL) showed that the presence of the plasmid restored the
ability of S2 to produce A-band LPS without delay (Fig.
6). Interestingly, the A
wzx mutant randomly chosen for further analysis (X14)
appears to have a secondary mutation affecting wbpL, since
it was rendered A+ by the addition of pFV110
(wbpL) alone (see below).
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FIG. 6.
Alleviation of the AL phenotype by
wbpL in trans. The AL
wzxs mutant S2 was transformed with pFV110, a
high-copy-number plasmid containing wbpL (Fig. 1). Analysis
of LPS from 12-h cultures of the transformants showed that they were
producing A-band LPS without delay, suggesting WbpL was limiting in
S2.
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From these data, it appears that A-band biosynthesis per se is not
impeded by the
wzx mutation and that WbpL is expressed
and
functional in A
L wzx mutants, since A-band LPS
can eventually be detected. Typically,
some WbpL molecules would
initiate A-band LPS biosynthesis; however,
the frequency of the
initiation event likely relies upon the number
of WbpL molecules
available. Possible explanations for the A
L phenotype
include a reduction in the amount of
wbpL expression
in
wzx mutants, a decrease in the normal rate of initiation of
A-band LPS, possibly due to the presence of B-band O units on
C
55P, or interference with normal WbpL function or
availability.
We are currently analyzing the transcription of the
wbpG-wbpL operon in both PAO1 and S2 to explore the first
possibility. However,
provision of
wbpL expressed in
multicopy from an unregulated promoter
could overcome any of these
difficulties.
Complementation analysis of wzx A+ and
A mutants.
We thought that emergence of
A+ or A derivatives of wzx mutants
could be due to the selection for strains with spontaneous secondary
mutations in genes necessary for LPS biosynthesis, perhaps due to a
reduction in free C55P. Liu and coworkers
(25) showed that wzx mutants appeared to
accumulate a single O-antigen unit on C55P. In
P. aeruginosa, interruption of B-band biosynthesis after formation of the O-antigen unit, but prior to its translocation, may cause C55P to be sequestered. Removal of
C55P from the cellular pool would be deleterious for other
cell functions, such as peptidoglycan formation. The strong pressure to
overcome the reduction in availability of C55P may lead to
accumulation of second-site mutations. The most likely sites for such
mutations would be in the B-band O-antigen genes or in housekeeping
pathways which feed into LPS synthesis in order to prevent formation of
the B-band O unit altogether. For example, a second-site mutation in
the B-band cluster would prevent formation of the O-antigen unit on
C55P, allowing the synthesis of A-band LPS to continue
normally (A+ cells) in the presence of the wzx
mutation (unless they occurred within wbpL itself).
In support of this hypothesis, a number of
wzx mutants with
A-band LPS expression atypical of a
wzx mutant were
isolated.
This subset included five A
+ wzx
mutants and at least three A
wzxx
mutants, examples of which are shown in Fig.
5. The
inability
to complement either A
+ or A
wzx mutants using pFV162-26 (
wzx)
alone, a construct that rendered
isogenic A
L wzx
strains A
+B
+, confirmed that
A
+/A
strains had likely accumulated
additional mutations leading to
loss of expression of A band, B band,
or
both.
Complementation of the atypical strains with the entire gene clusters
necessary for either A- or B-band biosynthesis identified
the B-band
LPS genes as the site of secondary mutations. Introduction
of pFV3
(
23), carrying the A-band LPS biosynthetic genes, into
the
A
wzx mutant X14 was not able to restore
A-band biosynthesis, locating
its secondary mutation(s) elsewhere (Fig.
7). In contrast, pFV100
(
6,
24) carrying the
wbp (B-band) gene cluster could
restore
B-band LPS biosynthesis in both A
+ and
A
wzx mutants as well as A-band synthesis in
the
wzx A
mutant (Fig.
7). These results imply
that, in these mutants,
a secondary mutation(s) affecting LPS
biosynthesis was in the
B-band genes. Further analysis showed that the
A
wzx mutant X14 could be complemented to
A
+B
+ by pFV114 (Fig.
1), which contains both
wzx and
wbpL, and to
A
+ but not
B
+ by pFV110, containing
wbpL (Fig.
7). Taken
together, these results
show that X14 contains the original
wzx mutation as well as a
secondary mutation in
wbpL.
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|
FIG. 7.
Complementation analysis using atypical wzx
mutants. Complementation of representative A+ and
A wzx mutants with cosmid clones containing
the A-band (pFV3) (23) and B-band (pFV100) (6,
24) LPS gene clusters. None of these mutants could be
complemented to A+B+ by pFV162-26 (not shown).
However, pFV100 complements both A-band (in the A
wzx mutant) and B-band synthesis (in all mutants), showing
that these wzx::Gmr strains contain an
additional mutation(s) affecting LPS synthesis that maps within the
B-band genes. The A wzx mutant X14 could be
rendered A+ by pFV110 (containing wbpL) (Fig.
1), implying that it contains mutations within, or polar upon,
wbpL. pFV110 cannot complement the original
wzx::Gmr mutation, so the cells remain
B. Complementation of X14 with pFV114 (wzx
through wbpL) (Fig. 1) restores it to
A+B+.
|
|
Generation of double knockout mutants.
We attempted to
replicate the phenotype of A+ wzx mutants by
introducing a second knockout mutation of the B-band LPS biosynthetic genes into an AL wzx mutant, S2. We reasoned
that prevention of synthesis of the first sugar of the O unit, Fuc2NAc,
would prevent accumulation of any material on C55P.
The highly conserved gene, wbpM, that lies at the 3' end of
the B-band LPS gene cluster is implicated in Fuc2NAc biosynthesis
(6). The wbpM gene was inactivated with a
nonpolar carbenicillin resistance cassette, and the resulting construct
was introduced into the chromosome of the AL wzx
mutant, S2.
The PAO1 parent strain, the A
L wzx strain (S2),
and the S2
wbpM::Cb
r mutants were
grown for 12 h (the time point at which S2 had no
detectable
A-band LPS; Fig.
4). LPS was prepared by the method
of Hitchcock and
Brown (
15) and examined on silver-stained SDS-polyacrylamide
gels and Western immunoblots with LPS-specific MAbs. Introduction
of
the
wbpM::Cb
r mutation was not able to
relieve the A
L phenotype of the S2 mutant (not shown). This
result may mean
that the atypical A
+ and A
wzx mutants did not arise from an A
L
wzx background. Alternatively, since the function of WbpM in
Fuc2NAc biosynthesis has not yet been ascertained, it may not
be the
appropriate target for inactivation in order to recreate
the
A
+ wzx phenotype from an A
L
wzx mutant.
With the exception of ABC-type transporters (
19,
29), the
identification and analysis of components of the LPS transport
machinery have proven to be complicated. Problems related to the
low
copy number and integral membrane location of transport proteins
and
the deleterious effects on cell growth caused by their mutation
(
31) have hampered elucidation of this aspect of LPS
biosynthesis.
Despite the precedent, we have successfully generated
wzx chromosomal
knockout mutants and did not experience the
reported difficulties
encountered during attempts to clone
wzx in the absence of other
LPS genes (
26).
P. aeruginosa is a unique model system in which two
forms of LPS with independent pathways of biosynthesis are coproduced.
The only analogous system that has been studied in enterics involves
the concomitant production of a homopolymeric O8 or O9 O antigen
with a
heteropolymeric lipid A-core-linked "capsular" exopolysaccharide
in
E. coli (called K
LPS) (
2,
10,
12).
Although a
wzx homologue
has been identified in the K40
cluster of
E. coli O8 and both
polymers are WecA dependent
(
2),
wzx mutants are not yet available
(
1). Therefore, it is not clear whether a
wzxK40 mutation would
affect production of the
O8 or O9 O
antigens.
This study has demonstrated that
wzx is essential in
the synthesis of the heteropolymeric B-band O antigen of
P. aeruginosa O5 and that its mutation can affect the
synthesis of the homopolymer,
A band. In addition, the key role of WbpL
in initiation of both
A- and B-band synthesis has been reemphasized. By
generating chromosomal
wzx mutants, we have laid the
foundation for understanding the
role of Wzx in translocation of O
units and the effect of
wzx mutations on the activities of
the initial glycosyltransferase,
WbpL.
| |
ACKNOWLEDGMENTS |
Funding for the study was provided to J.S.L. from the Medical
Research Council of Canada (grant MT14687). L.L.B. is the recipient of
a Canadian Cystic Fibrosis Foundation Fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 3823. Fax: (519) 837-1802. E-mail: jlam{at}uoguelph.ca.
| |
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Journal of Bacteriology, February 1999, p. 973-980, Vol. 181, No. 3
0021-9193/99/$04.00+0
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Abstract
Introduction
