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Journal of Bacteriology, August 1999, p. 4746-4754, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Influence of a Putative ECF Sigma Factor on Expression of the
Major Outer Membrane Protein, OprF, in Pseudomonas
aeruginosa and Pseudomonas fluorescens
Fiona S. L.
Brinkman,1
Geert
Schoofs,2
Robert E. W.
Hancock,1,* and
René
De Mot2
Department of Microbiology and Immunology,
University of British Columbia, Vancouver, British Columbia, Canada V6T
1Z3,1 and F. A. Janssens Laboratory
of Genetics, Catholic University of Leuven, Heverlee, Belgium B-30012
Received 7 April 1999/Accepted 25 May 1999
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ABSTRACT |
The gene encoding OprF, a major outer membrane protein in
Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a
single sigma 70 promoter immediately upstream of the gene. We now
report the identification of a novel putative ECF (extracytoplasmic
function) sigma factor gene, sigX, located immediately
upstream of oprF in both Pseudomonas aeruginosa
PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In
P. aeruginosa, Northern analysis demonstrated that this
reduction was a result of an effect on transcription of monocistronic
oprF combined with a polar effect due to termination of a
transcript containing sigX and oprF. Comparison
of sigX-disrupted and wild-type cell transcripts by primer
extension indicated that monocistronic transcription of
oprF occurs from two overlapping promoters, one that is
SigX-dependent and resembles ECF sigma factor promoters in its minus-35
region and another promoter that is independent of SigX and is
analogous to the sigma 70-type promoter previously reported.
Complementation of the P. aeruginosa sigX-disrupted mutant
with plasmid-encoded OprF did not resolve the phenotypes associated
with this mutant, which included a markedly reduced logarithmic-phase
growth rate in rich medium (compared to that in minimal medium),
further reduction of the growth rate in a low-osmolarity environment,
secretion of an unidentified pigment, and increased sensitivity to the
antibiotic imipenem. This indicates that SigX is involved in the
regulation of other genes in P. aeruginosa. Disruption of
the sigX gene in P. fluorescens also had an
effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas
syringae isolate and six P. aeruginosa clinical
isolates. Collectively, these data indicate that an ECF sigma factor
plays a role in the regulation and expression of OprF and also affects
other genes.
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INTRODUCTION |
OprF, a major outer membrane protein
in type I Pseudomonas spp., is a nonspecific porin that
plays a role in the maintenance of cell shape and is required for
growth in a low-osmolarity environment (8, 10, 19, 29).
Clinically derived mutants of the opportunistic human pathogen
Pseudomonas aeruginosa which are multiply antibiotic resistant (MAR) and are deficient in the major outer membrane protein
OprF have been isolated (20). Sequencing of the
oprF gene in such a clinical isolate has shown that the
oprF gene and promoter are intact, suggesting that a
possible regulatory mutation is involved (21, 21a). In plant
root-colonizing Pseudomonas fluorescens, regulation of
OprF is of interest because of the in vitro-demonstrated role of OprF
in adhesion to plant roots (4).
Previous transcriptional analysis of P. aeruginosa oprF by
primer extension, S1 nuclease mapping, and Northern blot analysis indicated that there was a single transcriptional start site 57 bp
upstream of oprF (5). A putative rho-independent
transcription terminator was identified immediately downstream of
oprF, followed by a gene transcribed in a convergent
direction. The promoter upstream of oprF was found to share
similarity with other sigma 70-type promoters (5), and
changes in OprF expression were not observed under a variety of growth
conditions. Therefore, oprF was thought to be constitutively
transcribed as a monocistronic unit, and thus, studies of its
regulation, or upstream genes, were not pursued further.
However, we now report the identification of sigX, a new
putative ECF (extracytoplasmic function) sigma factor gene located upstream of oprF in both P. aeruginosa PAO1 and
P. fluorescens OE 28.3, and show that SigX plays a role in
OprF expression. Characterization of sigX-disrupted mutants,
and the mutant complemented with oprF on a plasmid,
demonstrated that this probable sigma factor also affects the
expression of other genes in P. aeruginosa.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
All
strains and plasmids used are listed in Table
1. Strains were grown in Mueller-Hinton,
brain heart infusion, chocolate agar, or Luria Bertani (LB) medium (1%
tryptone, 0.5% yeast extract, pH 7.0; Difco). BM2 medium
(9) containing succinate as a carbon source was used as a
minimal medium for P. aeruginosa, and either BM2 or M9
medium (containing glucose as a carbon source for M9 [15]) was used for P. fluorescens. Varying
concentrations of NaCl were added to LB or minimal medium (no-salt,
low-salt, normal-salt, high-salt, and very-high-salt medium represented
additions of 0, 8, 85, 200, and 500 mM NaCl, respectively). In some
experiments, 171 mM NaCl was added. Antibiotics were used when
necessary at the following concentrations unless otherwise indicated:
streptomycin, 500 µg/ml; ampicillin, 100 µg/ml; carbenicillin, 300 µg/ml; chloramphenicol, 25 µg/ml; kanamycin (Km), 50 µg/ml;
gentamycin (Gm), 12 µg/ml; and spectinomycin, 50 µg/ml. For growth
curve studies, frozen aliquots of cells were first grown overnight on
agar containing appropriate antibiotics for maintenance of the strain
genotype, and then the cells were grown overnight in the appropriate
antibiotic-free broth medium. This overnight culture was then diluted
1:100 into 20 ml of fresh medium in a 250-ml sidearm flask. Growth of
the cells in this 20-ml culture was then monitored by determining the
optical density either with a Klett spectrophotometer with a red filter
or by determination of absorbance at 610 nm with a standard UV and
visual spectrophotometer. The culture was subjected to constant shaking
to provide aeration and incubated at 37°C for P. aeruginosa or 30°C for P. fluorescens unless
otherwise described. Some growth curves of P. fluorescens were determined with a Bioscreen growth analyzer
(Labsystems, Helsinki, Finland) with constant shaking at 30°C and
absorbance measurements at 600 nm.
General DNA procedures and sequencing.
Most common DNA
procedures were performed as described by Sambrook et al.
(22). For PCR, either Vent or Taq DNA polymerase (Fisher Scientific) was used under the standard conditions suggested by
the manufacturer, unless otherwise described. Most PCR experiments were
performed with an MJ Research thermal cycler or a Trio-thermoblock PCR
apparatus (Biometra) with the following thermal profile repeated 30 times: 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min. DNA
was sequenced (both strands) with either an ABI 373A automated sequencing system (Perkin-Elmer, Norwalk, Conn.) or an ALF sequencer (Pharmacia) according to the manufacturer's instructions, and oligonucleotides were synthesized on an ABI 392 DNA-RNA synthesizer as
described by the manufacturer. For primer extension experiments, sequencing was performed with the fmol sequencing kit (Promega) as
described by the manufacturer with [
-32P]ATP end-labeled primer (see "RNA analysis" below). For P. fluorescens OE 28.3, the complete sequences of the inserts in plasmids pFAJ2001, pFAJ2511,
and pFAJ2672 (described in Table 1) were determined, resulting in a
total of 5.6 kb of sequence determined upstream of oprF. For
P. aeruginosa, the complete sequences of the inserts in
pWW1901, pWW1701, and pWW2300 were determined (providing a total of
3,020 bp of sequence information upstream of oprF) and
additional sequence information (to a total of 5,519 bp of sequence
upstream of oprF) was obtained from the
Pseudomonas Genome Project sequence data (20a).
The sigX genes of P. syringae pv. syringae LMG
1247 and of P. fluorescens M114 were also amplified by PCR
with, as a forward primer,
5'-ATAGGATCAAGGAGGACTTGTTATGAATAAAGCCCAAACG-3' (italics indicate the added BamHI tag and ribosome
binding site), and with
ATAGGATCCGCACTAAGTTTCAGTCTCGCC-3' as a reverse
primer. Note that the possible TTG start codon in the forward primer
was replaced with ATG (represented in boldface), to assist in
prospective expression studies in Escherichia coli. The
resulting BamHI-digested PCR amplicons obtained from
P. syringae pv. syringae LMG 1247 and P. fluorescens M114 were cloned for sequence analysis in pUC18 and
pUC19 to generate pFAJ2448 and pFAJ2432, respectively. The sequence of
sigX was also determined from six P. aeruginosa
clinical isolates, H246, H344, H397, H411, H580, and H813, by direct
sequencing of PCR amplicons of the sigX gene obtained with
primers FL37 (5'-GGCCAACCGTCTACTGCTCG-3') and FL9
(5'-TTGTCCAACAATCAGCCGCA-3'), which flank the gene.
RNA analysis.
For RNA analysis, cells were grown in
high-salt LB medium as described above to early log phase, mid-log
phase, or late log phase or were grown overnight (stationary phase).
Total P. aeruginosa RNA was extracted from the cells with
the protocol and materials supplied in the RNeasy RNA isolation kit
from Qiagen (Hilden, Germany). All RNA preparations were treated with
RNase-free DNase and repurified by the Qiagen protocol before
electrophoresis, reverse transcription (RT)-PCR, or primer extension
experiments. Five micrograms of this RNA was subjected to
electrophoresis in 1% formaldehyde agarose gels as described by
Sambrook et al. (22). Total RNA from P. fluorescens was prepared according to the method of Nagy et al.
(18). For Northern analysis, RNA was transferred to
positively charged nylon membranes (Boehringer Mannheim), and hybridization and probe preparation were performed with the AlkPhos direct DNA labeling and detection system from Amersham. This method for
direct labeling of the probe bypasses the need for antibody detection
and is reported to be quantitative by the manufacturer. A probe
containing oprF sequences was PCR amplified from the plasmid pRW5, which contains only 60 bp of sequence upstream of oprF
(plus a mutated promoter region) and so provided a good template for preparation of an oprF probe that was free of any
sigX sequences. The sigX probe was prepared by
PCR amplification from pWW2300 using one set of primers within the
gene, followed by a second round of PCR using the resulting amplicon as
a template and primers internal to the first-round PCR primers. This
also produced a probe that, according to control experiments, was not
contaminated with upstream (cmpX) or downstream
(oprF) gene sequences. For RT-PCR experiments, RNA was first
reverse transcribed with specific primers and Moloney murine leukemia
virus reverse transcriptase (Promega or Roche, Basel, Switzerland) or
avian myeloblastosis virus reverse transcriptase (Promega) according to
the manufacturer's instructions. The resulting cDNA was then subjected
to amplification by PCR with a second set of internal primers. For
primer extension experiments, 5 µg of RNA was reverse transcribed
with SuperscriptII (Life Technologies) according to the manufacturer's
instructions with the following end-labeled primer which is
complementary to the reverse strand of the 5' end of oprF:
5'-CAACCAGCGAGCCGATGACA-3'. The primer was end-labeled with
Redivue [-32P]ATP (Amersham). A corresponding sequencing reaction
was performed as described above with the same end-labeled primer. RNA
for the primer extension experiments was obtained from mid-log-phase
cells that had been grown on high-salt LB medium as described above.
Protein procedures.
Outer membranes were prepared by the
one-step sucrose gradient method of Hancock and Carey (9).
Cell envelopes were prepared by subjecting the cells (resuspended in 10 mM Tris-HCl, 5 mM MgSO4, pH 7.5) to breakage in a French
pressure cell (twice at 15,000 lb/in2), followed by
centrifugation of the lysate at low speed (1,200 × g)
to remove unbroken cells and debris. The supernatant was then subjected
to one high-speed centrifugation at 151,000 × g, and
the pellet, containing cell envelope proteins, was resuspended in
water. The proteins were solubilized in solubilization buffer at
100°C for 10 min and then separated by electrophoresis in 12.5% polyacrylamide gels as previously described (9).
Two-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) of whole-cell extracts of P. fluorescens was carried out as described previously (2,
3). The gels were stained with Coomassie brilliant blue R250
(Bio-Rad) for visualization of proteins. Western immunoblot analysis
for the detection of OprF proteins was performed as described
previously, using the monoclonal antibody MA7-1 for P. aeruginosa (17) or MA28B9 for P. fluorescens
(2). Densitometric analysis of both protein gels and
Northern blots was performed with small amounts of sample analyzed with
the AlphaImager 1200 documentation and analysis system (Alpha
Innotech Corporation). To roughly confirm the densitometric analysis,
different amounts of sample loaded on a gel were compared with each
other to determine the amounts required to obtain the same band intensity.
Construction of gene disruptions.
For the construction of a
P. fluorescens OprF-deficient mutant, the 3.3-kb
SmaI fragment carrying the P. fluorescens OE 28.3 oprF gene (1) was cloned into the
HindIII site of pCL1921 (12) to generate
pFAJ2052. The Tn903 kanamycin resistance gene, obtained as a
1.26-kb HindIII fragment from pUC4K (Pharmacia Biotech), was then inserted in the NruI site of the oprF
gene in pFAJ2052. From this construct, pFAJ2073, with the
aminoglycoside 3'-phosphotransferase gene in the opposite orientation
to oprF, the interrupted oprF gene was recovered
as the BamHI-HindIII fragment and cloned into pSUP202 (24). The resulting plasmid, pFAJ2082, was
transferred from E. coli S17-1 to P. fluorescens
OE 28.3 by biparental conjugation. Among the kanamycin-resistant
Pseudomonas transconjugants, a strain (FAJ2026) sensitive to
chloramphenicol was selected, since this antibiotic resistance pattern
would correspond to a double crossover, resulting in loss of the
vector-associated chloramphenicol resistance. The anticipated genomic
rearrangement interrupting the oprF gene in strain FAJ2026
was confirmed by Southern blot analysis (using vector-, kanamycin
cassette-, and oprF-specific probes). No OprF protein was
detectable in mutant FAJ2026 by Western blotting with the OprF-specific
monoclonal antibody MA28B-9 (2).
For the creation of the
P. fluorescens sigX disruption, the
Tn
903 kanamycin resistance gene was inserted in an opposite
orientation
into pFAJ2052, which was linearized at an
Asp700
site within
sigX by partial digestion. A
BamHI
(complete) and
HindIII (partial)
digest of this clone
was used to obtain a fragment containing
the disrupted
sigX
gene, which was cloned into
BamHI-
HindIII-digested
pSUP202. This plasmid,
pFAJ2431, was mobilized from
E. coli S17-1
into
P. fluorescens OE 28.3, and a putative
sigX mutant
resulting
from double homologous recombination (FAJ2030) was selected
as
described above for the
oprF::Km
r
mutant
FAJ2026.
The
P. aeruginosa sigX disruption was constructed with a
2.4-kb
xylE-gentamicin resistance cassette from pX1918GT,
which was
cloned into a unique
EcoRV site within
sigX present in pWW1701.
A 3.4-kb
FspI-
SmaI fragment from the resulting plasmid,
pFB2e1,
was cloned into the
SmaI site of pEX100t
(
23), producing pFB2e1a3.
This plasmid, which encodes a
counterselectable
sacB marker, was
transformed into
E. coli S17-1 for mobilization into
P. aeruginosa H103.
After conjugation, colonies which grew on BM2 minimal medium
containing
78 mM salt, 4 µg of gentamicin/ml, and 150 µg of carbenicillin/ml
were plated onto LB medium containing 5% sucrose and 4 µg of
gentamicin/ml.
Sucrose-resistant colonies were screened for sensitivity
to 300
µg of carbenicillin/ml, indicating a double-crossover event
had
occurred. Each colony was confirmed to be a
sigX::Gm
r mutant by its gentamicin
resistance and production of a yellow
color when exposed to catechol
(indicating
xylE function) by PCR
with primers yielding
appropriately sized fragments containing
both
sigX and the
gentamicin cassette sequences and by Southern
blots probed with the
gentamicin cassette. Four separately obtained
cultures were subjected
to preliminary phenotypic analysis, and
all had identical phenotypes
under the conditions observed (OprF
expression levels and growth rate
in low-salt and high-salt media).
One such culture was given the strain
name
H814.
For mobilization of the pRW5 plasmid into strain H814, the cells were
electroporated as described by Farinha and Kropinski
(
6),
except that high-salt medium containing 50 mM MgCl
2 was
used for the recovery period after
electroporation.
MIC and other phenotypic tests.
MICs were determined as
previously described (29) for P. aeruginosa H103
(wild type), P. aeruginosa H814
(sigX::Gmr), and P. aeruginosa H636 (oprF::Strr) by
using the following compounds: tetracycline, chloramphenicol, ciprofloxacin, nalidixic acid, enoxacin, carbenicillin, ceftazidime, cefpirome, gentamicin, imipenem, rifampin, polymyxin B, erythromycin, SDS, HgCl2, ZnSO4, CuCl2,
Co(NO3)2, K2CrO4,
Ni(NO3)2, and AgNO3. The
Biolog GN Microplate (Biolog Inc., Hayward, Calif.), and API20 NE
(BioMérieux, Marcy l'Étoile, France) metabolic
tests were conducted as described by the manufacturers. Uptake of the
hydrophobic probe 1-N-phenyl-1-naphthylamine was
examined with a fluorometer as described previously (13).
Nucleotide sequence accession numbers.
The accession numbers
for the sequences reported in this paper are AF027290, AF115334,
AF115335, and AF115338.
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RESULTS |
Upstream of oprF is a putative ECF sigma factor
gene, sigX.
DNA sequencing upstream of the oprF
gene in P. fluorescens OE 28.3 and P. aeruginosa
H103 revealed the existence of an open reading frame (ORF) 108 bp
upstream of oprF that shared significant similarity with
sigma factor genes of the ECF family (approximately 40 to 45% amino
acid similarity; BLASTP Expect values of up to 8e-12). This similarity
was most pronounced in regions known to be highly conserved among
well-studied ECF sigma factors and included putative helix-turn-helix
and RNA polymerase-binding domains (Fig. 1). This ORF was named sigX in
both P. fluorescens and P. aeruginosa, since the
high degree of similarity between these two putative genes (94% amino
acid similarity) and the conserved gene order surrounding the genes
(Fig. 2) suggests that these genes are
orthologs. Of note, sigX, and most of the sigma factor genes
it closely resembled, did not have a downstream (or upstream)
regulatory gene, as is seen for some ECF sigma factors (14).
Interestingly, the sigX genes were most similar to known or
putative ECF sigma factor genes from gram-positive rather than
gram-negative bacteria.
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FIG. 1.
Alignment of the deduced amino acid sequences from the
sigX genes from P. aeruginosa H103 (PaeSigX),
P. fluorescens OE 28.3 (PflSigXOE2), P. fluorescens M114 (PflSigXM11), and P. syringae pv.
syringae LMG 1247 (PsySigX), as well as four other selected ECF sigma
factors that have had their sigma factor function confirmed (P. aeruginosa AlgU [PaeAlgU], M. tuberculosis SigE
[MtuSigE], Streptomyces coelicolor SigE [ScoSigE], and
Bacillus subtilis SigX [BsuSigX]). Note that for P. fluorescens M114 and P. syringae pv. syringae LMG 1247, the N-terminal sequence MNKAQT and the C-terminal sequence GETET
correspond to parts of the primers used to amplify the genes and so do
not necessarily reflect actual deduced SigX sequence for these
organisms. The asterisk marks a possible alternate ATG start site for
sigX. The bar with arrowheads marks the proposed location of
the polymerase core binding region, and the bar with diamond ends is
above the proposed helix-turn-helix motif region. Residues conserved in
50 to 74% of the shown sequences are shaded in grey, residues
conserved in 75 to 99% of the sequences are shaded in dark grey, and
residues conserved in 100% of the sequences are shaded in black.
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FIG. 2.
Schematic diagram of the organization of genes
surrounding sigX and oprF in P. aeruginosa H103 and P. fluorescens OE 28.3. The same
gene organization is observed in both species, and a combination of PCR
and sequence data suggests that cmpX (putative cytoplasmic
membrane protein X), sigX, and oprF are also
conserved in size and gene order in P. syringae pv. syringae
(data not shown). ORFs are shown as thick arrows, and the open boxes
mark the locations of putative rho-independent transcription
terminators. The sequence of the cobA gene in P. fluorescens has been previously reported (accession no. U09566),
but the P. aeruginosa cobA gene was deduced from the
Pseudomonas Genome Project sequence (20a). All
other genes are putative, and similarity of cmaX,
menG, estX, and ppsA was observed to
an unidentified ORF from E. coli (accession no. AE000232),
the E. coli S-adenosyl-methione-2-demethyl menaquinone
methyl transferase gene menG, human gastric lipase, and
phosphoenol pyruvate synthase, respectively. The diagram is not drawn
accurately to scale.
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Based on sequence similarity with certain other ECF sigma factor genes
(e.g., the genes for
P. aeruginosa AlgU and
Mycobacterium tuberculosis SigE in Fig.
1), the start site
for
sigX in both
organisms was designated as a TTG codon,
585 bp upstream of the
gene's stop codon. However, upstream of this
start codon is a
very weak ribosome binding site. Another very likely
start codon
is an in-frame ATG that is 468 bp upstream of the stop
codon and
has a better ribosome binding site upstream that is well
conserved
in both
Pseudomonas species.
We were also able to PCR amplify a similar
sigX sequence
from upstream of
oprF in
P. syringae pv.
syringae,
P. fluorescens M114 (Fig.
1), and six clinical
isolates of
P. aeruginosa. SigX
was highly conserved in all
of these pseudomonads. The six
P. aeruginosa clinical
isolates (H246, H344, H397, H411, H580, and
H813) had identical
sigX sequences, except for H411, which had
a single silent
T-to-C transition mutation 369 bp upstream of
the proposed stop codon
for
sigX.
Transcriptional linkage and analysis of other genes upstream of
oprF.
Other probable genes in the same orientation as
sigX-oprF that were similar in sequence and gene order in
both P. aeruginosa H103 and P. fluorescens OE
28.3 were identified by sequencing the region upstream of
sigX. A schematic diagram of this region is shown in Fig. 2,
with the genes described in the respective GenBank sequence submissions.
To examine possible transcriptional linkage among these genes, RT-PCR
experiments were performed with both
P. aeruginosa and
P. fluorescens with primers that flanked the intergenic
sequences
within this region (data not shown). A primer complementary
to
a
sigX sequence and a primer complementary to the 5' end
of
oprF were able to successfully amplify a PCR product of
the expected
size from reverse-transcribed total RNA isolated from
log-phase
wild-type cells. Primers flanking the intergenic region
between
cmpX and
sigX and primers coupling
cmaX-crfX,
crfX-cmpX,
menG-cmaX,
and
estX-menG were also able to produce a PCR amplicon. No PCR
amplicon or, in one instance, a very faint PCR amplicon was obtained
for primers coupling
ppsA and
estX in
P. aeruginosa (this region
contained a putative transcription
terminator in
P. aeruginosa).
These results are consistent
with the concept that there is some
degree of transcriptional linkage
between neighboring genes in
this region from
estX to
oprF.
Disruption of sigX reduces OprF expression: protein and
transcriptional analysis.
In both P. fluorescens OE
28.3 and P. aeruginosa H103, we constructed disruptions of
sigX. These disruptions also terminated any transcript
produced from a promoter upstream of sigX, since rho-independent transcription terminators flank the inserted antibiotic resistance cassettes. Examination of outer membrane protein
preparations, cell envelope preparations (Fig.
3), and whole-cell lysates revealed that
there was an estimated 2- to 10-fold reduction in the levels of OprF as
a result of this disruption. This was confirmed by a Western immunoblot
probed with an anti-OprF monoclonal antibody (data not shown). A slight
increase in another unidentified outer membrane protein (approximately
47 kDa) was also observed for the sigX-disrupted mutant
versus wild type for both species (Fig. 3) (also observed in outer
membrane preparations). This change was not observed in the
OprF-deficient mutant for each species. Comparative
two-dimensional SDS-PAGE analysis of whole-cell proteins of the
sigX::Kmr mutant and wild-type strains
of P. fluorescens did not reveal other quantitative changes
or qualitative changes (data not shown).
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FIG. 3.
Coomassie blue-stained SDS-PAGE gel of cell envelope
preparations of P. aeruginosa (A) and P. fluorescens (B) cells. (A) Lane 1, molecular weight marker
(103, from top to bottom) 94, 67, 43, 30, 20.1, and 14.4;
lane 2, P. aeruginosa H103 (wild type); lane 3, P. aeruginosa H814 (sigX disrupted); lane 4, P. aeruginosa H636 (oprF disrupted). (B) Lane 1, molecular
weight marker (same as for panel A); lane 2, P. fluorescens
OE 28.3 (wild type); lane 3, P. fluorescens FAJ2030
(sigX disrupted). The position of the band corresponding to
OprF is indicated with an arrow next to both gels. Note that OprF in
P. fluorescens OE 28.3 does not contain the "disulfide
bridge" region (a 23-amino-acid insertion containing two disulfide
bonds) found in OprF proteins in some pseudomonad strains, including
P. aeruginosa H103, and so migrates further when subjected
to SDS-PAGE. Note also that the cell envelope preparations shown were
picked to indicate the variability in the level of reduction of OprF.
The P. aeruginosa sigX mutant would frequently have a more
marked reduction in OprF expression than is observed here. An asterisk
marks the location of a 47-kDa protein that is increased in expression
in the sigX mutant.
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To determine whether the reduction in OprF in the
sigX-disrupted mutant was due to a polar effect (because of
cotranscription
of
sigX-oprF) or to a role of SigX in the
transcription of monocistronic
oprF, Northern blots of total
RNA from
P. aeruginosa cells grown
to different stages of
growth were probed with PCR-amplified DNA
fragments containing either
sigX or
oprF sequences. The probes
were prepared
(see Materials and Methods) to ensure that they
were free of any
contaminating flanking sequences. For cells grown
overnight (stationary
phase) in LB medium with 200 mM salt, a
1.2-kb transcript predominated
with the
oprF probe, corresponding
to monocistronic
transcription of
oprF (Fig.
4), though faint
transcripts of sizes
possibly corresponding to
cmpX-sigX-oprF and to
sigX-oprF were visible upon overexposure of the Northern
blot. However, cells grown to the early logarithmic or mid-logarithmic
stages of growth clearly contained the predominant 1.2-kb transcript
plus an additional minor transcript that hybridized with both
the
sigX and
oprF probes, indicating cotranscription
of these
genes and confirming their linkage by RT-PCR (Fig.
4). The
size
of this putative
sigX-oprF transcript seemed too small
to accommodate
the size of
sigX predicted from the TTG start
site mentioned above,
consistent with the proposed alternate ATG start
site for the
gene; however, the size was not accurately determined. For
similarly
probed blots containing RNA from the
sigX-disrupted mutant, the
sigX-oprF transcript
was not observed and there was a reproducible,
marked reduction in the
amount of the 1.2-kb monocistronic
oprF transcript (Fig.
4).
The lack of
sigX-oprF transcript was also
confirmed by
RT-PCR (with the same primers used above to show
linkage between these
genes in the wild-type strain). Densitometric
analysis of these data
(as well as comparisons of different amounts
loaded on a gel) suggests
that these reductions in
sigX-oprF and
moncistronic
oprF transcript levels could account for the reduction
in
OprF expression observed. These data therefore suggest that
the
reduction of OprF expression in the
sigX mutant is due to
an
effect of SigX on transcription of monocistronic
oprF
(either
directly or indirectly) combined with a more minor polar effect
of the knockout on the downstream
oprF gene.
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|
FIG. 4.
Northern blots of P. aeruginosa wild-type and
(SigX+) and sigX mutant (SigX ) RNA obtained from log-phase
(log) and stationary-phase (stat.) cells. The RNA was probed with
sigX (A) and oprF (B) sequences. The arrows mark
the bands corresponding to a proposed sigX-oprF transcript.
The thicker bands observed in the blots probed with oprF
correspond to the monocistronic oprF transcripts.
|
|
The effect of SigX on transcription of monocistronic
oprF
was further examined through primer extension analysis. Experiments
were performed with a primer complementary to the reverse strand
of the
5' end of
oprF and with
P. aeruginosa RNA
extracted from
wild-type and
sigX mutant cells grown in LB
medium with 200 mM
salt to mid-logarithmic stages of growth. A major
primer extension
product (Fig.
5), of
equal intensity for both wild-type and
sigX mutant cells,
was observed that corresponded to the sigma 70 transcriptional
start
site previously reported by Duchêne et al. (
5) 57 bp
upstream of
oprF. However, an additional primer extension
product
was also observed 40 bp upstream of oprF that was present for
wild-type cells but absent from the
sigX mutant (Fig.
5).
These
results, therefore, suggest that there are two overlapping
promoters
upstream of
oprF (Fig.
6), one which is SigX independent and one
which is SigX dependent. Upstream of the SigX-dependent start
site in
both the
P. aeruginosa and
P. fluorescens
sequences were
possible
35 regions (GAAGTT in
P. aeruginosa and CAAGTT in
P. fluorescens)
which shared notable similarity to consensus
35
sequences for other
ECF sigma factors (
11,
16) (Fig.
6). The
10 region is not
normally conserved among different ECF sigma-type
promoters and in this
case was proposed to be GTTGTG and GTTGTC
in
P. aeruginosa and
P. fluorescens, respectively
(Fig.
6).
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|
FIG. 5.
Primer extension analysis of transcriptional start sites
immediately upstream of oprF in wild-type (H103) (SigX+) and
sigX mutant (H814) (SigX) P. aeruginosa. Cells
were harvested from high-salt LB broth at mid-log phase. The band
corresponding to a SigX-dependent transcriptional start site is marked
40, indicating the start site's position upstream of the initiation
codon of oprF. The band corresponding to the
SigX-independent start site is at location 57 from the start of
oprF. The cutting and pasting for this figure was
necessitated by the fact that it required different exposure times to
optimally visualize the sequencing ladder (G, A, T, C) and primer
extension products.
|
|
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|
FIG. 6.
Alignment of P. aeruginosa H103 (P. aerug)
and P. fluorescens OE 28.3 (P. fluor) sequence between
sigX and oprF (boxed), showing the location of
putative promoter sequences. Identical bases are indicated with a
colon. The two transcriptional start sites, as determined from primer
extension analysis (Fig. 5), are indicated with arrows, and the
nucleotides (35 and 10 sites) proposed to be associated with the
SigX-dependent promoter and the SigX-independent promoter are marked
with underlined italics and underlined boldface characters,
respectively. The 35 site for the SigX-independent promoter proposed
by Duchêne et al. (5) seems somewhat unlikely based on
the lack of alignment between the P. aeruginosa and P. fluorescens sequences and the nonoptimal 20-nucleotide spacing
from the 10 site, but it was not further investigated in this
paper.
|
|
Growth rate of the sigX-disrupted mutant: evidence that
SigX regulates genes other than oprF.
The most noticeable
phenotype associated with the sigX disruption was a marked
reduction in logarithmic-phase growth rate and/or an increase in lag
phase for cultures of the mutant grown in various media (Fig.
7 and Table
2). For P. aeruginosa, this reduction in growth rate was particularly apparent under low-salt conditions (8 mM), and the mutant did not grow at all in medium containing no salt. OprF-deficient P. aeruginosa is also
unable to grow in medium containing no salt (30); however,
an oprF-disrupted strain grew with a shorter doubling time
than the sigX knockout in LB medium containing low salt (8 mM [Table 2]). The inclusion of 200 mM salt in LB medium restored the
growth of the OprF-deficient strain to near wild-type levels, but only
partially reversed the growth defect of the
sigX::Gmr mutant. When the
sigX mutant was complemented with oprF on a plasmid (by using a cloned oprF gene with a mutated
promoter, resulting in expression of relatively normal levels of OprF,
as confirmed for the complemented sigX mutant), the growth
rate was not restored to wild-type levels under any growth condition
examined, including low-salt media (Table 2). This indicated that
factors other than the reduced expression of OprF played a role in the inability of the sigX-disrupted mutant to grow like its
parent wild type in low-salt medium.
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|
FIG. 7.
Growth of P. aeruginosa (A) and P. fluorescens (B) SigX mutants (circles) and parent wild-type
(squares) and OprF-deficient strains (triangles) in LB medium. Solid
symbols denote cultures grown in high-salt (200 mM) media, and open
symbols are used for cultures grown in low-salt (8 mM) media.
OD600, optical density at 600 nm.
|
|
View this table:
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[in a new window]
|
TABLE 2.
Influence of OprF and SigX on doubling times of P. aeruginosa and P. fluorescens grown in different liquid
media with constant aeration
|
|
A reduction in growth rate was observed regardless of whether the
sigX mutant was grown in LB, Mueller Hinton, or brain heart
infusion broth, and smaller colonies were observed for the mutant
and
the wild type when grown for the same length of time on chocolate
agar,
indicating that even very rich medium did not complement
this
slow-growth phenotype. In minimal medium (BM2), the differences
in
doubling time between the wild type and the
sigX mutant were
less pronounced but still apparent when the medium contained high
salt
(200 or 171 mM
both concentrations produced essentially identical
results). With the addition of very high salt to the minimal medium
(500 mM) the growth rate of the wild type and the
sigX
mutant
became similar (Table
2). While there was slower growth of the
wild type in this very-high-salt environment, it is notable that
the
growth rate of the
sigX mutant did not correspondingly
decrease,
as it did when grown under other stress conditions, such as
extremes
of pH (data not shown) or low temperature (Table
2). A similar
growth rate for the wild type and the
sigX::Gm
r mutant was also observed
when the high salt in this minimal medium
was replaced by high sucrose
or KCl (both 500 mM [Table
2]).
This suggests that it was not high
salt, but rather high osmolarity,
that allowed the mutant to grow at
the same rate as the wild type.
However, the mutant still grew more
slowly than the wild type
in LB medium containing 500 mM salt (Table
2), suggesting that
osmolarity was not the only factor affecting growth
of the
sigX mutant in rich media. Addition of succinate to
the LB medium containing
500 mM salt did not restore the
sigX mutant to wild-type growth,
showing that the
differences between growth in LB and minimal
media under these
very-high-salt conditions were not solely due
to differences in carbon
sources that the bacterium could utilize
in each
medium.
For
P. fluorescens, the growth rate differences between the
wild type and the
sigX mutant grown in rich medium (LB) were
less
pronounced than that observed for the corresponding
P. aeruginosa strains; however, differences were still apparent
(Table
2).
A notable difference in lag phase was observed, with smaller
changes
in growth rate, suggesting that the
sigX knockout
may affect different
genes (or possibly fewer genes) in
P. fluorescens than in
P. aeruginosa.
Other phenotypes resulting from the disruption of sigX
in P. aeruginosa.
Some small but notable MIC differences for
some antimicrobials and metals were observed for the P. aeruginosa sigX mutant versus its parent wild type. The
sigX knockout was 4-fold more susceptible to imipenem,
2-fold more susceptible to polymyxin B, and 1.5- to 2-fold more
resistant to chromate ions and the fluoroquinolone enoxacin (see
Materials and Methods for a full list of antimicrobials and metals
examined). A MAR phenotype was not seen for this sigX
mutant, indicating that it does not play a direct role in determining
the phenotype of MAR OprF-deficient clinical isolates (20).
Another phenotypic change involved the production of an unidentified
bright-yellow pigment in the
P. aeruginosa sigX mutant.
When
grown in LB, this pigment appeared as the culture reached
an optical
density at 610 nm of 0.7, while the wild type remained
colorless at
this stage of growth; after overnight growth, the
mutant always had a
more intense yellow color. This pigment, which
was secreted into the
culture supernatant, was not fluorescent
and, in aqueous solution at pH
7.5 to 8.0, had an absorption maximum
of 380 nm. However, we are
uncertain of the significance of the
pigment, since alterations in the
levels of
Pseudomonas pigments
are commonly associated with
different growth conditions and mutations
(
27).
The magnitude of differences in growth rate between the
P. aeruginosa wild type and the
sigX mutant remained
similar during
growth at low or high temperatures (8, 15, 22, 42, and
45°C),
at low or high pH (pHs 3, 4, 5, 9, 10, 11, and 12), or under
anaerobic
conditions. Similarly, no significant differences were
observed
for uptake of the hydrophobic probe
N-phenyl-1-naphthylamine (
13),
which is often
used to measure changes in outer membrane permeability
to hydrophobic
compounds and/or efflux changes. The
sigX mutant
was motile
and had metabolic profiles similar to those of the
wild type, according
to Biolog GN Microplate and API20 NE strip
tests. When examined under a
microscope at ×100, the cells did
not appear to be different from the
wild type (while OprF-deficient
cells are noticeably shorter
[
30]).
| |
DISCUSSION |
Our results indicate that OprF in P. aeruginosa is not
just expressed constitutively from a sigma 70 promoter but rather the sigX gene product, a probable ECF sigma factor, plays a role
in its expression. Transcriptional linkage of sigX and
oprF was detected, and there appear to be two overlapping
promoters immediately upstream of oprF, one independent of
SigX and resembling the sigma 70 consensus sequence and another that is
SigX dependent and resembles an ECF sigma promoter. This SigX-dependent
transcription was not previously identified in the study by
Duchêne et al. (5); however, their results are
consistent with our results obtained for late-log- or stationary-phase
cells, when sigX is not well expressed. The transcript
initiating from the sigma 70-like promoter is still a significant
source of oprF transcript; however, clearly whatever conditions effect sigX transcription may also have an impact
on OprF expression.
OprF, and its deficiency in some clinical isolates of P. aeruginosa, has been previously correlated with significant
antibiotic resistance; however, the level of antibiotic resistance in
both an oprF knockout (29) and the
sigX knockout described here is modest and does not match
the resistance observed in an OprF-deficient clinical isolate
(20). Since OprF is apparently subject to more complex
regulation than was previously thought, it is possible that the MAR-
and OprF-deficient clinical phenotype is due to a regulatory mutation
that affects both OprF and MAR determinants. The one clinical isolate
studied in detail to date is notable for its frequent reversion to
normal antimicrobial sensitivity and an OprF+ phenotype in
a single step, and it contains no significant mutations in the
oprF gene or in the upstream promoter region (20, 21, 21a). However, our studies demonstrate that this regulatory
mutation would not likely be within the sigX gene, since
sigX disruption did not result in complete OprF deficiency
and the MAR phenotype was not observed for the sigX mutant.
Though a MAR phenotype was not observed, the increased imipenem
sensitivity of the P. aeruginosa sigX mutant is interesting,
since there was increased expression of a 47-kDa protein in the
sigX mutant that is approximately the size of OprD, an outer
membrane protein known to be involved in uptake of imipenem.
Other genes subject to control by SigX have not yet been identified;
however, phenotypic analysis of the two sigX knockout mutants, and the P. aeruginosa sigX mutant complemented with
oprF on a plasmid, clearly indicated that other genes
require SigX for expression. The sigX mutant showed a marked
inability to grow in low-osmolarity medium, even more so than an
OprF-deficient mutant, and was only able to grow at the same rate as
the wild type in a very-high-osmolarity environment. This would suggest that SigX is involved, directly or indirectly, in growth and/or survival in low-osmolarity environments. SigX was clearly required for
optimal growth in a low-osmolarity environment, a notable factor for
both P. aeruginosa and P. fluorescens, given that
their niches include water. SigX in P. aeruginosa also
seemed to be required for utilization of nutrients in rich medium such
as LB medium), since growth of the mutant was always markedly slower than that of the wild type in rich media, even under the very-high-salt conditions in which the wild type and mutant grew at similar rates in
minimal medium.
In conclusion, we have presented evidence that a probable ECF sigma
factor plays a role in OprF expression and that this putative sigma
factor also influences other genes. These results shed new light on the
nature of the regulation and expression of OprF, which could impact on
our understanding and study of this protein's involvement in
rhizosphere colonization by P. fluorescens and its
relationship with clinical antibiotic resistance in P. aeruginosa.
| |
ACKNOWLEDGMENTS |
This work was funded in part by the Canadian Cystic Fibrosis
Foundation (CCFF), the Medical Research Council (MRC) of Canada, and
the Biotechnology Program of the European Union (BIO2-CT93-0196). R.E.W.H. is a recipient of the MRC Distinguished Scientist Award. F.S.L.B. is the recipient of a fellowship from the CCFF. R.D. is a
Senior Research Associate with the Fund for Scientific Research (Flanders).
We acknowledge Colin Crist for studies of the conservation of the genes
in P. syringae, Rick Heffernan for help with phenotypic analysis of the SigX mutant in P. aeruginosa, Alex Beysen
for constructing P. fluorescens FAJ2026, and Margaret Pope
for aid with primer extension experiments with P. aeruginosa.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, 300-6174 University Blvd., University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3. Phone:
(604) 822 2682. Fax: (604) 822 6041. E-mail:
bob{at}cmdr.ubc.ca.
| |
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0021-9193/99/$04.00+0
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Top
Abstract
Introduction
