From the Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, United Kingdom
Received for publication, October 9, 2002
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ABSTRACT |
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Mutation of the
cyanide-insensitive terminal oxidase of Pseudomonas
aeruginosa leads to pleiotropic effects. A cio mutant and strains, including the wild-type, carrying the cioAB
genes on a multicopy plasmid were temperature-sensitive and had a cell division defect, leading to the formation of non-septate,
multinucleated filaments. Such strains of this intrinsically
antibiotic-resistant bacterium were more sensitive to a range of
antibiotics including chloramphenicol, Respiratory adaptability is likely to be an important component of
the growth and survival of Pseudomonas aeruginosa in the diverse ecological niche in which it is found. This Gram-negative, opportunistic pathogen is able to respire and grow under a variety of
aerobic and anaerobic conditions (1). Although it preferentially obtains energy via aerobic respiration, it is well adapted to conditions of limited oxygen supply (2). It is capable of anaerobic growth with nitrate as a terminal electron acceptor, and in its absence
it is able to ferment arginine, generating ATP by substrate level
phosphorylation (1-3).
Examination of the genome sequence of P. aeruginosa suggests
that its aerobic respiratory chain is extensively branched and is
terminated by at least 5 terminal oxidases
(4).1 Of four putative
terminal oxidases belonging to the heme-copper oxidase superfamily,
three are predicted to be cytochrome c oxidases, and one is
a quinol oxidase. The fifth oxidase is the cyanide-insensitive oxidase
(CIO),2 which is encoded by
the cioAB operon (5, 6). CioA and CioB are homologous to the
two subunits of the cytochrome bd quinol oxidases, CydA and
CydB, of Escherichia coli, Azotobacter
vinelandii, and other bacteria (7) and as such show no homology to
members of the ubiquitous heme-copper oxidase superfamily (8).
Histidine and methionine residues identified in E. coli
cytochrome bd as being ligands to the low spin heme
b558 and high spin b595
are conserved, as is a periplasmic loop, the Q-loop, that contains a
putative quinol-oxidizing site, although the Q-loop is significantly shorter in CioA than in CydA (5). However, the distinctive absorption
bands of a cytochrome bd quinol oxidase have never been
observed in numerous detailed studies of the cytochrome composition of
P. aeruginosa membranes (2). Consequently, this bacterium had not been considered to contain a cytochrome bd-type
quinol oxidase. Further spectral studies and respiratory activity
measurements of membranes from various P. aeruginosa strains
were carried out, but no cytochrome d-like signals were
found in wild-type strains (5, 6). An atypical cytochrome
d-like signal was seen but only in strains in which
the cioAB genes were present in high copy number on a
recombinant plasmid. The appearance of these cytochrome
d-like spectral signals did not quantitatively parallel an
increase in CIO activity. The heme d signals had absorption maxima at different wavelengths from those of the E. coli
cytochrome bd, particularly in CO-difference spectra.
The E. coli d2+-CO complex has a
maximum at 644 nm and a minimum at 621 nm, compared with a maximum at
635 nm and a minimum at 594 nm in P. aeruginosa membranes.
In reduced minus oxidized spectra an absorption maximum was observed at
623 nm, which shifted from the 628-nm maximum of E. coli
cytochrome d. Based upon these data it was argued that the
CIO of P. aeruginosa does not contain heme d and
suggested that the heme d-like signals may arise from the
promiscuous incorporation of heme d1 when CioA and CioB are
overexpressed. These data raised the probability that there is a family
of bacterial quinol oxidases related to the cytochrome bd of
E. coli and of other bacteria.
The cioAB genes are present on a 2.7-kb transcript, the 5'
and 3' ends of which have been mapped, indicating that cioA
and cioB are co-transcribed to form an operon (5).
Preliminary data indicate that the CIO is required for full
pathogenicity in a Caenorhabditis elegans
model.3 P. aeruginosa produces hydrogen cyanide as a metabolic product under
low O2 conditions at concentrations that inhibit terminal oxidases of the heme-copper superfamily (9). Therefore, it has been
proposed that the production of the CIO is an important adaptation,
allowing aerobic respiration under cyanogenic conditions (5). Cyanide
has been detected in tissue samples infected with P. aeruginosa (10), and it has been shown recently (11) that cyanide
poisoning is responsible for nematode killing in one of the C. elegans virulence models. It is established that defects in the
cytochrome bd of E. coli can lead to a range of
phenotypes, including temperature sensitivity for growth and difficulty
in exiting stationary phase (12-16). Therefore, we were interested in whether mutation of the CIO had pleiotropic effects on P. aeruginosa. We show that mutation or introduction of the
cioAB genes on a multicopy plasmid leads to a range of
phenotypes in P. aeruginosa including a cell division defect
and multiple antibiotic sensitivity via loss or damage to a
Bacterial Strains and Plasmids--
P. aeruginosa
PAO6049 met-9011 amiE200 strA was used as the wild-type
strain in these studies, together with the cio mutant PAO7701 cio::miniD-171, which has been described
previously (5, 6). The plasmids used to complement these strains are
described in Table I.
Growth Conditions--
Bacteria were grown in LB medium (17)
with agar added to 1.5% (w/v) for solid medium. As required,
antibiotics were added at the following concentrations: streptomycin (1 mg/ml), tetracycline (300 µg/ml), and carbenicillin (500 µg/ml).
Starter cultures were prepared by inoculating a single colony from a LB
plate into 5 ml of LB broth in a 25-ml sterile universal bottle and
incubating overnight at 30 °C in an orbital shaker at 200 rpm.
Overnight cultures were diluted 1:100 (into 50 ml of LB in a 250-ml
conical flask, to give an A600 of 0.02-0.04)
for growth at 30 and 37 °C or diluted 1:25 (to give an
A600 of 0.1) for growth at 42 °C, and
cultures were incubated with shaking at 200 rpm. Membranes were
prepared as described previously (5). Protein content was determined by
the method of Markwell et al. (18).
Phosphate-buffered saline contained (in g liter Investigation of the Temperature-sensitive (Ts) Growth
Phenotype--
Strains were grown aerobically on LB agar plates
supplemented with appropriate antibiotics and incubated at 30, 37, or
42 °C for 24 h. For broth cultures bacteria were grown
overnight in LB broth at 30 °C, diluted 1:100 into LB to give an
A600 of 0.02-0.04, and incubated at 30, 37, or
42 °C. The effects of various reducing agents and non-reducing
agents on the Ts phenotype was determined as described by Goldman
et al. (19). The following compounds were used at the
concentrations described previously (19): reduced glutathione,
methionine, alanine, L-cysteine, D-cysteine,
glutamine, oxidized glutathione, mercaptoethanesulfonic acid, a
combination of dithiothreitol and oxidized glutathione, bovine
catalase, and bovine superoxide dismutase. They were added to 1-cm
filter disks at the center of seeded plates, and the plates were
incubated at 42 °C for 24 h.
Hydrogen Peroxide Sensitivity Assay--
Cultures were grown in
LB medium until mid-exponential phase (A600 = 0.6) at 37 °C before challenging with a final concentration of 10 mM hydrogen peroxide. Samples were collected at different time points, and after dilution in LB they were plated onto LB agar and
incubated at 30 °C to obtain viable cell counts.
Microscopy--
A 1-ml sample of a stationary phase culture was
fixed and stained with 4',6-diamidino-2-phenylindole as described by
Hiraga et al. (20) and observed using a Zeiss Axiovision 3.0 microscope, and images were captured using an AxioCam digital camera
and processed with Zeiss Axiovision software.
Determinations of Minimal Inhibitory Concentrations
(MIC)--
The cultures were grown to stationary phase at 30 or
37 °C. They were harvested and diluted to 5 × 104
cell ml Assay of Chloramphenicol Accumulation in Intact Cells--
Cells
from stationary phase cultures at 30 and 37 °C were harvested and
diluted in fresh LB. The cultures were grown to mid-exponential phase,
when the culture density had reached 0.25-0.4 mg (dry weight) ml Hydrogen Peroxide Production--
Cells from 2.5 ml of a culture
grown in LB to early stationary phase were harvested and resuspended in
25 ml of phosphate-buffered saline. After 20 min, samples were
centrifuged at 4500 × g for 5 min to pellet the cells,
and the hydrogen peroxide concentration of the supernatant was
determined using the scopoletin assay as described (22), except that
the reaction mix contained 0.1 µM scopoletin instead of
0.2 µM scopoletin.
Determination of Catalase Activity--
Catalase activity was
determined in total cell lysates (4 µg of protein per sample) using a
Clark-type oxygen electrode (23). Catalase activity in polyacrylamide
gels was localized as described previously (24).
Oxidative Protein Modification Assays--
The carbonyl groups
in the protein side chains were derivatized, using the OxyBlot kit
(Intergen), to 2,4-dinitrophenylhydrazone by reaction with
2,4-dinitrophenylhydrazine. Total lysates were prepared from cultures
grown in LB to early stationary phase and slot-blotted using a 48-well
apparatus (Schleicher & Schuell) onto nitrocellulose membranes.
Oxidatively modified proteins were detected with
anti-2,4-dinitrophenylhydrazone antibodies using the ECL detection
reagents (Amersham Biosciences).
Mutation or Overexpression of the Cyanide-insensitive Oxidase Leads
to Temperature-sensitive Growth--
The wild-type (PAO6049), a
cio mutant (PAO7701), and a complemented mutant
(PAO7701/pLC8) all grew similarly at 30 °C. We have shown previously
that pLC8 complements PAO7701 for its respiratory defect (5). Both the
mutant and complemented mutant grew more slowly than the wild-type at
37 °C, but on solid medium they form similarly sized colonies
(~1.5-2.0-mm diameter). The wild-type grew more rapidly at
42 °C, but both the mutant and the complemented mutant grew very
poorly at 42 °C, forming only pinprick-sized colonies on solid
medium (~<0.1-mm diameter compared with 1.5-2.0-mm diameter for the
wild-type). In broth cultures both the cio mutant (PAO7701)
and the complemented mutant (PAO7701/pLC8) failed to grow promptly at
42 °C when our usual inoculum (A600 ~0.02)
was used (Fig. 1). However, if a larger
inoculum (A600 ~0.1) was used then the mutant
grew in liquid culture, albeit after a significant lag (data not
shown). However, the Ts growth phenotype was not because of a
polar effect of the mini-D171 insertion used to construct the
cio mutant strain (6), as introducing pLC8 or pLC2 conferred the Ts growth phenotype upon the wild-type (Fig. 1) (data not shown).
Introduction of the cioAB genes into the wild-type on the
plasmid pLC1, which is derived from a different plasmid backbone (Table I), led to an intermediate
growth phenotype at 42 °C (Fig. 1). The plasmid pLC8 carries a
2.7-kb fragment in which the only open reading frames are the
cioA and cioB genes. We have shown previously
that complementation of the mutant with the cioAB genes on a
multicopy plasmid or introduction of the plasmid into the wild-type led
to an overexpression of CIO activity compared with the wild-type strain
(5, 6), whereas the plasmid pLC1 had a much less marked effect.
Therefore, it seemed plausible that overexpression of CIO activity may
lead to the defective growth phenotype at 42 °C. Most of these
earlier experiments were done on late exponential phase cultures, and
we now know that CIO expression is increased rapidly and up to 5-fold
once the culture enters stationary phase.1 Therefore, we
repeated these activity measurements on the strains used in this study,
taking particular care to grow cultures to mid-exponential phase (well
before CIO expression increases) and into stationary phase (where CIO
has been induced). These data, determined as
succinate-dependent CIO activity on whole cells, are
reported in Table I. What is clear is that pLC2 and particularly pLC8
have a marked effect on CIO activity (up to a 2-fold increase) in
wild-type and mutant backgrounds in exponential but not in stationary
phase cultures. In contrast pLC1 had little or no effect on CIO
activity in exponential phase cultures. No effects were observed with
the pUCP18 control plasmid. Therefore, we conclude that either mutation
or the presence of cioAB in trans at levels that lead to
overexpression of CIO activity in growing cultures leads to a
temperature-sensitive growth phenotype in P. aeruginosa.
As has been shown previously for the Ts growth phenotype of E. coli cytochrome bd mutants (19), the Ts defect of the
cio mutant and pLC2/pLC8 containing strains of P. aeruginosa could be suppressed by thiol-reducing agents and
catalase. The thiol-reducing agents L-cysteine, reduced
glutathione, cio Mutant and cioAB Plasmid-containing Strains Form Non-septate,
Multinucleated Filaments--
While looking at the Ts phenotype of
cio mutant- and cioAB plasmid-containing strains,
we observed clumps in cultures, the numbers of which increased as the
growth temperature was raised. Microscopic examination of cultures
following 4',6-diamidino-2-phenylindole staining indicated that both
mutation and the presence of the cioAB genes on a multicopy
plasmid led to the formation of non-septate, multinucleated filaments
in a temperature-sensitive manner (Fig. 2). Wild-type cultures contained small
rods at all growth temperatures (Fig. 2, A-C).
However, the cio mutant formed occasional short filaments at
30 °C (Fig. 2F), but these become longer and more frequent as the growth temperature was raised to 37 °C. At 42 °C
most cells in cio mutant cultures were filamentous and
indeed fell to the bottom of a static culture vessel (Fig.
2H), as was also the case in pLC8 complemented mutant
cultures. Introduction of the cioAB-containing plasmids pLC8
or pLC2 into the wild-type also induced filamentation in a
temperature-dependent manner (data not shown), with
filamentation most pronounced at 42 °C (Fig. 2E).
However, as with the Ts phenotype, introduction of pLC1 into the
wild-type had little effect on cell morphology and a comparatively minor effect in a cioAB mutant background (Fig. 2,
D and I). No effect on cellular morphology was
seen with pUCP18 control plasmids (data not shown). We conclude that
strains mutated in cio or carrying the cioAB
genes on a multicopy plasmid have a cell division defect that lies in
one of the processes associated with septum formation. These strains
also had a problem exiting stationary phase. If cultures were grown to
stationary phase at 37 °C, subcultured into fresh medium, and
incubated at 30 °C then the wild-type resumed exponential growth
immediately, whereas the cioAB mutant or
pLC2/pLC8-containing strains showed a significant lag phase, often of
many hours, before resuming growth. Microscopic observation showed that
during this lag period the filaments broke down, and exponential growth
only commenced when the population was almost totally comprised of single cells (data not shown).
Mutation of cioAB Leads to Multiple Antibiotic
Sensitivity--
Given the pleiotropic effects of cioAB
mutation on P. aeruginosa, we looked at its effect on a
critical biological property of this bacterium; its efflux
pump-mediated multiple antibiotic resistance (25). We found that the
cio mutant and cioAB plasmid-containing strains
had a multiple antibiotic-sensitive phenotype, which was temperature-dependent (Table
II). Strains mutated in cioAB
or with cioAB on multicopy plasmids were more sensitive to a
range of antibiotics including chloramphenicol,
Protonmotive force-dependent multidrug efflux
pumps, many with unusually broad specificity, play a major role in
intrinsic antibiotic resistance (25). Four have been described in
P. aeruginosa, of which one, the MexAB-OprM efflux system,
which pumps out cio Mutant and cioAB Plasmid-containing Strains Are Oxidatively
Stressed--
We investigated further the possibility that increased
levels of reactive oxygen species might be a common cause of the
diverse phenotypes observed. Consistent with this was the finding that the cio mutant and plasmid pLC8-containing strains were more
sensitive to H2O2 than the wild-type (Fig.
4). Furthermore, we found these strains
contained higher levels of oxidatively modified proteins (Fig.
5). Protein carbonyls can be used as a
general measure of oxidative damage to proteins (30). At 37 and
42 °C protein carbonyl levels were higher in the cio
mutant and in the strains PAO7701/pLC8 and PAO6049/pLC8 than in the
wild-type, and this was most apparent at 42 °C (Fig. 5B).
These data suggest that in strains mutated in cioAB or
carrying plasmid-borne copies of the cioAB genes there is
increased oxidative damage to proteins, particularly at higher temperatures.
cio Mutant and cioAB Plasmid-containing Strains Have Reduced
Catalase Activity--
Oxidative stress levels in a cell can increase
as a consequence of increased rates of production or decreased rates of
decomposition of reactive oxygen species. Therefore, we looked at
hydrogen peroxide production rates and steady state concentrations in
cells, as well as catalase activity. The rates of
H2O2 production by cytoplasmic membrane
fractions were similar in wild-type, mutant, and cioAB plasmid-containing strains (data not show). However, there were small
differences in the steady state levels of H2O2
in cultures of these strains, but these changes were not significant at
a p < 0.005 (data not shown). However, there was a
statistically significant lower catalase activity in the mutant and
cioAB plasmid-containing strains at all three growth
temperatures (Fig. 6A).
Furthermore, localization of catalase activity in stained native gels
indicated that the catalase expression pattern was
temperature-dependent and different among wild-type,
mutant, and plasmid-containing strains (Fig. 6B). Two bands
were visible for the wild-type growing at 37 and 42 °C (Fig.
6B, lanes 6 and 10), but only one band
was visible for the cioAB mutant and the complemented mutant
and wild-type/pLC8. In the wild-type the faster migrating band was
temperature-induced, as it was absent from the wild-type grown at
30 °C, was only very faint, but reproducibly so, at 37 °C, and
was most evident in 42 °C cultures (Fig. 6B). As these
catalase bands were not paraquat-inducible neither can be assigned as
the P. aeruginosa KatB (31) (data not shown). Therefore, the
catalase bands observed must result from the major catalase in P. aeruginosa, KatA (32), probably the slower migrating band that is
present at all temperatures, and KatC. KatC is a third catalase
predicted to be encoded by the P. aeruginosa genome (4). We
suggest it might be KatC whose levels are affected in the CIO-defective
strains, as the temperature-inducible catalase is barely detectable at
37 °C in the wild-type, a temperature at which KatA is expressed at
significant levels (32).
The important finding of this study is that mutation of the
cyanide-insensitive terminal oxidase of P. aeruginosa leads
to a remarkable range of phenotypes. These include temperature
sensitivity for growth, a phenotype that has been shown previously to
be associated with mutation of genes required for the production of a
functional cytochrome bd oxidase in E. coli
(12-16, 19). However, the phenotypes elicited by mutation of
the CIO in P. aeruginosa are significantly more broad
ranging. These include defects in two critical biological properties
of P. aeruginosa, its cell division cycle and multiple antibiotic resistance, and are brought about by the presence of multiple, plasmid-borne copies of the cioAB structural
genes, as well as mutation of the oxidase.
The cloned cioAB genes on the plasmids pLC2 and pLC8
complement the cioAB mutant for its defect in CIO activity
but also lead to overexpression of oxidase activity compared with the
wild-type (5, 6) (Table I). Although there is clear evidence that the
cioAB genes form an operon (5), the plasmids pLC2 and pLC8 did not complement the Ts, cell division, or multiple
antibiotic-sensitive phenotypes. Introduction of these plasmids
into the wild-type led to similar and often more severe
phenotypic changes than in the cio mutant, which indicates
that the phenotypes did not result from a polar effect of the
cio mutation but rather depended on the presence of
plasmid-borne copies of the cioAB. In contrast, strains
containing the plasmid pLC1 showed modest (6) or insignificant changes
in CIO activity compared with the wild-type (Table I) and were only
slightly affected in growth at 42 °C and showed little of no
filamentation (Fig. 2). Therefore, it is possible that the phenotypes
result in part from the increased exponential phase CIO activity
present in strains containing the plasmids pLC2 and pLC8 (Table I).
The temperature-dependent formation of non-septate,
multinucleated filaments was indicative of a defect in cell division
following chromosome replication and segregation but before septum
formation (33). This may have resulted from the targeting by an unknown mechanism (see below) of a key component of the apparatus associated with septum formation (33). The cell division defect also explains why
CIO-defective strains of P. aeruginosa have difficulty
exiting stationary phase as exponential growth only commences once all of the filaments have broken down into single cells. A cell division defect may also explain the stationary phase exit phenotype of cytochrome bd mutants of E. coli (14).
P. aeruginosa is renowned for its intrinsically high levels
of resistance to a wide range of antibiotics (25). A combination of the
relatively low permeability of its cell envelope and the presence of
multidrug efflux pumps, with a broad specificity for different classes
of antimicrobial agents, explains this resistance (25). The
cio mutant and cioAB plasmid-containing strains
were markedly more sensitive to a range of antibiotics, a pattern
consistent with a loss of activity of the constitutively expressed
MexAB-OprM efflux pump of P. aeruginosa (21, 27, 34). The
loss of uncoupler-dependent chloramphenicol uptake in
CIO-defective strains as the growth temperature was increased was
consistent with the impairment of or loss of efflux pump function. What
is uncertain is whether this effect reflected a change in efflux pump
expression, stability, or damage to pre-existing pumps components.
Three further efflux pumps have been characterized in P. aeruginosa. These include MexCD-OprJ, MexEF-OprN, whose expression
is de-repressed in nfxB and nfxC mutant
backgrounds, respectively (35), and MexXY-OprM, which is induced in
response to the presence of certain antibiotics (36). It will be
interesting to determine whether CIO defects also lead to multiple
antibiotic sensitivity in nfxB and nfxC strains.
So how do cio mutation and the presence of multiple copies
of cioAB in trans on multicopy plasmids both lead
to similar phenotypes, and is the same mechanism involved in each case?
Overexpression of cioAB leads to a new spectral signal
appearing in cytoplasmic membrane preparations, which may result from
abhorrent heme incorporation (5). This may result in the presence of
non-functional forms of the oxidase in the membrane that disrupt
electron transfer in vivo. Alternatively, an inappropriate
complement of terminal oxidases, whether this be through the absence of
an oxidase through mutation or through the presence of non-ideal
levels, may interfere with optimal respiratory performance. One of the
issues that is clear from this study is the need for P. aeruginosa, and perhaps other bacteria with similar oxidases, to
carefully control the levels of the CIO, as inappropriate levels of
expression can have significant consequences for the cell. Evidence
from complementation studies suggests that the mini-D171 insertion used
to construct PAO7701 is in
cioB.4 Therefore,
a further possibility is that the phenotypes (and perhaps those
of E. coli cytochrome bd mutants) might
arise from truncated subunits or imbalances in the amounts (or
function) of CioA or CioB.
A key question is how does a defect in the CIO lead to such a range of
phenotypes, and do the CIO phenotypes result from a common mechanism or
from multiple causes? As has been found for E. coli
cytochrome bd mutants (14, 11, 19) the Ts phenotypes of
mutated and cioAB plasmid-containing strains of P. aeruginosa can be suppressed by reducing agents and catalase.
Furthermore, the antibiotic sensitivity of P. aeruginosa can
also be partially restored in the presence of catalase. The increased
sensitivity of the cio mutant and
cioAB-containing plasmids to H2O2
also supported the idea that these strains are oxidatively stressed.
Although we could not show statistically significant differences in the steady state H2O2 levels between strains, the
protein carbonyl levels were higher at each of the three temperatures
tested in the CIO-defective strains, indicating increased oxidative
protein damage (30). So oxidative modification to key proteins with their likely loss of function may explain some of the phenotypes observed. The absence of a specific catalase isoenzyme and subsequent less efficient removal of H2O2 is consistent
with the decreased H2O2 resistance of
CIO-defective strains and the increased oxidative protein damage
observed. The temperature dependence of this catalase activity also may
explain the Ts nature of the phenotypes. However, two outstanding
questions are the identity of this catalase and how its production is
affected in CIO mutants. Although oxidative damage to proteins may be
an important factor it is likely to be a secondary consequence of the
loss of catalase activity. An understanding of the mechanism behind the
failure of CIO-defective strains to produce a functional catalase will
provide a more complete and all-encompassing explanation of the
molecular basis of the phenotypic properties of CIO-defective strains
of P. aeruginosa.
-lactams, quinolones,
aminoglycosides, and macrolides. The effect of cio mutation
on
p-dependent accumulation of chloramphenicol suggested
that antibiotic sensitivity resulted from loss of or damage to a
multidrug efflux pump. The ability of reducing agents and catalase to
suppress the temperature-sensitive phenotype and of catalase to
partially suppress antibiotic sensitivity suggested that increased
levels of reactive oxygen species might be the cause of the observed
phenotypes. Consistent with this was the increased sensitivity of
strains to H2O2 and their increased protein carbonyl content, an indicator of oxidative protein modification. The
temperature-dependent synthesis of a specific catalase was absent in the cio mutant and in strains carrying multiple
plasmid-borne copies of cioAB. We propose that reduced
catalase levels result in oxidative modification and consequent loss of
function of proteins involved in a range of cellular functions.
How mutation or overexpression of the cyanide-insensitive
terminal oxidase leads to a loss of catalase activity is unknown at present.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
p-dependent efflux pump.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1)
NaH2PO4·H2O (2.6),
Na2HPO4 (11.5), and NaCl (8.5).
1 in medium containing the test antibiotic (1 ml
of medium in a 10-ml tube). Tubes were incubated overnight at 30 or
37 °C with shaking. The MIC corresponded to the minimum
concentration of drug that could prevent the growth of cells, as
determined by visible inspection. As the cio mutant and
strains carrying pLC2 or pLC8 grown at 42 °C have difficulty exiting
stationary phase a modified procedure was used to determine MICs at
this temperature. Cultures were initially grown to stationary phase at
37 °C and then diluted into LB to a cell concentration of 2.5 × 105 cell ml
1 containing appropriate
concentrations of the test antibiotic and then incubated at 42 °C.
The MICs were read after 24 h of incubation.
1. To assay drug accumulation at 42 °C, a culture
was first grown to stationary phase at 37 °C, subcultured, and grown
to mid-exponential phase at 42 °C. Chloramphenicol uptake
experiments were performed on these cells using
[3H]chloramphenicol as described by Li et al.
(21), except that a different mixture of silicone oils (a 4:6 (v/v)
mixture of Dow Corning silicone oils 510 and 550) was used to separate
cells from the supernatant fraction prior to determination of
radioactivity in the cell fraction by liquid scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mutation and overexpression of
cioAB leads to temperature-sensitive growth of
P. aeruginosa. Cultures were grown into early
stationary phase in LB at 37 °C, diluted into fresh medium, and
incubated at 42 °C. , PAO6049;
, PAO6049/pLC1;
,
PAO6049/pLC8;
, PAO7701; *, PAO7701/pLC1;
, PAO7701/pLC8.
CIO activity of Pseudomonas aeruginosa strains
-mercaptoethanesulfonic acid, and dithiothreitol and
catalase suppressed the Ts phenotype. This was shown by dense halos of
growth on agar plates around filter disks containing these compounds
for the cio mutant and complemented mutant, when grown at
42 °C (data not shown).
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Fig. 2.
Fluorescence photomicrographs of
4',6-diamidino-2-phenylindole-stained P. aeruginosa strains. Cultures were grown at
30 °C (A and F), 37 °C (B and
G), and 42 °C (C-E and
H-J) and stained as described under
"Materials and Methods." Wild-type strain PAO6049
(A-C), PAO6049/pLC1 (D), PAO6049/pLC8
(E), PAO7701 (F-H), PAO7701/pLC1
(I), and PAO7701/pLC8 (J) are shown.
-lactams,
quinolones, and macrolides at all growth temperatures. At 30 °C
(data not shown) and 37 °C (Table II) there was a 2-8-fold decrease
in the MIC for a range of different antibiotics. However, a much
greater decrease in MIC for the same antibiotics was found in cultures grown at 42 °C. Antibiotic sensitivity was also found in
wild-type/pLC8 but not in strains containing the control plasmid pUCP18
(data not shown). The antibiotic sensitivity of the strains carrying pLC2/pLC8 was greater than of the cio mutant strain. The
addition of exogenous catalase (~500 units) resulted in a 5-10-fold
increase in the MIC of the cio mutant and PAO7701/pLC2
strains at 37 and 42 °C but no change in that of the wild-type
(Table III). This suggests that damage
caused by reactive oxygen species is a contributory factor to the
multiple antibiotic-sensitive phenotype.
Susceptibility of P. aeruginosa to antimicrobial agents
Chloramphenicol sensitivity is corrected by addition of catalase
-lactams, tetracycline, chloramphenicol, and
quinolones, as well as trimethoprim and sulfamethoxazole, is the major
contributor to the high intrinsic antibiotic resistance of P. aeruginosa (21, 26-29). The multiple antibiotic-sensitive
phenotypes observed could result from damage to a multidrug efflux
pump. Therefore, we determined the
p-dependent accumulation of chloramphenicol in various strains as a function of
temperature (Fig. 3). There was a
progressive impairment of [3H]chloramphenicol
accumulation in the PAO7701 and PAO7701/pLC2 strains, as the growth
temperature was increased (Fig. 3), which parallels the
temperature-dependent changes in multiple antibiotic sensitivity demonstrated (Table II). PAO7701 and PAO7701/pLC2 grown at
42 °C immediately accumulated [3H]chloramphenicol to
levels only observed in the wild-type following treatment with the
protonophore carbonyl cyanide
m-chlorophenylhydrazone. This is consistent with a
complete loss of
p-dependent chloramphenicol efflux in
both the cio mutant and strains containing cioAB
on a multicopy plasmid at this temperature.
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Fig. 3.
Accumulation of
[3H]chloramphenicol by CIO-defective strains.
Cultures were grown at 30 °C (A), 37 °C
(B), and 42 °C (C) to mid-exponential phase,
harvested, and resuspended in phosphate
buffer/MgSO4/glucose. [3H]Chloramphenicol
accumulation was determined as described under "Materials and
Methods." Dashed and solid lines indicate
samples treated and untreated with carbonyl cyanide
m-chlorophenylhydrazone, respectively. , PAO6049;
,
PAO7701;
, PAO7701/pLC2.
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Fig. 4.
Sensitivity of P. aeruginosa strains to
H2O2. Strains were grown in LB medium to
mid-exponential phase (A600 = 0.6) at 37 °C.
Cells were challenged with 10 mM
H2O2 as described under "Materials and
Methods," and samples were taken periodically, diluted, and plated
onto LB medium to determine cell viability. , PAO6049;
,
PAO6049/pLC1;
, PAO6049/pLC8;
, PAO7701;
, PAO7701/pLC8.
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Fig. 5.
Protein carbonyl levels in P. aeruginosa strains grown at different temperatures.
Strains were grown in LB medium to early stationary phase at 37 °C
(A) or 42 °C (B), and protein carbonyl levels
were determined as described under "Materials and Methods."
Slot 1, wild-type PAO6049; slot 2, PAO6049/pLC8;
slot 3, PAO7701; slot 4, PAO7701/pLC8. 7.5 µg
of protein was loaded per slot.
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Fig. 6.
Catalase levels in P. aeruginosa
strains. A, total catalase activity at different
growth temperatures. Open bars, PAO6049; hatched
bars, PAO7701; solid bars, PAO7701/pLC8. Units are nmol
of O2 evolved per min 1 mg
protein
1. Error bars are means ± S.E.
B, catalase activity gels of cultures grown at 30 °C
(lanes 1-3), 37 °C (lanes
4-6), and 42 °C (lanes
7-10). Lanes 3, 6, and
10, PAO6049; lanes 2, 5, and
8, PAO7701; lanes 1, 4, and
7, PAO7701/pLC8; and lane 9, PAO6049/pLC8.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-020-75945383;
Fax: 44-020-75842056; E-mail: h.d.williams@ic.ac.uk.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M210355200
1 M. Cooper, G. R. Tavankar, and H. D. Williams, unpublished data.
3 G. R. Tavankar, D. Mossialos, and H. D. Williams, unpublished data.
4 G. R. Tavankar and H. D. Williams, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CIO, cyanide-insensitive oxidase; Ts, temperature-sensitive; MIC, minimal inhibitory concentrations.
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REFERENCES |
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1. | Palleroni, N. J. (1984) in Bergey's Manual of Systematic Bacteriology (Krieg, N. R. , and Holt, J. G., eds) , pp. 141-219, Williams and Wilkins, Baltimore, MD |
2. | Zannoni, D. (1989) Biochim. Biophys. Acta 975, 299-316[Medline] [Order article via Infotrieve] |
3. | Davies, K. J., Lloyd, D., and Boddy, L. (1989) J. Gen. Microbiol. 135, 2445-2451[Medline] [Order article via Infotrieve] |
4. | Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J., and Lagrou, M. (2000) Nature 406, 959-964[CrossRef][Medline] [Order article via Infotrieve] |
5. | Cunningham, L., Pitt, M., and Williams, H. D. (1997) Mol. Microbiol. 24, 579-591[CrossRef][Medline] [Order article via Infotrieve] |
6. | Cunningham, L., and Williams, H. D. (1995) J. Bacteriol. 177, 432-438[Abstract] |
7. | Junemann, S. (1997) Biochim. Biophys. Acta 1321, 107-127[Medline] [Order article via Infotrieve] |
8. | Garcia-Horsman, J. A., Barquera, B., Rumbley, J., Ma, J., and Gennis, R. B. (1994) J. Bacteriol. 176, 5587-5600[Medline] [Order article via Infotrieve] |
9. | Blumer, C., and Haas, D. (2000) Arch. Microbiol. 173, 170-177[CrossRef][Medline] [Order article via Infotrieve] |
10. | Goldfarb, W. B., and Margraf, H. (1967) Ann. Surg. 165, 104-110[Medline] [Order article via Infotrieve] |
11. |
Gallagher, L. A.,
and Manoil, C.
(2001)
J. Bacteriol.
183,
6207-6214 |
12. | Delaney, J. M., Wall, D., and Georgopoulos, C. (1993) J. Bacteriol. 175, 166-175[Abstract] |
13. | Poole, R. K., and Cook, G. M. (2000) Adv. Microbiol. Physiol. 43, 165-224[Medline] [Order article via Infotrieve] |
14. | Siegele, D. A., and Kolter, R. (1993) Genes Dev. 7, 2629-2640[Abstract] |
15. | Siegele, D. A., Imlay, K. R., and Imlay, J. A. (1996) J. Bacteriol. 178, 6091-6096[Abstract] |
16. | Wall, D., Delaney, J. M., Fayet, O., Lipinska, B., Yamamoto, T., and Georgopoulos, C. (1992) J. Bacteriol. 174, 6554-6562[Abstract] |
17. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
18. | Markwell, M. A., Haas, S. M., Bieber, L. L., and Tolbert, N. E. (1978) Anal. Biochem. 87, 206-210[Medline] [Order article via Infotrieve] |
19. | Goldman, B. S., Gabbert, K. K., and Kranz, R. G. (1996) J. Bacteriol. 178, 6348-6351[Abstract] |
20. | Hiraga, S., Niki, H., Ogura, T., Ichinose, C., Mori, H., Ezaki, B., and Jaffe, A. (1989) J. Bacteriol. 171, 1496-1505[Medline] [Order article via Infotrieve] |
21. | Li, X. Z., Nikaido, H., and Poole, K. (1995) Antimicrob. Agents Chemother. 39, 1948-1953[Abstract] |
22. |
Soballe, B.,
and Poole, R. K.
(2000)
Microbiology
146,
787-796 |
23. | Rorth, M., and Jensen, P. K. (1967) Biochim. Biophys. Acta 139, 171-173[Medline] [Order article via Infotrieve] |
24. | Wayne, L. G., and Diaz, G. A. (1986) Anal. Biochem. 157, 89-92[Medline] [Order article via Infotrieve] |
25. |
Nikaido, H.
(1996)
J. Bacteriol.
178,
5853-5859 |
26. | Kohler, T., Michea-Hamzehpour, M., Henze, U., Gotoh, N., Curty, L. K., and Pechere, J. C. (1997) Mol. Microbiol. 23, 345-354[Medline] [Order article via Infotrieve] |
27. | Li, X. Z., Livermore, D. M., and Nikaido, H. (1994) Antimicrob. Agents Chemother. 38, 1732-1741[Abstract] |
28. |
Mine, T.,
Morita, Y.,
Kataoka, A.,
Mizushima, T.,
and Tsuchiya, T.
(1999)
Antimicrob. Agents Chemother.
43,
415-417 |
29. | Poole, K., Gotoh, N., Tsujimoto, H., Zhao, Q., Wada, A., Yamasaki, T., Neshat, S., Yamagishi, J., Li, X. Z., et al.. (1996) Mol. Microbiol. 21, 713-724[Medline] [Order article via Infotrieve] |
30. | Levine, R. L., and Stadtman, E. R. (2001) Exp. Gerontol. 36, 1495-1502[CrossRef][Medline] [Order article via Infotrieve] |
31. | Brown, S. M., Howell, M. L., Vasil, M. L., Anderson, A. J., and Hassett, D. J. (1995) J. Bacteriol. 177, 6536-6544[Abstract] |
32. |
Hassett, D. J.,
Alsabbagh, E.,
Parvatiyar, K.,
Howell, M. L.,
Wilmott, R. W.,
and Ochsner, U. A.
(2000)
J. Bacteriol.
182,
4557-4563 |
33. | Rothfield, L., Justice, S., and Garcia-Lara, J. (1999) Annu. Rev. Genet. 33, 423-448[CrossRef][Medline] [Order article via Infotrieve] |
34. | Poole, K., Krebes, K., McNally, C., and Neshat, S. (1993) J. Bacteriol. 175, 7363-7372[Abstract] |
35. |
Masuda, N.,
Sakagawa, E.,
Ohya, S.,
Gotoh, N.,
Tsujimoto, H.,
and Nishino, T.
(2000)
Antimicrob. Agents Chemother.
44,
2242-2246 |
36. | Masuda, N., Sakagawa, E., and Ohya, S. (1995) Antimicrob. Agents Chemother. 39, 645-649[Abstract] |
37. | Darzins, A., and Cassadaban, M. J. (1989) J. Bacteriol. 171, 3917-3925[Medline] [Order article via Infotrieve] |