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INTRODUCTION |
Pseudomonas aeruginosa is
ubiquitous, being found in diverse environments such as soil,
freshwater, and marine environments. It is also an opportunistic
pathogen of the airways of cystic fibrosis patients and in
immunocompromised hosts including cancer, AIDS, and burn patients
(16). Similar to other pathogens and gram-negative
bacteria, P. aeruginosa has a global regulatory system known
as quorum sensing (QS) that controls expression of numerous genes, many
of which are associated with virulence (11, 37). Bacterial
QS, or cell-to-cell communication, is a process in gram-negative and
some gram-positive bacteria where low-molecular-weight diffusible
molecules synthesized by one cell trigger gene activation in other
cells (17). In gram-negative bacteria, the signaling molecules are either homoserine lactone/acyl side chain based (HSLs;
autoinducers), diketopiperazine (29), or via
2-heptyl-3-hydroxy-4-quinolone (45), while gram-positive
bacteria use small peptides. Because of its aforementioned ubiquity in
nature and its importance in disease, P. aeruginosa is a
model organism for QS study.
QS is viewed as a cell density-dependent phenomenon that allows
bacteria to communicate, sense population density, and ultimately coordinate transcription of many genes. Bacteria monitor their population by sensing the level of autoinducer signal molecules (17). HSL-based QS in P. aeruginosa is a
multitiered process governed by two gene tandems, lasR-lasI
and rhlR-rhlI (41-43). The las
system is composed of LasR, a transcriptional regulator protein, and
LasI, an autoinducer synthase that produces one of the three known
Pseudomonas HSLs, PAI-1
[N-(3-oxododecanoyl)-L-homoserine lactone].
The second tier consists of RhlR, which is also a transcriptional regulator, and RhlI, an autoinducer synthase that catalyzes the synthesis of a second HSL, PAI-2
(N-butyryl-L-homoserine lactone). PAI-1
interacts with the regulator LasR to activate transcription of target
genes (42). The LasR-PAI-1 complex will activate the transcription of lasI and several genes important in defense
against oxidative stress such as those coding for the major catalase, KatA, and the manganese-cofactored superoxide dismutase (Mn-SOD) (27). Further, recent work by Whiteley et al. has
identified many other QS-regulated genes that were previously
unrecognized (52).
Whether considered in either disease or environmental settings, an
important aspect of P. aeruginosa ecology is its propensity to form biofilms. P. aeruginosa biofilms have high cell
densities and an architecture that typically consists of highly ordered mushroom- and pillar-like structures (7). This important
aspect of P. aeruginosa biology has also been shown to be
influenced by QS (8). QS-deficient mutants form a thin,
tightly packed biofilm, differing markedly from wild-type biofilm
architecture, suggesting that particular aspects of biofilm cell
physiology are under control of QS and are important for normal biofilm
formation. The physiology of bacterial biofilms is viewed to be
different from that of planktonic cultures (7), but the
true extent of such potential differences is still poorly understood.
We are examining P. aeruginosa biofilm responses to
environmental stimuli as a means of studying gene expression and
physiology of biofilm bacteria. We have elected to focus on the
oxidative stress response because our knowledge of the antioxidant
responses in this organism is firmly grounded genetically and
physiologically (20-24) and because antioxidant enzymes
are of central importance to the pathogenicity of this organism
(26). Further, we have recently found that key components
of the oxidative stress response are regulated by QS (27).
Curiously, earlier studies had also implicated iron availability as a
significant controlling factor in expression levels of antioxidant
enzymes in P. aeruginosa (20-27). As QS has
thus far been found to exert its effects when cell densities are high,
a condition which is found in biofilms and which can lead to localized
areas of high nutrient demand, we have hypothesized that nutrient
limitation may also be an important factor to consider in studies aimed
at understanding QS and biofilm biology (28). The
availability of well-defined QS mutants offers an excellent opportunity
to examine and compare gene expression in biofilms and planktonic cells
under conditions where the availability of a specific nutrient can be
conveniently and reliably manipulated.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
P.
aeruginosa wild-type strain PAO1 (30), the
lasI::Tn10 mutant PAO-JP1
(44), and the lasR mutant PAO-R1
(42) were used in this study. Plasmids pDJH201 (contains
sodA::lacZ [27]) and pPCS223 (contains lasI::lacZ
[51]) were used in reporter gene experiments. These
plasmids were introduced into the different strains by electroporation
and maintained with carbenicillin (300 mg · liter
1). In each case, plasmid transformation was
verified by restriction enzyme analysis of plasmid preparations
(47) of the different transformants. In some experiments,
iron bioavailability in the medium was manipulated by the addition of
iron (25 µM FeCl3) or the iron-specific chelator
2,2-dipyridyl (500 µM for Trypticase soy broth [TSB] or 50 µM for
1/10 TSB).
Planktonic cultures were grown in TSB at 37°C in a rotary shaker at
300 rpm. Culture volumes did not exceed 10% of the flask volume to
ensure maximum aeration. Biofilms were cultured using a drip flow
reactor system previously described by Huang et al. (31)
and included 316L stainless steel slides (1.3 by 7.6 cm) as the
substratum. Briefly, 10 ml of 1/10 TSB was added to each chamber (four
chambers per reactor), followed by inoculation with 1 ml of
stationary-phase culture of the test strain grown in TSB. The reactor
was then incubated horizontally at 37°C for 24 h to allow
bacterial attachment to the substratum. Following the attachment period, the reactor was inclined 10° and a constant drip of 1/10 TSB
was allowed to flow over the slides at a rate of 50 ml · h1. Biofilms were cultured in a 37°C incubator. To
achieve this, the sterile 1/10 TSB contained in the external carboy was
preequilibrated to 37°C prior to entry into the drip flow reactor. To
accomplish this, the medium was first preheated to 42°C by pumping
through masterflex silicone tubing coiled in a 42°C water bath fixed
atop the incubator chamber. The feed tubing leaving the 42°C water bath was foam insulated (to reduce thermal loss) and channeled through
the heat vent hole of the culture incubator. A mercury thermometer was
attached to the medium flow tubing via aluminum tape, providing for
isothermic association with the tubing and verifying that the
temperature of medium was 37°C as it entered the incubator chamber. A
final temperature equilibration step designed to guarantee appropriate
temperature involved additional coiling (3-m flow length) of the feed
tubing in distilled water (2-liter beaker) that was equilibrated at
37°C within the chamber. This ensured a final medium temperature of
37°C prior to entry into the drip flow reactor. Silicone tubing
exiting each reactor chamber was used to pump the waste out of the
chamber and into external waste carboys.
Cell extract preparation, nondenaturing gel electrophoresis, and
enzyme and reporter activity.
Nondenaturing gel electrophoresis
methods were as described by Hassett et al. (26). Briefly,
cell extracts were prepared from cultures harvested by centrifugation
at 10,000 × g for 10 min at 4°C. Bacteria were
washed twice in ice-cold 50 mM potassium phosphate buffer (pH 7.0) and
sonicated in an ice water bath for 30 s. The sonicate was
clarified by centrifugation at 13,000 × g for 10 min
at 4°C. Extract protein content was estimated by the Bradford assay
(2). SOD activity stains in nondenaturing gels were used
to semiquantatively follow induction of Mn-SOD and to assess its
activity separately from the constitutively expressed Fe-SOD (coded by
sodB) (26). Gel images were computer scanned
and stored as Powerpoint image files. LacZ reporter enzyme expression
was measured as 
-galactosidase activity as previously described by
Miller (38).
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RESULTS |
Mn-SOD regulation.
Initial experiments compared Mn-SOD levels
in the P. aeruginosa wild-type strain PAO1 and the
lasI mutant PAO-JP1. In PAO1, Mn-SOD activity was detectable
in stationary-phase cells (t = 12 h) (Fig.
1A), which correlates with the production
and accumulation of the QS autoinducer molecule PAI-1
(27). Mn-SOD was absent from stationary-phase TSB cultures
of the lasI mutant JP1, which cannot synthesize PAI-1
(44), and the lasR mutant PAO-R1, which lacks
the regulatory protein essential to QS (12, 13) (results not shown). All of these observation were consistent with our previous
findings, which showed that Mn-SOD production is controlled by QS in
this organism (27).
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FIG. 1.
SOD activity in planktonic cells cultured in TSB. Cell
extracts (50 µg of protein per lane) of each strain were prepared at
different time points corresponding to different growth stages, 6 h (mid-log phase) and 10 h (early stationary phase). SOD activity
stains of wild-type strain PAO1 cultured in TSB (A), PAO1 in TSB
amended with the iron-specific chelator 2,2-dipyridyl (B), and the
lasI mutant PAO-JP1 in TSB amended with the iron-specific
chelator 2,2-dipyridyl (C) were prepared as described in Materials and
Methods. Results are of one of three independent experiments
demonstrating this response.
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Assessment of iron effects on QS-controlled gene expression was
initiated in experiments where either TSB was supplemented with 25 µM
FeCl3 or 500 µM 2,2-dipyridyl was added to render the iron unavailable. In wild-type cells cultured to stationary phase in
iron-supplemented TSB, no Mn-SOD activity was observed in native gels
(results not shown). When 2,2-dipyridyl was included in the culture
medium, Mn-SOD was evident in mid-log-phase and stationary-phase PAO1
cells (Fig. 1B), but only in stationary phase in the lasI mutant PAO-JP1 (Fig. 1C) and the lasR mutant PAO-R1 (results
not shown). The iron-sensitive Mn-SOD activity in PAO1 is consistent with prior work demonstrating iron-dependent sodA expression
(23-25) and demonstrated that either excess or limiting
iron can override QS control of sodA expression in P. aeruginosa.
To establish whether the iron effect on Mn-SOD levels occurs at the
transcriptional level, a plasmid containing a
sodA::lacZ transcriptional fusion was
transformed into PAO1, PAO-JP1, and PAO-R1, and the above experiments
were repeated. As shown in Fig. 2, iron
limitation increased sodA expression in PAO1 mid-log-phase cells. Iron limitation also caused equivalent levels of sodA
up-regulation in mid-log-phase cells of PAO-JP1 (Fig. 2), even though
the isozyme was not apparent in activity stains of JP1 cell extracts
(Fig. 1C). Reporter gene activity was also increased in iron-starved mid-log-phase cells of PAO-R1, but the increase was smaller than in
strain PAO1 or PAO-JP1 (Fig. 2). In stationary-phase cells, up-regulation of sodA in response to iron starvation also
occurred. -Galactosidase reporter enzyme levels measured in PAO1,
PAO-JP1, and PAO-R1 were approximately 4-, 11-, and 7-fold greater,
respectively, under iron-limiting growth conditions compared to cells
with ample iron. The reporter gene results with both mid-log and
stationary-phase cells again demonstrated that QS control of
sodA could be overridden by the iron starvation response but
also showed that the autoinducer PAI-1 and the regulatory protein LasR
were still required for maximal sodA expression, regardless
of iron availability. Further, these studies suggested that Mn-SOD
activity in the lasI mutant was attenuated by abnormal
posttranscriptional activity because iron starvation-based induction of
sodA in mid-log-phase cells did not result in increased
Mn-SOD levels in native gel SOD activity stains.
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FIG. 2.
Transcription of sodA in mid-log-phase and
stationary-phase cells of PAO1, PAO-JP1, and PAO-R1 as affected by iron
limitation. Relative transcription levels were determined by measuring
the reporter enzyme -galactosidase as described by Miller
(38) in planktonic cells harvested at mid-log (6 h) and
late stationary (16 h) phases. Open bars, cells grown in TSB; filled
bars, cells grown in TSB containing the iron-specific chelator
2,2-dipyridyl. The data represent the mean of three independent
experiments (two to three cultures per experiment). Where visible,
error bars denote 1 standard error.
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Effects of iron concentration on lasI expression.
Additional experiments were conducted to determine if the iron
starvation effect on sodA expression could be linked to
las gene expression. Over three independent experiments,
iron limitation increased lasI expression in the wild-type
strain by approximately 30 to 35% (Fig.
3). The increase, while relatively small,
was highly reproducible and statistically significant. However, no increase in lasI::lacZ reporter
activity (range, 90 to 150 Miller units) could be measured under the
same culture conditions for the lasI mutant and the
lasR mutant (results not shown), implying again that the
iron-based response appeared to require LasR and PAI-1 for the maximal
effect to be observed. The lack of an iron effect on lasI
induction in the LasI mutant also demonstrated that
potential alternative autoinducers were most likely not participating
in the iron stress response under the conditions used for cell culture
in these experiments.
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FIG. 3.
Expression of lasI in response to iron
deprivation. Wild-type strain PAO1 was transformed with pPCS223, which
contains the lasI::lacZ reporter fusion
(50), and then cultured to mid-log or stationary phase in
TSB (open bars) or TSB amended with the iron-specific chelator
2,2-dipyridyl (filled bars). The data represent the average of the
means of three independent experiments (two cultures per experiment).
Error bars, where visible, represent 1 standard error of the three
experimental means. Differences between iron treatments for each
culture stage are statistically significantly different (P = 0.01).
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Production of Mn-SOD in biofilms.
An additional important
motivation for this study was to investigate potential differences in
gene expression and regulation between biofilm cells and planktonic
cells. The combination of mutants and the use of Mn-SOD as a native
reporter enzyme provided an opportunity to assess the same regulatory
issues when the cells were grown as biofilms and to determine if
protein expression patterns were different. Surprisingly different
Mn-SOD expression patterns were encountered between wild-type and QS
mutant biofilms. Mn-SOD was observed only in mature (6-day) PAO1
biofilms (Fig. 4A), whereas it was
expressed within the first 24 h of lasI mutant biofilm
formation (Fig. 4B). In identical biofilm experiments with the
lasR mutant, Mn-SOD levels were the same as observed with
the lasI mutant (results not shown). Additional biofilm
experiments were then conducted to assess whether biofilm cells would
respond to iron manipulation as was observed with planktonic cultures. When 2,2-dipyridyl was included in the biofilm flow medium, Mn-SOD was
evident in PAO1 biofilms; Mn-SOD activity stains obtained from 24-h
biofilm cell extracts appeared weak, but by day 2, they were roughly
equivalent to the lasI and lasR mutant biofilms
(Fig. 5) in the other experiments. Given
the apparent constitutive sodA expression in lasI
and lasR biofilms, further biofilm experiments sought to
establish whether sodA expression in JP1 and PAO-R1 biofilms
was still sensitive to environmental iron concentrations. This was
confirmed by supplementing the biofilm medium with iron (25 µM
FeCl3). When iron was provided at such ample levels, Mn-SOD was absent in biofilms of wild-type and both mutant strains for the
duration of the experiments (up to 6 days [results not shown]). During the course of all biofilm experiments, no apparent changes in
the Fe-SOD activity band occurred (results not shown).
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FIG. 4.
SOD activity stains of cell extracts (50 µg of protein
per lane) of the wild-type strain PAO1 (A) and the lasI
mutant PAO-JP1 (B) obtained from biofilms cultured with 1/10 TSB for up
to 6 days (6d). The data are representative of duplicate experiments,
and locations of the Mn-SOD and Fe-SOD bands are as shown.
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FIG. 5.
Mn-SOD expression in PAO1 biofilms as affected by iron
starvation. SOD activity was detected using activity stains as
described in Materials and Methods. Cell extracts (50 µg per lane)
were obtained from PAO1 biofilms grown for 1 or 2 days in 1/10 TSB
containing the iron-specific chelator 2,2-dipyridyl and from 1- and
2-day PAO-JP1 biofilms grown in 1/10 TSB. The data are representative
of duplicate experiments, and locations of the Mn-SOD and Fe-SOD bands
are as shown.
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DISCUSSION |
Nutritional override of QS.
It is clear that HSLs are critical
for activation of the QS regulatory circuitry in P. aeruginosa. However, prior to the discovery of QS in this
organism, the production of several gene products now known to be QS
regulated was initially found to be up-regulated by nutritional
factors. An example of overlap between a nutrient limitation response
and QS-regulated activity in P. aeruginosa is pyocyanin
production. This exoproduct is typically observed in stationary-phase
cells and is controlled by QS (4) but can also be induced
by phosphate starvation (20). The effects of other
nutrients such as iron have been observed more frequently. Elastase
synthesis has been shown to be QS controlled (41, 53); however, its production occurs maximally when P. aeruginosa
is iron limited but is restricted in the presence of iron (1, 32,
49). Expression of sodA was also previously shown to
be iron regulated (23-25) and then later found to be
controlled by QS (27). Reporter experiments in the present
study established that sodA can be induced by iron
limitation in either log-phase or stationary-phase cells of the
wild-type strain (Fig. 1A, 1B, and 2) and QS mutants (Fig. 2). However,
relative to the wild-type strain, maximal iron stress-dependent
sodA induction was much lower in mid-log-phase
lasR cells and in stationary-phase cells of both
las mutants (Fig. 2). This implies that the autoinducer PAI-1 and the LasR regulatory protein were still required for maximum
iron-starved sodA induction under these experimental
conditions. This was particularly apparent in stationary-phase cells,
where QS is thought to be most active in gene regulation. It is perhaps important that even though relatively quite low,
sodA::lacZ expression in the
LasR and LasI mutants was still inducible
by iron starvation (Fig. 2). This suggests the possibility that
QS-targeted promoters such as that governing the
fagA-fumC-orfX-sodA operon can still be binding targets for
RNA polymerase in the absence of positive regulatory complexes (such as
LasR-PAI-1), if the repressor protein is also not present.
In addition, we bring attention to the observation that apparent
wild-type sodA transcription occurred in mid-log-phase cells of the LasI mutant (Fig. 2), but it did not translate
into mature Mn-SOD enzyme as measured by activity stains in native gels
(Fig. 1C). While activity stains in native gels are only
semiquantitative, cell extract sample loading in these experiments was
adequate to detect meaningful SOD enzyme levels, provided for
reasonable strain and growth condition comparisons, and allowed for
sensitive detection of this isozyme in a wild-type background. This
basic observation implies that posttranscriptional processing of the sodA mRNA was altered in the las mutants.
Further, it suggests that LasI, or the autoinducer it synthesizes
(PAI-1), is involved in some as yet unknown but essential cellular
activity at low cell densities.
The overall iron effect on lasI expression (Fig. 3) was
small relative to increases in lasI transcription previously
observed in stationary-phase cells (35), but we note that
the increase was highly reproducible and thus directly links the iron
stress response with QS circuitry. Integration of QS with nutrient
availability should not be unexpected, and it is perhaps no coincidence
that QS-based regulation is most often observed in stationary-phase cultures when cell densities approach levels that represent a significant nutrient sink and indeed growth rates are declining due to
limitation of some nutrient(s). Nutrients such as iron are subject to
biotic and abiotic redox reactions, typically yielding insoluble
precipitates under aerobic conditions. Thus, the bioavailability of
such nutrients can be sparingly low regardless of local biological demand. In biofilms where cell densities can approach 1010
to 1012 cells per cm3 (6), low
nutrient bioavailability coupled with high localized nutrient demand
could result in a nutrient stress response. Lazazzera (36)
has recently reviewed a similar concept for Bacillus
subtilis, and there are other reports that suggest QS and nutrient
sensing in gram-negative bacteria are integrated. Kjelleberg and
colleagues have connected carbon starvation, QS, and the stringent
response in a marine Vibrio isolate (14, 16, 39,
50). Other, similar interactions integrating nutrient
limitation, cell-to-cell communication, and the stringent response have
also been reported for Myxococcus xanthus (19, 33, 34,
48) and thus serve to further demonstrate the complexity of
cell-to-cell signaling systems. When possible strategies for
manipulating QS for controlling bacterial infections are considered,
the effects of multiple control mechanisms or nutritionally based
regulatory override systems must be recognized. The onset of
QS-regulated gene expression relative to the accumulation of
autoinducers or to other metabolites, and the timing of their accumulation relative to changes in the cell nutritional state, represents a critical area of research. To account for the observations made in this work, we examined the promoter region of the operon that
contains sodA (Fig. 6). Iron
boxes (Fur-Fe2+ binding sites [24]) and
putative LasR-PAI-1 binding sites (designated Lux boxes) were found
and are located such that under high iron conditions, the Fur repressor
protein would bind as a dimer to the iron boxes, inhibiting binding and
thereby reducing transcriptional activation by the LasR-PAI-1 complex.
The occurrence of two (or more) regulatory elements in the promoter
region of other QS-regulated genes has not been studied but represents
an important issue for understanding and manipulating QS in bacteria.
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FIG. 6.
A potential mechanism for dual control of
sodA by iron-sensitive and QS circuitry. (A) Gene
arrangement of the fagA-fumC-orfX-sodA operon. (B) DNA
sequence directly upstream of fagA, which is promoter
proximal in the depicted operon (24, 25). The Fur
regulatory protein (encoded by fur, which is itself
regulated by iron availability) utilizes Fe2+ as a
corepressor (23). The Fe(II)-Fur complex directly binds to
the promoter region of genes that contain a specific regulatory
sequence known as the iron box (9). Sequence analysis
suggests the presence of iron boxes (orientation of each shown as a
dashed line), which is the binding site for Fe(II)-Fur. Note that
Mn-SOD production is elevated in fur mutants
(25). Iron boxes are located at nucleotide positions -18 to -37 and -21 to -42. Positions of putative Lux boxes, the binding
site for the PAI-1-LasR complex, are also shown at -11 to -29 and at
-228 to -247. Nucleotide positions that are homologous with the
consensus Vibrio Lux box are indicated by the connecting
lines. Under high iron conditions, binding of the iron box by the
Fe(II)-Fur complex would inhibit the LasR-PAI-1 complex from binding
to the Lux box 2 region and would also inhibit transcription possibly
originating upstream due to potential activation from binding at Lux
box 1. Upon iron starvation, the Fe(II)-Fur complex would not be
present, and thus the LasR-PAI-1 complex would be free to activate
transcription. The bold thick line indicates a potential ribosome
binding site.
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Biofilm gene expression.
Another important issue addressed in
this study included P. aeruginosa biofilm cell physiology.
The use of defined regulatory mutants and an endogenous reporter enzyme
that had been shown to be controlled by both QS and nutritional effects
presented an opportunity to assess basic protein expression in biofilms and make relevant comparisons to planktonic cells. P. aeruginosa has been used as a model organism for studying biofilm
behavior and for the development of biofilm control strategies
(7), and though recent progress has been significant,
P. aeruginosa biofilm cell physiology is still only poorly
understood. Particularly lacking is information regarding gene
expression and regulation. Gradients in metabolic activity have been
shown to exist in P. aeruginosa biofilms, and some
information regarding adaptive gene regulation has also been recently
published (31). These studies examined P. aeruginosa gene regulation in response to environmental stress,
showing, for example, that induction of phoA in response to
phosphate limitation occurred only in the upper region (ca. 20%) of
the biofilm (31).
In the present study, differences in Mn-SOD levels between wild-type
and QS mutant biofilms were significant. Whereas Mn-SOD activity was
not detected in wild-type biofilms until after 6 days, it was
detectable within the first 24 h of biofilm formation by both
las mutants. The notable lack of Mn-SOD in wild-type
biofilms implies that the cells were not limited for iron and is
consistent with an analysis of iron availability under these growth
conditions. Chemical analysis of TSB showed an iron concentration of 15 µM. The iron content in the 1/10 TSB used for biofilm experiments would be proportionately lower, but the constant flow would
continuously deliver fresh medium over the developing biofilm. Over the
course of such experiments, the total iron made available to the
developing biofilm would exceed batch conditions at least eightfold,
while total biomass generated in the different growth modes would be similar (T. R. McDermott and D. J. Hassett, unpublished
data). The appearance of Mn-SOD in 6-day-old wild-type biofilms likely represents iron limitation resulting from the reduced iron in 1/10 TSB
being insufficient for the high cell numbers accumulated during this
time period (~5.0 × 1010 total on the stainless steel
slide) and/or potential diffusion limitations.
The constitutive presence of Mn-SOD in the las mutant
biofilms reveals a potentially important behavior difference between wild-type and las mutant cells and an as yet undefined (but
not necessarily unexpected) important characteristic of QS regulation. As implied by the Mn-SOD activity in the las mutant
biofilms, the absence of normal QS regulatory components resulted in
the apparent unrestricted expression of sodA, suggesting
that in biofilm cells QS regulation may exert negative gene control. An
alternative explanation is that developmentally immature biofilms have
an increased iron requirement that results in an iron stress response. However, adding the iron-specific chelator 2,2-dipyridyl to the medium
of wild-type biofilms resulted in the induction of Mn-SOD activity,
implying that such biofilms were not otherwise iron limited (also see
above). However, there are other potentially important issues to
consider in assessing the constitutive expression of Mn-SOD in
lasI mutant biofilms. There are alternative transcriptional start sites within fagA and fumC
(24) that could result in sodA expression and
represent biofilm-specific gene regulation, and it has been established
that synthesis of the P. aeruginosa iron chelator pyoverdine
is responsive to iron availability (46) and also under QS
control (51). In the biofilm experiments conducted with
the lasI mutant, lack of the iron chelator pyoverdine could conceivably result in significantly reduced iron acquisition by the
mutant and thus result in an iron starvation-like response. Regardless,
the constitutive presence of Mn-SOD in las mutant biofilms
suggests that a biofilm-specific regulatory system that is independent
of the Las system is present and can affect gene expression in the
absence of LasR or PAI-1. This may be an important consideration when
evaluating autoinducer analogs as an alternative chemotherapy for
biofilm-related infections.
Of additional importance, the experiments in this study demonstrated
that biofilm cells respond like planktonic cells to environmental effects
in this case, iron concentration. The addition of iron to the
biofilm medium caused las mutant biofilms to predictably repress Mn-SOD levels, and likewise the addition of 2,2-dipyridyl to
the medium caused wild-type biofilm cells to up-regulate Mn-SOD activity (Fig. 5). This suggests that at least in the case of sodA control, basic iron sensor-response circuitry is
unchanged for cells growing in either setting. The basis for the
apparent up-regulation of sodA in the QS mutant biofilms
under conditions that are not obviously iron limiting is the focus of
continuing experiments.
In summary, this study demonstrated that (i) the nutritional status of
the cell must be taken into account when evaluating QS-based gene
expression, (ii) QS-based gene regulation models based on studies with
planktonic cells must be modified in order to explain biofilm gene
expression behavior, and (iii) gene expression in biofilms is dynamic.
In addition, the results from the las mutant biofilms
implied that QS regulation can exert negative regulatory control.
Determining physiological differences between biofilms and planktonic
cultures is critical to the understanding and eventual treatment of
P. aeruginosa infections such as that found in the cystic
fibrosis lung or for removing problematic biofilms from industrial or
environmental settings. Previous studies showed evidence that bacteria
in lung tissue grow under iron-limited conditions (5, 10,
18). Further experiments aimed at understanding in situ
conditions and gene expression, particularly as related to P. aeruginosa infections, should offer significant opportunity to
improve our understanding of disease and its control.
This material is based on work supported by National Science
Foundation Center for Biofilm Engineering Cooperative Agreement EEC-8907039 (T.R.M.) and National Institutes of Health grants AI-40541
(D.J.H.).
| 1.
|
Bjorn, M. J.,
P. A. Sokol, and B. H. Iglewski.
1979.
Influence of iron on yields of extracellular products in Pseudomonas aeruginosa cultures.
J. Bacteriol.
138:193-200[Abstract/Free Full Text].
|
| 2.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[CrossRef][Medline].
|
| 3.
|
Braun, V.,
S. Schaffer,
K. Hantke, and W. Troger.
1990.
Regulation of gene expression by iron, p. 164-179.
In
G. Hauka, and R. Thauer (ed.), The molecular basis of bacterial metabolism. Springer-Verlag, Heidelberg, Germany.
|
| 4.
|
Brint, J. M., and D. E. Ohman.
1995.
Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family.
J. Bacteriol.
177:7155-7163[Abstract/Free Full Text].
|
| 5.
|
Brown, M. R. W.,
H. Anwar, and P. A. Lambert.
1984.
Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions.
FEMS Microbiol. Lett.
21:113-117.
|
| 6.
|
Characklis, W. G.,
K. C. Marshall, and G. A. McFeters.
1990.
The microbial cell, p. 131-160.
In
W. G. Characklis, and K. C. Marshall (ed.), Biofilms. John Wiley & Sons, New York, N.Y.
|
| 7.
|
Costerton, J. W.,
Z. Lewandowski,
D. E. Caldwell,
D. R. Korbe, and H. M. Lappin-Scott.
1995.
Microbial biofilms.
Annu. Rev. Microbiol.
49:711-745[CrossRef][Medline].
|
| 8.
|
Davies, D. G.,
M. R. Parsek,
J. P. Pearson,
B. H. Iglewski,
J. W. Costerton, and E. P. Greenberg.
1998.
The involvement of cell-to-cell signals in the development of a bacterial biofilm.
Science
280:295-298[Abstract/Free Full Text].
|
| 9.
|
de Lorenzo, V.,
S. Wee,
M. Herrero, and J. B. Neilands.
1987.
Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor.
J. Bacteriol.
169:2624-2630[Abstract/Free Full Text].
|
| 10.
|
Emery, T.
1982.
Iron metabolism in humans and plants.
Am. Sci.
70:626-631[Medline].
|
| 11.
|
Fuqua, C.,
S. C. Winans, and E. P. Greenberg.
1996.
Census and conscensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators.
Annu. Rev. Microbiol.
50:727-51[CrossRef][Medline].
|
| 12.
|
Gambello, M. J., and B. H. Iglewski.
1991.
Cloning and characterization of the Pseudomonas aeruginosa lasR gene: a transcriptional activator of elastase expression.
J. Bacteriol.
173:3000-3009[Abstract/Free Full Text].
|
| 13.
|
Gambello, M. J.,
S. Kaye, and B. H. Iglewski.
1993.
LasR of Pseudomoas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression.
Infect. Immun.
61:1180-1184[Abstract/Free Full Text].
|
| 14.
|
Givskov, M.,
R. de Nys,
M. Manefield,
L. Gram,
R. Maximilien,
L. Eberl,
S. Molin,
P. D. Steinberg, and S. Kjelleberg.
1996.
Eukaryotic interference with homoserine lactone-mediated prokaryotic signaling.
J. Bacteriol.
178:6618-6622[Abstract/Free Full Text].
|
| 15.
|
Govan, J. R. W., and V. Deretic.
1996.
Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.
Microbiol. Rev.
60:539-574[Abstract/Free Full Text].
|
| 16.
|
Gram, L.,
R. de Nys,
R. Maximillen,
M. Givskov,
P. D. Steinberg, and S. Kjelleberg.
1996.
Inhibitory effects of secondary metabolites from the red alga Delisea pulchra on swarming mobility of Proteus mirabilis.
Appl. Environ. Microbiol.
62:4284-4287[Abstract].
|
| 17.
|
Greenberg, E. P.
1997.
Quorum sensing in gram-negative bacteria.
ASM News
63:371-377.
|
| 18.
|
Haas, B.,
J. Kraut,
J. Marks,
S. C. Zanker, and D. Castigenetti.
1991.
Siderophore presence in sputa of cystic fibrosis patients.
Infect. Immun.
59:3997-4000[Abstract/Free Full Text].
|
| 19.
|
Harris, B. Z.,
D. Kaiser, and M. Singer.
1998.
The guanosine nucleotide (p)ppGpp initiates development and A-factor production in Myxococcus xanthus.
Genet. Dev.
12:1022-1035.
|
| 20.
|
Hassett, D. J.,
L. Charniga,
K. A. Bean,
D. E. Ohman, and M. S. Cohen.
1992.
Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase.
Infect. Immun.
60:328-336[Abstract/Free Full Text].
|
| 21.
|
Hassett, D. J.,
W. A. Woodruff,
D. J. Wozniak,
M. L. Vasil,
M. S. Cohen, and D. E. Ohman.
1993.
Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria.
J. Bacteriol.
175:7658-7665[Abstract/Free Full Text].
|
| 22.
|
Hassett, D. J.,
H. P. Schweizer, and D. E. Ohman.
1995.
Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism.
J. Bacteriol.
177:6330-6337[Abstract/Free Full Text].
|
| 23.
|
Hassett, D. J.,
P. Sokol,
M. L. Howell,
J.-F. Ma,
H. P. Schweizer,
U. Ochsner, and M. L. Vasil.
1996.
Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake and altered aerobic metabolism.
J. Bacteriol.
178:3996-4003[Abstract/Free Full Text].
|
| 24.
|
Hassett, D. J.,
M. L. Howell,
U. Ochsner,
M. Vasil,
Z. Johnson, and G. E. Dean.
1997.
An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator (Fur) in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels.
J. Bacteriol.
179:1452-1459[Abstract/Free Full Text].
|
| 25.
|
Hassett, D. J.,
M. L. Howell,
P. A. Sokol,
M. L. Vasil, and G. E. Dean.
1997.
Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of the native FumC.
J. Bacteriol.
179:1442-1450[Abstract/Free Full Text].
|
| 26.
|
Hassett, D. J.,
J. G. Elkins,
D.-J. Ma, and T. R. McDermott.
1999.
Pseudomonas aeruginosa biofilm sensitivity to biocides: use of hydrogen peroxide as a model antimicrobial agent for examining resistance mechanisms.
Methods Enzymol.
310:599-608[Medline].
|
| 27.
|
Hassett, D. J.,
J.-F. Ma,
J. G. Elkins,
T. R. McDermott,
U. A. Ochsner,
S. E. H. West,
C.-T. Huang,
J. Fredericks,
S. Burnett,
P. S. Stewart,
G. McFeters,
L. Passador, and B. H. Iglewski.
1999.
Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide.
Mol. Microbiol.
34:1082-1093[CrossRef][Medline].
|
| 28.
| Hassett, D. J., U. A. Ochsner, T. de Kievit,
B. H. Iglewski, L. Passador, T. S. Livinghouse, J. A. Whitsett, and T. R. McDermott. Genetics of quorum sensing
circuitry in Pseudomonas aeruginosa: implications for
control of pathogenesis, biofilm formation and antibiotic/biocide
resistance. In U. Streips and R. Yasbin (ed.), Modern
molecular genetics, in press. Wiley-Liss, New York, N.Y.
|
| 29.
|
Holden, M. T.,
S. Ram Chhabra,
R. de Nys,
P. Stead,
N. J. Bainton,
P. J. Hill,
M. Manefield,
N. Kumar,
M. Labatte,
D. England,
S. Rice,
M. Givskov,
G. P. Salmond,
G. S. Stewart,
B. W. Bycroft,
S. Kjelleberg, and P. Williams.
1999.
Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other gram-negative bacteria.
Mol. Microbiol.
33:1254-1266[CrossRef][Medline].
|
| 30.
|
Holloway, B. W.
1969.
Genetics of Pseudomonas.
Bacteriol. Rev.
33:419-443[Free Full Text].
|
| 31.
|
Huang, C.-T.,
K. D. Xu,
G. A. McFeters, and P. A. Stewart.
1998.
Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilm in response to phosphate starvation.
Appl. Environ. Microbiol.
64:1526-1531[Abstract/Free Full Text].
|
| 32.
|
Iglewski, B. H.,
L. Rust, and R. Bever.
1990.
Molecular analysis of Pseudomonads elastase, p. 36-53.
In
S. Silver, A. M. Chakrabarty, B. H. Iglewski, and S. Kaplan (ed.), Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology. American Society for Microbiology, Washington, D.C.
|
| 33.
|
Kuspa, A.,
L. Kroos, and D. Kaiser.
1986.
Intercellular signaling is required for developmental gene expression in Myxococcus xanthus.
Dev. Biol.
117:267-276[CrossRef][Medline].
|
| 34.
|
Kuspa, A.,
L. Plamann, and D. Kaiser.
1992.
Identification of heat-stable A-factor from Myxococcus xanthus.
J. Bacteriol.
174:3319-3326[Abstract/Free Full Text].
|
| 35.
|
Latifi, A.,
M. Foglino,
K. Tanaka,
P. Williams, and A. Lazdunski.
1996.
A hierarchial quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhlR (VsmR) to expression of the stationary-phase sigma factor RpoS.
Mol. Microbiol.
21:1137-1146[CrossRef][Medline].
|
| 36.
|
Lazazzera, B. A.
2000.
Quorum sensing and starvation: signals for entry into stationary phase.
Curr. Opin. Microbiol.
3:177-182[CrossRef][Medline].
|
| 37.
|
Mekalanos, J. J.
1992.
Environmental signals controlling expression of virulence determinants in bacteria.
J. Bacteriol.
174:1-7[Free Full Text].
|
| 38.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 39.
|
Östling, J.,
K. Flärdh, and S. Kjelleberg.
1995.
Isolation of a carbon starvation regulatory mutant in a marine vibrio.
J. Bacteriol.
177:6978-6982[Abstract/Free Full Text].
|
| 40.
|
Östling, J.,
L. Holmquist, and S. Kjelleberg.
1996.
Global analysis of the carbon starvation response of a marine vibrio disrupted in genes homologous to relA and spoT.
J. Bacteriol.
178:4901-4908[Abstract/Free Full Text].
|
| 41.
|
Passador, L.,
J. M. Cook,
M. J. Gambello,
L. Rust, and B. H. Iglewski.
1993.
Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication.
Science
260:1127-1130[Abstract/Free Full Text].
|
| 42.
|
Pearson, J. P.,
K. M. Gray,
L. Passador,
K. D. Tucker,
A. Eberhard,
B. H. Iglewski, and E. P. Greenberg.
1994.
Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.
Proc. Natl. Acad. Sci. USA
91:197-201[Abstract/Free Full Text].
|
| 43.
|
Pearson, J. P.,
L. Passador,
B. H. Iglewski, and E. P. Greenberg.
1995.
A second N-acylhomoserine lactone produced by Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. USA
92:1490-1494[Abstract/Free Full Text].
|
| 44.
|
Pearson, J. P.,
C. P. Everett, and B. H. Iglewski.
1997.
Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes.
J. Bacteriol.
179:5756-5767[Abstract/Free Full Text].
|
| 45.
|
Pesci, E. C.,
J. B. Milbank,
J. P. Pearson,
S. McKnight,
A. S. Kende,
E. P. Greenberg, and B. H. Iglewski.
1999.
Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. USA
96:11229-11234[Abstract/Free Full Text].
|
| 46.
|
Rombel, I. T.,
B. J. McMorran, and I. L. Lamont.
1995.
Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads.
Mol. Gen. Genet.
246:519-528[CrossRef][Medline].
|
| 47.
|
Sambrook, J.,
E. F. Fristch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 48.
|
Singer, M., and D. Kaiser.
1995.
Ectopic production of guanosine penta- and tetraphosphate can initiate early developmental gene expression in Myxococcus xanthus.
Genes Dev.
9:1633-1644[Abstract/Free Full Text].
|
| 49.
|
Sokol, P. A.,
C. D. Cox, and B. H. Iglewski.
1982.
Pseudomonas aeruginosa mutants altered in their sensitivity to the effect of iron on toxin A or elastase yields.
J. Bacteriol.
151:783-787[Abstract/Free Full Text].
|
| 50.
|
Srinivasan, S.,
J. Ostling,
T. Charlton,
R. de Nys,
K. Takayama, and S. Kjellberg.
1998.
Extracellular signal molecule(s) involved in the carbon starvation response of marine Vibrio sp. strain S14.
J. Bacteriol.
180:201-209[Abstract/Free Full Text].
|
| 51.
|
Van Delden, C.,
E. C. Pesci,
J. P. Pearson, and B. H. Iglewski.
1998.
Starvation selection restores elastase and rhamnolipid production in a Pseudomonas aeruginosa quorum-sensing mutant.
Infect. Immun.
66:4499-4502[Abstract/Free Full Text].
|
| 52.
|
Whiteley, M.,
K. M. Lee, and E. P. Greenberg.
1999.
Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. USA
96:13904-13909[Abstract/Free Full Text].
|
| 53.
|
Winson, M. K.,
M. Camara,
A. Latifi,
M. Foglino,
S. R. Chhabra,
M. Daykin,
M. Bally,
V. Chapon,
G. P. Salmond,
B. W. Bycroft,
A. Lazdunski,
G. S. A. B. Stewart, and P. Williams.
1995.
Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. USA
92:9427-9431[Abstract/Free Full Text].
|
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Abstract
Introduction
