Applied and Environmental Microbiology, March 2001, p. 1375-1379, Vol. 67, No. 3
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1375-1379.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Factors Affecting Catalase Expression in Pseudomonas
aeruginosa Biofilms and Planktonic Cells
Jesse R.
Frederick,1
James G.
Elkins,1
Nikki
Bollinger,1
Daniel J.
Hassett,2 and
Timothy R.
McDermott1,3,*
Center for Biofilm
Engineering1 and Department of Land
Resources and Environmental Sciences,3 Montana
State University, Bozeman, Montana 59717, and Department of
Molecular Genetics, Biochemistry, and Microbiology, University of
Cincinnati College of Medicine, Cincinnati, Ohio
45267-05242
Received 29 August 2000/Accepted 14 December 2000
 |
ABSTRACT |
Previous work with Pseudomonas aeruginosa showed
that catalase activity in biofilms was significantly reduced
relative to that in planktonic cells. To better understand biofilm
physiology, we examined possible explanations for the differential
expression of catalase in cells cultured in these two different
conditions. For maximal catalase activity, biofilm cells required
significantly more iron (25 µM as FeCl3) in the medium,
whereas planktonic cultures required no addition of iron. However,
iron-stimulated catalase activity in biofilms was still only about
one-third that in planktonic cells. Oxygen effects on catalase activity
were also investigated. Nitrate-respiring planktonic cultures produced
approximately twice as much catalase activity as aerobic cultures grown
in the presence of nitrate; the nitrate stimulation effect could also
be demonstrated in biofilms. Cultures fermenting arginine had reduced
catalase levels; however, catalase repression was also observed in
aerobic cultures grown in the presence of arginine. It was concluded
that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.
| |
TEXT |
Microbial biofilms have been the
subject of significant interest because of accumulating evidence
suggesting that surface-associated growth and proliferation are normal
and preferable for microorganisms. The implications of this simple
observation are beginning to emerge, with the most important being that
bacteria growing in biofilms are much more resistant to antimicrobial
agents than are bacteria growing as planktonic cultures.
Antibiotic killing models thus far developed have used planktonic cells
(i.e., batch cultures) extensively but are likely invalid for biofilms.
It has been suggested that the physiology of bacteria in a biofilm is
different from that in planktonic cells (3), yet there are
few defined examples and even fewer explanations for described differences.
We have initiated a series of studies focused on examining biofilm cell
physiology and response to environmental stimuli; we are using elements
of the oxidative stress response in this work. The oxidative stress
response is well suited for these experiments because (i) the genes and
enzymes involved are well understood (9, 10, 11, 12, 14),
(ii) it is an important defense mechanism of recalcitrant bacterial
biofilms in infections against the oxidative burst of phagocytic cells
(16), and (iii) oxidative biocides are also used in
industry to remove problematic biofilms from piping systems. Further,
in Pseudomonas aeruginosa, key enzymes of the oxidative
stress response, such as the major catalase, KatA, and a
manganese-cofactored superoxide dismutase (Mn-SOD), are controlled by
quorum sensing (16, 17); in addition, the activities of
these enzymes are also known to be very sensitive to iron and phosphate
availability (9, 12, 13, 14). Therefore, these enzymes and
the genes that encode them represent effective models for assessing
certain important aspects of bacterial physiology in the biofilm environment.
In recent work, we observed that catalase activity in P. aeruginosa biofilm cells was considerably lower than that in
planktonic cells (16). The most obvious environmental
factors that could conceivably influence catalase levels in biofilms
and potentially account for this difference include iron and oxygen
availability and H2O2
production. Iron limitation significantly reduces KatA activity
(13), and it is possible that movement of a cationic micronutrient into a biofilm is limited by anionic carboxyl or hydroxyl
groups on bacterial surface polysaccharides. We also considered oxygen
availability as a possible explanation for the depressed levels of
catalase in biofilms. Studies have shown that oxygen tension is reduced
in bacterial biofilms (3, 21). We postulated that if
oxygen respiration is reduced, endogenous generation of
H2O2 also will be reduced,
thus reducing the requirement for elevated catalase levels. In an
attempt to better understand the basis for the differential
expression of catalase in these two growth modes, we conducted a series
of experiments to determine the extent to which catalase levels can be
manipulated. In so doing, we also hoped to gain a better understanding
of the physiological differences between biofilm and planktonic bacteria.
Iron effects on catalase activity.
Planktonic cultures and
biofilms of P. aeruginosa wild-type strain PAO1
(18) were grown in tryptic soy broth (TSB) as previously described (16). Briefly, batch (planktonic) cultures were
grown to stationary phase in 1/10-strength TSB and then assayed for catalase activity (which is comprised of only KatA under these conditions [2]) using previously described methods
(15). Biofilms were cultured on stainless steel slides
(1.3 by 7.6 cm) in a drip-flow reactor (5) using
1/100-strength TSB at a constant flow of 50 ml per h. To assess and
compare the response of biofilm catalase activity to iron availability,
the TSB medium for both planktonic cultures and biofilms was amended
with various amounts of FeCl3. To impose iron
starvation conditions (12, 13, 14), the iron-specific
chelator 2,2-dipyridyl (500 µM for planktonic cultures and 50 µM
for biofilm cultures) was added.
Under iron starvation conditions, catalase activities in planktonic
cells and biofilm cells were essentially the same and were very low
(approximately 125 U per mg of protein) (Fig.
1A). In contrast, when iron limitation
was removed, catalase levels in planktonic cells were about 10-fold
higher than those observed in biofilms, consistent with our previous
observations (16). The inclusion of additional iron had no
effect on catalase activity in planktonic cells. However, supplemental
iron significantly and reproducibly increased catalase activity in
biofilm cells. Catalase activity in biofilm cells increased
approximately threefold in response to added iron but was still
significantly lower than that in planktonic cells.
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FIG. 1.
P. aeruginosa biofilms require more iron
for maximal catalase activity. (A) Comparison of catalase activities in
biofilms ( ) and stationary-phase planktonic cultures ( ) grown in
the presence of the iron-specific chelator 2,2-dipyridyl, without added
iron, or with increasing iron concentrations. Catalase activity is
shown as units of activity per milligram of protein, where 1 U is
defined as the amount which decomposes 1 µmol of
H2O2 min 1. Error bars, where
visible, represent ±1 standard error of the mean for three separate
cultures. (B) SOD activity stain of planktonic and biofilm whole-cell
proteins (50 µg per lane) resolved in a native gel. The locations of
the isozymes Mn-SOD and Fe-SOD are designated to the left of the gel
image. Total protein concentration was determined using a Bio-Rad
protein assay kit with bovine serum albumin fraction V as a standard.
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|
To confirm the iron-limited status of the planktonic and biofilm cells,
we used SOD activity staining (15) of whole-cell proteins
separated by native gel electrophoresis to assess the expression of
Mn-SOD, which is strongly induced by iron starvation (12, 13,
14), and Fe-SOD, which is expressed constitutively but which is
predictably not enhanced by iron starvation (12, 13, 14).
As shown in Fig. 1B, biofilm cells expressed Mn-SOD when grown without
added iron but not when the medium was amended with iron. Planktonic
cells did not contain Mn-SOD unless 2,2-dipyridyl was included,
suggesting they were not iron limited under normal growth conditions.
Chemical analysis of 1/10-strength TSB medium showed an iron
concentration of 1.5 µM. The iron content in the 1/100-strength TSB
used for the biofilm experiments would be proportionately lower;
however, the constant flow over the biofilm cells would continuously deliver fresh medium over the developing biofilm. Over the
course of such experiments, the total iron made available to the
biofilm would exceed that in batch conditions by at least eightfold.
Regardless, in all subsequent experiments, 25 µM
FeCl3 was included in the medium.
Effect of oxygen availability on catalase activity.
To test
whether oxygen limitation might also partially account for the
reduced catalase levels in P. aeruginosa biofilms, planktonic cells were grown anaerobically. P. aeruginosa is
capable of anaerobic growth using nitrate as an electron acceptor
or arginine for substrate-level phosphorylation (8).
Strain PAO1 was cultured in sealed serum bottles containing
medium that had been amended with 100 mM potassium nitrate and
sparged with N2 gas prior to autoclaving. The
medium also contained the oxygen indicator 0.1% resazurin to verify
anaerobiosis. Cell growth was appreciably slower under these
conditions, requiring 5 days to reach stationary phase (results not
shown). However, when cells reached stationary phase, catalase activity
was about twofold higher than that in aerobic cultures amended
with nitrate and when measured at various times after stationary phase
was reached (Fig. 2). The addition of
nitrate had no effect on catalase activity in aerobic cultures (compare
to Fig. 1A). Also, as with the iron effects shown in Fig. 1A, the
increase in catalase activity was due to increased levels of KatA. In
native gel analysis, the oxidative-stress-inducible catalases KatB and
KatC were not evident (results not shown). The presence of nitrate had
no effect on Mn-SOD expression, which was absent in the cells grown in
these experiments (results not shown).
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FIG. 2.
Catalase activity is increased in nitrate-respiring
planktonic cultures of P. aeruginosa. Potassium nitrate
(100 mM) was included in cultures incubated aerobically (open bars) and
anaerobically (filled bars). Culture age is shown on the
x axis; anaerobic cultures grew more slowly. Error bars,
where visible, represent ±1 standard error of the mean for three
separate cultures. Results are from one representative experiment of
three independent experiments for each type of culture. Catalase
activity is shown as units of activity per milligram of protein, where
1 U is defined as the amount which decomposes 1 µmol of
H2O2 min1.
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|
The effect of nitrate was also tested with P. aeruginosa
biofilms. Catalase activity was significantly increased in mature biofilms that had been grown with 10 mM potassium nitrate (Fig. 3). The nitrate effect was not apparent
in young biofilms, but as with the anaerobic
NO3-respiring planktonic culture comparisons (Fig. 2), the presence of nitrate caused a doubling of catalase activity as the biofilms aged (Fig. 3).
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FIG. 3.
Catalase activity in P. aeruginosa
biofilms grown in the presence () or absence () of 10 mM nitrate.
Catalase activity is shown as units of activity per milligram of
protein, where 1 U is defined as the amount which decomposes 1 µmol
of H2O2 min1. Error bars, where
visible, represent ±1 standard error of the mean for at least two
biofilms at each time point.
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|
To assess the effects of anoxic substrate-level phosphorylation on
catalase levels, planktonic cells were cultured anaerobically in 100 mM
phosphate-buffered 1/10-strength TSB containing 40 mM arginine. Aerobic
cultures were included as controls. Surprisingly, catalase levels were
significantly depressed in both anaerobic and aerobic planktonic
cultures (Fig. 4A). As the anaerobic
metabolism of arginine results in increased culture pH (through the
release of NH3), we tested catalase levels
in aerobic cultures grown in 1/10-strength TSB buffered to pH 6 to pH 9 but observed no effect on catalase activity (results not shown). The
possibility that arginine acts as an iron chelator and thus limits
catalase activity was also tested by staining for SOD activity in
native gels (Fig. 4B). Iron-starvation-inducible Mn-SOD was strongly
expressed in cells growing aerobically in medium containing arginine,
but the addition of 10 µM FeCl3 repressed the
expression of this isozyme. Surprisingly, Mn-SOD was absent in
anaerobic arginine-fermenting planktonic cultures (Fig. 4B). When iron
was added to cells growing aerobically in TSB-arginine medium, catalase
activity was restored to nearly normal levels (Fig. 4C).
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FIG. 4.
Effect of arginine on catalase and SOD activities in
P. aeruginosa. (A) Catalase activity in planktonic
cultures grown aerobically with or without 40 mM arginine and in
planktonic cultures fermenting arginine. Catalase activity is shown as
units of activity per milligram of protein, where 1 U is defined as the
amount which decomposes 1 µmol of H2O2
min1. (B) SOD activity stain of a native gel used to
resolve Mn-SOD and Fe-SOD activities (50 µg of total cell protein per
lane). Oxygen and iron treatments are noted above each lane. (C)
Catalase activity in aerobic () and anaerobic () planktonic
cultures grown in the presence of 40 mM arginine and various
concentrations of added iron. For all assays, the cell extract protein
concentration was determined using a Bio-Rad protein assay kit with
bovine serum albumin fraction V as a standard. In panels A and C, error
bars, where visible, represent ±1 standard error of the mean for three
replicate cultures. Results are from one representative experiment for
each treatment. Each outcome was verified to be reproducible.
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|
Conclusions.
By measuring two different oxidative stress
enzymes that are affected in different ways by iron availability, we
found that P. aeruginosa biofilms exhibit behavior that is
consistent with an iron starvation response. Biofilms expressed high
levels of Mn-SOD (Fig. 1B), an enzyme that is strongly stimulated by
iron limitation (12, 13, 14). However, when iron was added
to the medium, Mn-SOD was not expressed and biofilm catalase levels increased (Fig. 1A). These two observations are consistent with each
other and suggest either that, relative to planktonic cells, biofilm
cells have a higher iron requirement or that iron movement within the
biofilm is somehow constricted, thus limiting iron bioavailability.
Previous studies provided evidence that bacteria colonizing cystic
fibrosis lung tissue grow under iron-limited conditions (1, 6,
7). Further experiments aimed at understanding in situ
conditions and gene expression, particularly as they are related
to P. aeruginosa infections, should offer significant opportunities to improve the understanding of diseases caused by this
organism. Further, determining the physiological differences between
biofilm cells and planktonic cells will likely be critical in the
understanding of and the eventual development of treatments for
P. aeruginosa biofilm infections like those found in cystic fibrosis lung tissue.
We found no evidence that depressed catalase activity in biofilms is
due to oxygen limitation per se. Catalase levels in planktonic cells
were actually significantly increased under anaerobic nitrate-respiring conditions (Fig. 2). The same effect was also apparent in biofilms grown with nitrate-amended medium. The latter observation is consistent with previous studies showing the oxygen-limited nature of the biofilm
interior (3) and the use of nitrate as an alternative electron acceptor for cells in a biofilm. Interestingly, catalase activity was significantly depressed under either aerobic and anaerobic
culture conditions when arginine was added to the medium (Fig. 4A). The
exact role of arginine (or products of arginine metabolism, e.g.,
ornithine) in catalase control is not clear at this time, but it is
likely linked somehow with iron availability, as the addition of iron
to the medium reversed the arginine effect (Fig. 4C). This result is
also in agreement with the presence of iron-starvation-inducible Mn-SOD
in aerobic cultures (Fig. 4B) but not under anaerobic conditions, where
Mn-SOD was absent. The latter observation is perhaps an important clue
about how sodA (the gene coding for Mn-SOD) may be regulated
while P. aeruginosa is involved in anaerobic arginine
metabolism. Under anaerobic culture conditions, P. aeruginosa metabolizes arginine via the arginine deiminase pathway
(20), with arginine being stoichiometrically converted to
ornithine (4). Ornithine derivatives are common features of microbial iron chelates (19); thus, it is
possible that subsequent metabolic alterations of the resulting
ornithine contribute to the synthesis of an iron-binding metabolite.
Work aimed at further understanding sodA expression under
anaerobic arginine-fermenting conditions is currently under way.
| |
ACKNOWLEDGMENTS |
We thank Irwin Fridovich for insightful discussions.
J.R.F. was supported by an undergraduate research fellowship from the
National Science Foundation Center for Biofilm Engineering (EEC-8907039), Montana State University. This work was also supported by National Institutes of Health grant AI-40541 to D.J.H.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717. Phone: (406) 994-2190. Fax: (406) 994-3933. E-mail:
timmcder{at}montana.edu.
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Applied and Environmental Microbiology, March 2001, p. 1375-1379, Vol. 67, No. 3
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.3.1375-1379.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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