JOURNAL OF BACTERIOLOGY, Aug. 2008, p. 5493–5501
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 15
Hydrogen Peroxide Linked to Lysine Oxidase Activity Facilitates
Biofilm Differentiation and Dispersal in Several
Anne Mai-Prochnow,1† Patricia Lucas-Elio,2† Suhelen Egan,1Torsten Thomas,1Jeremy S. Webb,1,3
Antonio Sanchez-Amat,2and Staffan Kjelleberg1*
School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, University of New South Wales, Sydney,
NSW 2052, Australia1; Department of Genetics and Microbiology, University of Murcia, 30100 Murcia, Spain2; and School of
Biological Sciences, University of Southampton, Southampton SO16 7PX, United Kingdom3
Received 21 April 2008/Accepted 16 May 2008
The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP,
which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal,
and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium
Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the
production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that
the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development
in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the
generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal
cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environ-
ments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two
of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide
was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and
the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly
linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death.
We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays
an important role in dispersal and colonization across a range of gram-negative bacteria.
Biofilm formation in microorganisms generally involves dif-
ferentiation processes leading to the formation of multicellular
three-dimensional structures. Most biofilms consist of micro-
colonies encased by an organic polymer matrix. Previously, it
has been shown that subpopulations of cells die during the
normal course of biofilm development. This phenomenon has
been observed for several organisms, including monospecies
biofilms of Pseudomonas aeruginosa (52), Serratia marcescens
(K. W. Lam, S. A. Rice, and S. Kjelleberg, unpublished data),
Caulobacter crescentus (13), and Pseudoalteromonas tunicata
(34), as well as mixed-species communities, including dental (4,
27) and river (30a) biofilm communities. Biofilm killing in P.
aeruginosa has been linked to the activity of a prophage (52),
and killing in P. tunicata is mediated by a large, autolytic
protein (AlpP) (34). In both organisms, cell death is localized
to the center of microcolonies and is controlled by specific
regulatory determinants, such as RpoN (52) and quorum sens-
ing in P. aeruginosa (J. S. Webb and S. Kjelleberg, unpublished
data) and a ToxR-like regulator, WmpR, in P. tunicata (11).
Mutants deficient in the production of AlpP show no cell death
despite the formation of similar biofilm architecture (10, 11,
34). Because it is not necessary for survival per se, it may be
suggested that cell death events represent an evolved capacity
of importance to the biofilm development in the organism.
AlpP-mediated biofilm cell death in P. tunicata has been
linked to the generation of a metabolically active and pheno-
typically diverse dispersal population (35). A major dispersal
event occurs in the wild-type strain after cell death events are
observed within the biofilm. It has been shown, using fluores-
cent staining for metabolic activity, that the dispersal popula-
tion of the P. tunicata wild type had higher activity than the
dispersal population of the ?AlpP mutant, which does not
show cell death (35). Moreover, P. tunicata wild-type dispersal
cells displayed a larger variation in motility, growth, and bio-
film formation than the ?AlpP mutant. A metabolically active
dispersal population with high variation in traits important to the
spread and colonization ability of the organism is hypothesized to
be advantageous under changing environmental conditions.
Since the first report of the antibacterial and autolytic pro-
tein in P. tunicata (28), a similar protein (LodA, previously
marinocine) has been reported to occur in the melanogenic,
marine bacterium Marinomonas mediterranea (32). Both pro-
teins show activity against both gram-negative and gram-posi-
tive bacteria from a diverse range of environments, as well as
displaying autotoxic activity. The antibacterial activity of LodA
has recently been shown to be due to the generation of hydro-
gen peroxide from L-lysine. LodA (EC 126.96.36.199) catalyzes the
following reaction: L-lysine ? O2? H2O 3 2-aminoadipate
6-semialdehyde ? NH3? H2O2(17).
* Corresponding author. Mailing address: School of Biotechnology
and Biomolecular Sciences and Centre for Marine Bio-Innovation,
Biological Sciences Building, University of New South Wales, Kens-
ington, Sydney, NSW 2052, Australia. Phone: 61 (2) 9385 2102/2276.
Fax: 61 (2) 9385 1779. E-mail: email@example.com.
† A.M.-P. and P.L.-E. contributed equally to this study.
?Published ahead of print on 23 May 2008.
In the present study we show that the P. tunicata AlpP
produces hydrogen peroxide from L-lysine, and evidence is
provided that hydrogen peroxide is responsible for cell death in
biofilms. Furthermore, it is demonstrated that the AlpP-homo-
logue, LodA, in M. mediterranea has a similar ecological func-
tion to AlpP during biofilm development. LodA production
leads to cell death of a subpopulation of cells within micro-
colonies of M. mediterranea biofilms, which is linked to the
generation of a phenotypically diverse dispersal population.
Moreover, it is demonstrated that the AlpP-homologues in
Chromobacterium violaceum (accession number NP_902938)
and in part in C. crescentus (accession number NP_419374) are
implicated in similar cell death events during biofilm forma-
tion. Our findings suggest that AlpP-mediated autotoxic events
may play an important role in biofilm development and differ-
entiation in a range of gram-negative bacterial groups.
MATERIALS AND METHODS
Strains and culture conditions. All of the bacterial strains and the respective
culture media used are shown in Table 1. P. tunicata and M. mediterranea were
grown at 25°C and C. violaceum and C. crescentus were grown at 30°C.
P. tunicata AlpP purification. AlpP was purified by using a method adapted
from James et al. (28). Briefly, P. tunicata was grown in 3 M for 48 h with shaking
at room temperature. Cells were then harvested by centrifugation at 15,000 ? g
for 15 min at 22°C. The cell pellet was resuspended in fresh medium (0.5 ml/2-g
pellet) and incubated for a further 24 h under static conditions. After centrifu-
gation for 1.5 h at 23,700 ? g and 4°C, the supernatant was filtered through a
0.22-?m-pore-size membrane. The filtrate was then dialyzed (12,000-kDa cutoff;
Sigma, Castle Hill, Australia) overnight against 2 liters of 20 mM Tris (pH 7.4).
A strong anionic ion-exchange matrix (High Q strong anion-exchanger; Bio-Rad,
Hercules, CA) was used to purify AlpP from the P. tunicata supernatant. The
protein was eluted between 250 and 350 mM NaCl in 20 mM Tris-HCl (pH 7.4).
The eluate was further purified by ultrafiltration with a 100-kDa molecular cutoff
filter (YM100; Diaflo; Amicon, Lexington, MA), and the retentate was saved.
The purity of the final sample was checked by running an 8% sodium dodecyl
sulfate polyacrylamide protein gel stained with Coomassie blue, yielding only the
two bands (60 and 80 kDa) previously identified as AlpP.
The total protein concentration was determined by using the BCA assay
(Sigma) according to the manufacturer’s instructions.
M. mediterranea LodA purification. LodA purification from M. mediterranea
cultures was performed as previously described (32). Briefly, M. mediterranea was
inoculated at an optical density at 600 nm of 0.05 in MN media (25). After 48 h
of culture, the supernatant was separated by centrifugation, and 2 volumes of
ethanol were added to precipitate LodA by incubation overnight at 4°C. The
compound was then recovered by centrifugation at 19,000 ? g at 4°C for 20 min.
The pellet was allowed to air dry and suspended in 0.1 M sodium phosphate
buffer (pH 7). Insoluble material was removed by centrifugation (13,000 ? g at
4°C for 20 min). The activity of the extract obtained was calculated by measuring
the halos formed in the antibiogram inhibition test against Escherichia coli DH5?
(32). Other proteins present in the sample were shown to have no antibacterial
activity (data not shown).
Lysine oxidase activity fluorometric measurements. The Amplex Red hydro-
gen peroxide/peroxidase assay kit (A22188; Molecular Probes, Eugene, OR)
assay was used to test the production of hydrogen peroxide from purified AlpP.
In the presence of horseradish peroxidase (HRP), the Amplex red reagent reacts
with hydrogen peroxide in a 1:1 stoichiometry to produce the red fluorescent
oxidation product resuforin. Resuforin fluorescence was measured at excitation
of 550 nm and an emission of 590 nm. Hydrogen peroxide (Univar, Kirkland,
WA) concentrations ranging from 2 to 12 ?M were used as standard. L-Lysine
(50 mM; Sigma) was used as a substrate for AlpP. The ability of AlpP to generate
hydrogen peroxide from L-lysine was tested with AlpP concentrations ranging
from 0.4 to 4 ng. In a negative control catalase (0.1 mg ml?1; Sigma, St. Louis,
MO) was added to the reaction of AlpP and L-lysine.
Lysine detection in biofilm effluent. Lysine was detected in biofilm effluent
samples of P. tunicata and M. mediterranea at 72 and 144 h of biofilm develop-
ment by using high-performance liquid chromatography (HPLC). Samples were
freeze-dried at ?50°C overnight. The freeze-dried samples were resuspended in
100 ?l of 0.1 mM methionine sulfone in 0.1 M HCl, and 50 ?l of each concen-
trated sample was used for lysine analysis. The sample was derivatized with
phenylisothiocyanate according to the protocol from Waters (Rydalmere, Aus-
tralia). Lysine HPLC analysis of the sample was carried out on HP1100 HPLC
unit (Hewlett-Packard, Australia) with an Agilent LC Chemstation (Agilent
Technologies, Inc.). A 1/20 volume of the sample was separated in the free amino
acids column by following the binary mobile-phase gradient table from Waters
(Rydalmere) at 46°C. The derivatized lysine was detected at 254 nm and quan-
tified by comparison to standard solutions.
Determination of the substrate affinity (Km) of AlpP for lysine. Purified AlpP
was used to perform fluorimetric measurements by using an Amplex Red hydro-
gen peroxide/peroxidase assay kit (A22188; Molecular Probes) to detect hydro-
gen peroxide production with D- or L-lysine as substrates for the reaction. The
assays were carried out in a fluorimetric enzyme-linked immunosorbent assay
reader, using an excitation filter of 550 nm and an emission filter of 590 nm. All
assays were performed in duplicate in 96-well plates. Background fluorescence in
the absence of lysine was subtracted to all data to minimize the effect of other
compounds that might be present in the extract. The Michaelis constant (Km) of
each reaction was calculated from a Lineweaver-Burk plot.
Flow cell experiments. All strains were grown in continuous-culture flow cells
(channel dimensions 1 by 4 by 40 mm) as previously described (41). Channels
were inoculated with 0.5 ml of early-stationary-phase cultures containing approx-
imately 109cells ml?1and incubated without flow for 1 h at 25°C. Flow was then
TABLE 1. Bacterial strains and culture media
Strain Culture mediuma
Minimal biofilm medium Source or reference
P. tunicata VNSS (36)3M supplemented with 0.01%
3M supplemented with 0.01%
Marine minimal medium MN (25)
Marine minimal medium MN (25)
P. tunicata ?AlpP mutant
M. mediterranea MMB-1
M. mediterranea SB1 lodA
VNSS ? Sm at 100 ?g ml?1? Km at
50 ?g ml?1
Marine medium 2216 (Difco)
Marine medium 2216 ? Km at
50 ?g ml?1(Difco)
Luria-Bertani broth (Difco)
M9 (39)UNSW culture collection
(accession no. 040100)
C. violaceum CVMUR1
C. crescentus CB15
Luria-Bertani broth ? Km at
40 ?g ml?1
PYE: peptone, 2 g liter?1; yeast
extract, 1 g liter?1; MgSO4? 7H2O,
0.2 g liter?1
PYE ? Sm at 5 ?g ml?1
M2 supplemented with 0.2%
C. crescentus CAUMUR1
E. coli DH5?
M2 supplemented with 0.2%
aSm, streptomycin; Km, kanamycin.
bACM, Australian Collection of Microorganisms.
5494MAI-PROCHNOW ET AL.J. BACTERIOL.
started with a mean flow velocity in the flow cells of 0.2 mm s?1, corresponding
to laminar flow with a Reynolds number of 0.02. Biofilms were run at 25°C for
P. tunicata and M. mediterranea and at 30°C for C. violaceum and C. crescentus.
Biofilm staining. To investigate cell death during biofilm development, bio-
films were stained by using a LIVE/DEAD BacLight bacterial viability kit (Mo-
lecular Probes). The two stock solutions of the stain (SYTO 9 and propidium
iodide [PI]) were diluted to 3 ?l ml?1in biofilm medium and injected into the
flow channels. Live SYTO 9-stained cells and dead PI-stained cells were visual-
ized with a confocal laser scanning microscope (CLSM; Olympus) using fluores-
cein isothiocyanate and tetramethyl rhodamine isocyanate optical filters, respec-
In order to provide a quantitative analysis of cell death within the biofilms, all
microcolonies (with or without dead cells in the center) were counted in at least
10 fields of view in three replicate flow cells. A percentage value of microcolonies
containing dead cells was then determined.
To visualize hydrogen peroxide production during biofilm development, bio-
films were stained with Amplex Red. HRP (0.14 U ml?1) and Amplex Red (25
?M) were diluted in the biofilm media and injected into the flow cell. Immedi-
ately after staining, biofilms were visualized with an epifluorescence microscope
(Leica Microsystem, Wetzlar, Germany). Amplex Red fluorescence was ob-
served at an excitation of 515 to 560 nm and an emission of 590 nm. Bright-field
images were taken from the same field of view.
Removal of hydrogen peroxide from biofilms. To remove hydrogen peroxide
from biofilms, catalase (Sigma) was added to the biofilm media. All biofilms were
allowed to establish for 24 h before catalase was added at a final concentration
of 100 ?M. This catalase concentration did not affect the planktonic growth rate
of the strains (data not shown). After 3 days of incubation, the biofilm was
stained with Amplex Red and observed with an epifluorescence microscope (see
Add back of LodA protein to SB1 mutant biofilms. M. mediterranea LodA was
prepared as described above, and approximately 60 ?g was added back to each
flow cell containing M. mediterranea SB1 mutant biofilms. The add-back exper-
iment was performed as described previously for P. tunicata (34). Briefly, LodA
was injected into the flow cells by using a syringe needle. Silicone tubing at either
side of the flow cell was then blocked off by using tubing clamps. As a control, 0.1
M phosphate buffer (pH 7.0) was inoculated into separate flow cell channels.
Biofilms were incubated at 25°C for 3 h before staining with the LIVE/DEAD kit
and visualization with the CLSM.
Add-back of hydrogen peroxide to ?AlpP mutant biofilms. ?AlpP mutant
biofilms were grown for 72 h before 1 mM hydrogen peroxide was injected into
the flow cells by using a syringe needle in a similar manner to that of the AlpP
add-back experiment (34). Silicone tubing at either side of the flow cell was then
blocked off by using tubing clamps. As a control, NSS buffer was inoculated into
separate flow cell channels. Biofilms were incubated at 25°C for 1 h before
staining with the LIVE/DEAD kit and visualization with the CLSM.
Phenotypic variation of M. mediterranea dispersal cells. To investigate the
hypothesis that cell lysis within microcolonies correlates with phenotypic varia-
tion in M. mediterranea, effluent was spread plated onto marine agar (Difco/
Becton Dickinson) at three time points during biofilm formation: 24 h (before
the onset of cell death), 72 h (shortly after the onset of cell death), and 144 h
(when cell death was more extended throughout the biofilm). Twenty colonies
derived from M. mediterranea wild-type and SB1 biofilms were randomly picked
from marine agar and screened for growth and biofilm formation. Portions (15
?l) of overnight cultures of the 20 colonies were inoculated into 1.5 ml of fresh
media in 24-well tissue culture plates. Plates were incubated at 25°C with agita-
tion (130 rpm). The optical density at 600 nm was measured after 24 h as an
indicator for growth ability. To measure biofilm-forming ability, the wells of the
tissue culture plates were stained with crystal violet for 20 min. After the wells
were washed twice, crystal violet was extracted in 95% ethanol, and the absor-
bance was read at 600 nm. The variation coefficient was calculated as the stan-
dard deviation of all measurements divided by the mean between all samples.
Creation of alpP/lodA homologue deletion strains in C. crescentus and C.
violaceum. Mutant strains with deletion of the genes homologous to M. mediter-
ranea lodA gene in C. crescentus, locus NP_419374, and C. violaceum, locus
NP_902938, were created via homologous recombination using the suicide vec-
tors pNPTS138 (M. R. Alley, unpublished results) and pFSVKCV, respectively.
The C. crescentus lodA deletion was created by a two-step recombination method
based on sacB counterselection (15). Two fragments of approximately 500 bp
upstream and downstream of the gene in C. crescentus were PCR amplified from
genomic DNA using the primers CAUDIR1pst and CAUREV1mlu for the
upstream fragment and CAUDIR2mlu and CAUREV2sal for the downstream
fragment (Table 2). Both fragments were cloned into pNPTS138, placing be-
tween them a streptomycin resistance cassette taken from pBSL299 (1). The
construct was mobilized into C. crescentus CB15 by electroporation, and strains
with integration of the vector by single recombination were selected by growth on
peptone-yeast extract with kanamycin at 25 ?g/ml before counterselection with
streptomycin at 5 ?g/ml and sucrose at 3% to select for double-crossover re-
combinants. Sucrose- and streptomycin-resistant colonies were screened for ka-
namycin sensitivity, and gene insertion confirmed by PCR with the primers
CAUDIR4-CAUREV5 (which gives a 670-bp fragment in wild-type and first
recombination mutants maintaining locus NP_419374) and CAUDIR3-CAU-
REV5 (which gives a 2.3-kb fragment in WT and a 2.1-kb fragment in CAU-
MUR1 as a result of the substitution of locus NP_419374 with the smaller
streptomycin resistance marker). Deletion of the C. violaceum alpP/lodA homo-
logue was achieved by using a previously described method (31). Briefly, a 500-bp
region of the gene containing the fourth and fifth conserved domains, out of a
total of eight in the homologous proteins, was amplified by PCR using the
primers CVDIR2nco and CVREV1 (Table 2). This PCR fragment was cloned
into a suicide vector containing a kanamycin resistance cassette to generate
pFSVKCV. The plasmid was then transferred to C. violaceum via conjugation
with E. coli S17-1?pir. Recombinants were selected on nutrient agar with kana-
mycin at 40 ?g/ml, and gene insertion was confirmed by PCR using the primers
CVDIR1 and CVREV1 to amplify a 1-kb fragment from domain 3 to domain 7
present in the wild-type strain and absent in the mutated strain (CVMUR1) and
the primers CVDIR1 and KmREV to amplify a 1.4-kb fragment only in the
mutant strain from the third conserved domain of the truncated gene to the
kanamycin resistance marker in the integrated plasmid (Table 2).
AlpP in P. tunicata produces hydrogen peroxide via lysine
oxidase activity. To investigate whether AlpP from P. tunicata
produces hydrogen peroxide from L-lysine, the fluorometric
Amplex Red assay was used. High fluorescence intensity
(390,000 relative counts) was detected after 6 min when AlpP
(0.13 mg ml?1) was incubated with the substrate L-lysine and
Amplex Red (Fig. 1). Similar fluorescence intensity was mea-
sured in the positive control containing Amplex Red and hy-
drogen peroxide (12 ?M). However, no fluorescence was de-
tected in the negative control (no AlpP) or when catalase was
added to the reaction of AlpP and L-lysine, thereby removing
the hydrogen peroxide produced (Fig. 1). These data strongly
suggest that AlpP has lysine oxidase activity.
In addition, the substrate affinity of AlpP to lysine was stud-
ied by using the Amplex Red assay. AlpP showed a remarkable
stereospecificity toward the L-isomer of lysine, showing a very
high affinity for this substrate (Km? 24.91 ? 0.48 ?M) com-
pared to the D-isomer (Km? 2.08 mM). No lysine oxidase
activity was also observed using other compounds (morpho-
linepropanesulfonic acid, trehalose, and tricine) at the concen-
tration used in the media (data not shown).
TABLE 2. Primer design for the creation of C. violaceum
CVMUR1 and C. crescentus CAUMUR1 strains
PrimerNucleotide sequence (5?–3?)
VOL. 190, 2008ROLE OF HYDROGEN PEROXIDE IN BIOFILM FORMATION 5495
Cell death occurs during biofilm development of M. medi-
terranea. The ability of M. mediterranea to form biofilms was
investigated. Particularly, it was explored whether the lysine
oxidase, LodA, in M. mediterranea is implicated in cell death
events during biofilm formation. Biofilms were allowed to form
in continuous culture flow cells and stained with the BacLight
LIVE/DEAD kit before visualization with a CLSM. Single
cells attached to the substratum and small microcolonies were
observed 24 h after inoculation (Fig. 2A). After 48 h, larger
microcolonies were formed, consisting only of viable cells.
However, at 3 days after inoculation, cell death started to occur
within microcolonies, and subpopulations of dead cells were
observed in 95% of the microcolonies (Fig. 2C and Table 3).
Cell death extended throughout the biofilms, before the bio-
film structure started to disrupt and detach, suggesting that,
similar to P. tunicata, cell death plays a role during biofilm
development and subsequent dispersal in M. mediterranea.
The SB1 mutant strain does not show cell death during
biofilm development. To assess whether biofilm cell death in
M. mediterranea is mediated by LodA, biofilm development of
a lodA mutant strain (SB1) (31) was investigated. The SB1
mutant also formed a biofilm with microcolony-based architec-
ture. However, similar to the P. tunicata ?AlpP mutant major
cell death events did not occur during any stage of biofilm
development (Fig. 2B and D and Table 3), and only a few
individual dead cells were observed in the SB1 mutant biofilm.
To further support the hypothesis that LodA causes cell death
in M. mediterranea, purified LodA was added back to mature
SB1 mutant biofilms. The add-back of purified LodA to mature
SB1 mutant biofilms induced cell death (Fig. 3A) in 78% of the
microcolonies, while the buffer control still showed only viable
cells, suggesting that LodA mediates cell death in M. mediter-
ranea biofilms (Fig. 3B).
FIG. 1. AlpP produces H2O2from L-lysine. The Amplex Red re-
agent reacts with H2O2in the presence of peroxidase (HRP) to pro-
duce the red fluorescent oxidation product resuforin. High fluores-
cence (390,000 counts) was detected in the presence of the H2O2
standard (12 ?M) (Œ). A similar fluorescence intensity was detected
when AlpP (0.13 mg ml?1) was added to the substrate L-lysine (50
mM) (f) in the presence of Amplex Red. However, no fluorescence
was detected in the presence of catalase (0.1 mg ml?1), AlpP (0.13 mg
ml?1), and L-lysine (50 mM) (?) and when no AlpP was added to the
FIG. 2. Cell death occurs during biofilm development of M. mediterranea wild-type but not the SB1 mutant. Biofilms were stained with the
BacLight LIVE/DEAD viability kit. (A) Wild-type, 24 h; (B) SB1 mutant, 24 h; (C) wild-type, 72 h; (D) SB1 mutant, 72 h.
5496MAI-PROCHNOW ET AL.J. BACTERIOL.
Hydrogen peroxide can be detected in biofilms of P. tunicata
and M. mediterranea. Because of the finding that AlpP and
LodA mediate cell death and produce hydrogen peroxide, we
hypothesized that hydrogen peroxide can be detected in bio-
films at the onset of killing. Amplex Red staining was used to
visualize hydrogen peroxide in biofilms of P. tunicata and M.
mediterranea. High red fluorescence was observed in wild-type
biofilms at the time of killing (Table 3), indicating the presence
of hydrogen peroxide in the biofilms. However, little or no
fluorescence was detected in the ?AlpP mutant of P. tunicata
or the SB1 mutant of M. mediterranea, where no cell death
occurs, supporting the hypothesis that hydrogen peroxide is
involved in biofilm killing. To provide further evidence for this
model, catalase was added to the biofilm media to remove
hydrogen peroxide. Biofilm cell death was almost entirely pre-
vented by the addition of catalase, and little or no hydrogen
peroxide was detected within the biofilms after the catalase
treatment (Table 3).
To further strengthen the notion that hydrogen peroxide is
involved in biofilm killing in organisms containing AlpP homo-
logues, ?AlpP mutant biofilms were exposed to hydrogen per-
oxide to induce cell death. The add-back of hydrogen peroxide
led to cell death within the center of the majority of micro-
colonies (78%) in a fashion similar to when the purified pro-
tein was added to the mutant biofilms (34) (Fig. 3C).
Lysine is produced in P. tunicata and M. mediterranea bio-
films. Because the biofilm medium used in the present study
does not contain lysine, it was investigated whether endoge-
nous lysine is produced by the cells in the biofilm to serve as a
substrate for AlpP or LodA, respectively. Lysine was detected
in the biofilm effluent of P. tunicata and M. mediterranea at 72
and 144 h of biofilm development by using HPLC analysis. The
TABLE 3. H2O2can be detected in biofilms at the onset of cell death, and the addition of catalase prevents biofilm cell death
In minimal medium PI fluorescence (dead
AR fluorescence (H2O2)
In minimal medium
PI fluorescence (dead
AR fluorescence (H2O2)
aTwo sets of triplicate biofilms were inoculated for each strain, one set in minimal medium and the other set in minimal medium with the addition of catalase (100
?g ml?1) after 24 h. Biofilms were allowed to establish for 4 days before staining with the BacLight LIVE/DEAD viability kit to detect cell death and with the Amplex
Red (AR) reagent to localize hydrogen peroxide, as indicated in column 2.
bThe fluorescence intensity was scored as follows: ???, high; ??, medium; ?, low; –, none.
FIG. 3. Add-back of purified LodA to M. mediterranea SB1 mutant and add-back of hydrogen peroxide to ?AlpP mutant biofilms can induce
cell death. Biofilms were stained with the BacLight LIVE/DEAD kit. (A) Add-back of LodA to 72-h SB1 mutant biofilms; (B) 72-h SB1 mutant
biofilm plus buffer control, (C) add-back of 1 mM H2O2to 72-h ?AlpP mutant biofilms; (D) 72-h ?AlpP mutant biofilm plus buffer control.
VOL. 190, 2008 ROLE OF HYDROGEN PEROXIDE IN BIOFILM FORMATION 5497
lysine concentration in the biofilm effluent at 72 h was 313.2
?M for P. tunicata and 165.5 ?M for M. mediterranea. Similar
lysine levels were measured at 144 h with 333.6 ?M in P.
tunicata and 126.4 ?M in M. mediterranea biofilm effluent.
Because these concentrations are at least 1 order of magnitude
above the Km’s of AlpP (see above) and LodA (17), we expect
to have maximum catalytic activity in the biofilm.
LodA-mediated cell death in M. mediterranea is linked to
phenotypic variation. In P. tunicata, AlpP-mediated cell death
has been linked to the generation of a metabolically active and
phenotypically diverse dispersal population (35). Detached cells
of the ?AlpP mutant showed significantly lower variation in mo-
tility, growth, and biofilm formation. In the present study, we
investigated whether LodA-mediated cell death in M. mediterra-
nea is also implicated in the generation of phenotypic variation
among biofilm dispersal cells. M. mediterranea wild-type and SB1
dispersal cells were tested for variation in growth and biofilm
formation at three different time points during biofilm develop-
ment. The M. mediterranea wild type showed higher variation in
growth ability at all time points investigated. Variation in growth
among the 20 randomly picked colonies was 19% in the wild-type
compared to only 5% in the SB1 mutant strain. Variation in
biofilm formation was highest in the wild-type at the 144-h time
point when cell death had occurred extensively throughout the
biofilm. At this time point variation was 45% in the wild-type
compared to only 25% in the mutant strain (Fig. 4). These results
suggest that LodA-mediated biofilm cell death in M. mediterranea
is linked to the generation of a phenotypically diverse dispersal
Hydrogen peroxide mediates cell death in biofilms of C.
violaceum and C. crescentus. Because AlpP homologues were
also identified in other gram-negative bacteria, including C.
violaceum (32% identity) and C. crescentus (27% identity), we
investigated whether biofilm cell death also occurs in these two
organisms. C. violaceum and C. crescentus formed a micro-
colony-based biofilm, and cell death occurred within the center
of mature microcolonies at 3 days postinoculation (Fig. 5A and
B). Ninety-three and ninety-five percent of the microcolonies
of C. violaceum and C. crescentus, respectively, showed dead
cells within the microcolony center. Similar to the observation
for P. tunicata and M. mediterranea, hydrogen peroxide was
detected at the time of killing associated with C. violaceum and
C. crescentus microcolonies using Amplex Red staining. More-
over, the addition of catalase removed hydrogen peroxide and
also prevented cell death in biofilms of C. violaceum and C.
crescentus, indicating that hydrogen peroxide is involved in the
killing (Table 3).
To investigate whether hydrogen peroxide in C. violaceum
and C. crescentus biofilms is a result of the lysine oxidase
activity of the AlpP-homologues, isogenic mutants (C. viola-
ceum CVMUR1 and C. crescentus CAUMUR1) were created
and tested for the occurrence of biofilm cell death and hydro-
gen peroxide. Both mutants formed a biofilm with a similar
architecture than the wild types consisting of microcolonies.
The CVMUR1 strain did not show cell death during biofilm
development, and no hydrogen peroxide could be detected,
indicating that its AlpP homologue is responsible for biofilm
killing in the wild type. In comparing the wild-type and the C.
crescentus CAUMUR1 strains, we noted that cell death oc-
curred in many but not all microcolonies of the mutant strain
(81%). Moreover, hydrogen peroxide was detected localized
into the microcolonies in CAUMUR1 biofilms, suggesting that
additional processes leading to hydrogen peroxide-mediated
cell death occur in this organism.
Selective lysis of cell subpopulations appears to be a highly
evolved and widespread mechanism among prokaryotes (3,
16), and several roles have been suggested for this process. It
was shown that killing of siblings in Streptococcus pneumoniae
leads to the release of a key virulence factor (Ply) and other
cytoplasmic contents during colonization in humans (16, 19).
Moreover, it was recently demonstrated that the suicide gene
spxB mediates cell death via hydrogen peroxide and confers a
survival advantage in colonization in S. pneumoniae (46). In B.
subtilis, sporulating cells can trigger the lysis of sibling cells and
feed on subsequently released nutrients, thereby delaying the
energy-costly sporulation process (18). The death of cell sub-
populations is also required during differentiation processes in
Streptomyces antibioticus (14, 38) and during the formation of
multicellular fruiting bodies of myxobacteria (53). In classically
nondifferentiating bacteria cell death occurs during biofilm
formation (4, 13, 27, 34, 52). The occurrence of cell death
events of subpopulations of cells in different microorganisms
and within diverse processes suggests a role for cell death for
enhanced survival of the remaining cells as seen, for example,
by the generation of phenotypically different biofilm dispersal
FIG. 4. M. mediterranea wild-type dispersal cells show a higher vari-
ation than M. mediterranea SB1 mutant. Variation coefficient of M.
mediterranea wild-type (f) and SB1 mutant ( ) biofilm dispersal cells
in growth (A) and biofilm formation (B). Variation coefficient (%)
calculated for 20 colonies for time points 24, 72, and 144 h after biofilm
5498 MAI-PROCHNOW ET AL.J. BACTERIOL.
AlpP and its homologues mediate cell death via the produc-
tion of hydrogen peroxide. The present study shows that AlpP
can produce hydrogen peroxide from the oxidation of L-lysine
(Fig. 1). Such a mode of action was first discovered for the
AlpP homologue LodA produced by M. mediterranea (31).
Interestingly, our results show that the substrate lysine is pro-
duced endogenously by the biofilm bacteria and consequently
allows for AlpP/LodA activity. The fact that the lysine concen-
tration detected in the biofilm effluent is significantly higher
than the Kmdetermined for AlpP (see results above) and
LodA (17) strongly suggests that there is enough substrate for
the enzyme to have maximum activity. A similar finding has
recently been reported in the sea hare Aplysia californica,
where a lysine oxidase and the amino acid are released from
the cells at the same time (29). Interestingly, endogenous pro-
duction of hydrogen peroxide was also shown in the human
bacterial pathogen S. pneumoniae, where the gene responsible
for hydrogen peroxide production induces an apoptosis-like
death that confers a survival advantage in colonization (46).
In eukaryotes L-lysine oxidases were first isolated from the
fungus Trichoderma sp. (30). Recently, an L-amino acid oxidase
with lysine oxidase activity was detected in the ink of the sea
hare A. californica (54). These enzymes have since become a
focus of research because of their unique properties, which
include cytotoxic, antitumor, antimetastatic, antiinvasive, anti-
viral, and antibacterial activities (5, 33). These characteristics
are suggested to be due to a decrease in the essential amino
acid L-lysine and the production of hydrogen peroxide. The
antibacterial activity of lysine oxidases was first demonstrated
using a rec mutant of Bacillus subtilis as a target strain. The rec
mutant was more sensitive to the antibacterial effect of the
lysine oxidase than the B. subtilis wild-type (30), suggesting that
the damaging effect of hydrogen peroxide on DNA is respon-
sible for the antibacterial activity, rather than the decrease of
L-lysine. Further evidence for this resulted from the ability of
catalase to protect the cells by removing hydrogen peroxide
The AlpP homologue LodA also mediates cell death in M.
mediterranea biofilms, leading to increased phenotypic varia-
tion among biofilm dispersal cells. M. mediterranea strain
MMB-1 is a melanogenic marine bacterium originally isolated
from a water column sample from the Mediterranean Sea (48).
Recently, however, new strains have been isolated from the
microbial communities associated with the sea grass Posidonia
oceanica in different Mediterranean areas (E. Marco-Noales,
unpublished data; E. Espinosa et al., unpublished data), sug-
gesting that this could be the microhabitat it occupies. The
results of the present study demonstrated, for the first time,
that M. mediterranea is able to form biofilms which may con-
tribute to its colonization ability and establishment of M. medi-
terranea on the plant surface. Similarly to P. tunicata, subpopu-
lations of cells die during its biofilm formation. Furthermore, it
was demonstrated that cell death in M. mediterranea biofilms is
mediated by its lysine oxidase LodA and that this process plays
a role in the dispersal of the organism.
Similar to the P. tunicata biofilm life cycle, cell death seems
FIG. 5. Cell death also occurs in C. violaceum and C. crescentus biofilms. Biofilms were stained with the BacLight LIVE/DEAD kit at 4 days
postinoculation. (A) C. violaceum wild-type; (B) C. crescentus wild-type; (C) C. violaceum CVMUR1; (D) C. crescentus CAUMUR1.
VOL. 190, 2008 ROLE OF HYDROGEN PEROXIDE IN BIOFILM FORMATION5499
to play a role in generating a phenotypically diverse dispersal
population from M. mediterranea biofilms. The present study
shows that the onset of LodA-mediated killing correlates with
the generation of high variation among dispersal cells of M.
mediterranea biofilms. However, because some variation also
occurs before the onset of cell death in wild-type dispersal
cells, it is possible that while LodA may be produced at low
undetectable levels at early stages of biofilm formation, these
concentrations are sufficient to induce variation before the
onset of cell death. Variation in biofilm formation also in-
creased with biofilm age among dispersal cells of the SB1
mutant; hence, other factors may play a role in inducing vari-
ation in M. mediterranea biofilms. Many organisms display vari-
ation in cells growing in biofilms, including P. aeruginosa (7, 9,
23, 51), Staphylococcus aureus (45, 47), Staphylococcus epider-
midis (8, 21), Vibrio cholerae (2, 37), Listeria monocytogenes
(42), and P. tunicata (35). Diverse mechanisms that may lead to
an increased genetic and phenotypic variation in biofilm-form-
ing bacteria have been investigated. These mechanisms include
phase variation (9), adaptive mutation (6, 44), enhanced gene
transfer through conjugation, transformation (22, 24, 40),
phage induction (51), and self-induced lysis (35). The process
of generating variation is hypothesized to be beneficial to the
population as the chances of thriving under different environ-
mental conditions are enhanced.
AlpP homologues are common proteins and may have similar
roles during biofilm development. Several AlpP homologues
exist in a range of gram-negative organisms, including Marino-
monas sp. strain MWYL1, M. mediterranea MMB-1, C. viola-
ceum, Magnetococcus sp. strain MC-1, C. crescentus CB15, Sh-
ewanella woodyi ATCC 51908, Hahella chejuensis KCTC 2396,
Roseovarius sp. strain HTCC2601, Burkholderia ambifaria
IOP40-10, Herpetosiphon aurantiacus ATCC 23779, Marinomo-
nas sp. strain MED121, Delftia acidovorans SPH-1, Rhodopseu-
domonas palustris CGA009, Synechococcus sp. strain WH 7805,
Rhodopirellula baltica SH 1, Alphaproteobacterium strain
BAL199, Ralstonia solanacearum UW551, Kordia algicida
OT-1, Nitrobacter hamburgensis X14, Plesiocystis pacifica
SIR-1, Saccharophagus degradans 2-40, Nitrobacter sp. strain
Nb-311A, and Laccaria bicolor S238NH82, suggesting that it is
a common protein among bacteria, where it may play a similar
role to the one described here.
Apart from P. tunicata and M. mediterranea, a possible role
for AlpP homologues in biofilm formation in two organ-
isms—C. violaceum and C. crescentus—was investigated. To
our knowledge, the present study is the first report describing
biofilm development and architecture of C. violaceum. We find
that C. violaceum also forms a differentiated, microcolony-
based biofilm, and cell death occurs in the center of micro-
colonies during the normal course of biofilm development. The
AlpP homologue was clearly linked to these cell death events
because the isogenic mutant C. violaceum CVMUR1 did not
undergo biofilm killing. Previously, biofilm development of C.
crescentus has been investigated, and the formation of micro-
colonies was observed. Moreover, the occurrence of cell death
within the center of microcolonies was described and suggested
to be due to senescence (13). Interestingly, biofilm cell death
was still observed in the C. crescentus CAUMUR1 mutant,
although to a lesser extent than in the wild type. Although our
results show that hydrogen peroxide causes cell death in C.
crescentus biofilms (because it can be detected at the time of
killing and catalase significantly reduces cell death), it remains
to be elucidated what processes, apart from a possible lysine
oxidation by the AlpP homologue, are involved in generating
hydrogen peroxide. Because C. crescentus is an obligate aerobe
bacterium and several oxidases have been identified in its ge-
nome, alternative ways for generating hydrogen peroxide dur-
ing biofilm formation are feasible.
Hydrogen peroxide plays a role in biofilm cell death of P.
tunicata. M. mediterranea, C. violaceum, and C. crescentus. Hy-
drogen peroxide is a low-molecular-weight compound that acts
on a wide range of molecules and is likely to facilitate cell
death and dispersal even in complex microbial communities
such as those on living marine surfaces. Hydrogen peroxide
was detected in biofilms of P. tunicata, M. mediterranea, C.
violaceum, and C. crescentus at the time when cell death occurs
(Table 3). Although the exact mechanism of hydrogen perox-
ide-mediated cell death remains to be elucidated, cells in the
center of microcolonies appear to be more susceptible because
the add-back of hydrogen peroxide leads to cell death within
the center of microcolonies. In the add-back experiment a
molar concentration of hydrogen peroxide three times higher
than the apparent lysine concentration was required to induce
cell death in the majority of microcolonies in the ?AlpP mu-
tant biofilms. It was previously shown that high catalase activity
can prevent penetration of hydrogen peroxide into biofilms of
P. aeruginosa at a concentration of 50 mM (49). The P. tunicata
genome also possesses three catalase genes (T. Thomas et al.,
unpublished results), which could be responsible for prevent-
ing penetration of exogenously added hydrogen peroxide, as
well as playing a role in different susceptibility of the cells to
In summary, the present study suggests that endogenous
hydrogen peroxide-mediated lysis of a subpopulation of cells
occurs in P. tunicata, M. mediterranea, C. violaceum, and C.
crescentus. AlpP homologue-mediated hydrogen peroxide ap-
pears to be fully responsible for autolysis of biofilm cells in P.
tunicata, M. mediterranea, and C. violaceum. We propose that
this process is an important mechanism for facilitating dis-
persal and generating diversity. It is suggested that hydrogen
peroxide allows (directly or indirectly) both killing of a sub-
population of cells (30) and a possible increase in DNA dam-
age and mutation frequency (50) of the remaining live cells,
which may lead to the observed phenotypic variation in the
dispersal cell population.
We acknowledge the assistance by Geoff Kornfeld and Abraham
Choy for the lysine analysis. We also thank Antonio A. Iniesta for the
plasmid pNPTS138 and helpful advice on the generation of the C.
crescentus CAUMUR1 mutant strain.
This research was supported by grants from the Australian Research
Council and the Centre for Marine Bio-Innovation at the University of
New South Wales (UNSW), Sydney, Australia. P.L.-E. was the recip-
ient of a grant from the Consejerı ´a de Educacio ´n y Cultura of the
Comunidad Auto ´noma de la Regio ´n de Murcia, Murcia, Spain, which
allowed her to visit UNSW.
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