APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2006, p. 5547–5555 Vol. 72, No.

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0099-2240/06/$08.00⫹0 doi:10.1128/AEM.00449-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Microbial Colonization and Competition on the Marine Alga Ulva australis

Dhana Rao, Jeremy S. Webb,† and Staffan Kjelleberg*
School of Biotechnology and Biomolecular Sciences and Centre for Marine Biofouling and Bio-innovation,
University of New South Wales, Sydney, New South Wales 2052, Australia

Downloaded from http://aem.asm.org/ on January 2, 2015 by BRITISH COLUMBIA INST OF TECH

Received 23 February 2006/Accepted 10 May 2006

Pseudalteromonas tunicata and Roseobacter gallaeciensis are biofilm-forming marine bacteria that are often
found in association with the surface of the green alga Ulva australis. They are thought to benefit the plant host
by producing inhibitory compounds that are active against common fouling organisms. We investigated factors
that influence the ability of P. tunicata and R. gallaeciensis to attach to and colonize the plant surface and also
the competitive interactions that occur between these organisms and other isolates from U. australis during
biofilm formation on the plant surface. A surprisingly high number of P. tunicata cells, at least 108 cells mlⴚ1,
were required for colonization and establishment of a population of cells that persists on axenic surfaces of U.
australis. Factors that enhanced colonization of P. tunicata included inoculation in the dark and pregrowth of
inocula in medium containing cellobiose as the sole carbon source (cellulose is a major surface polymer of U.
australis). It was also found that P. tunicata requires the presence of a mixed microbial community to colonize
effectively. In contrast, R. gallaeciensis effectively colonized the plant surface under all conditions tested. Studies
of competitive interactions on the plant surface revealed that P. tunicata was numerically dominant compared
with all other bacterial isolates tested (except R. gallaeciensis), and this dominance was linked to production of
the antibacterial protein AlpP. Generally, P. tunicata was able to coexist with competing strains, and each
strain existed as microcolonies in spatially segregated regions of the plant. R. gallaeciensis was numerically
dominant compared with all strains tested and was able to invade and disperse preestablished biofilms. This
study highlighted the fact that microbial colonization of U. australis surfaces is a dynamic process and
demonstrated the differences in colonization strategies exhibited by the epiphytic bacteria P. tunicata and R.
gallaeciensis.

Biofouling is caused initially by bacterial growth and biofilm
formation on natural and artificial surfaces. Bacterial biofilms
facilitate the attachment and growth of a range of other fouling
organisms, such as diatoms, invertebrate larvae, and algal
spores (18). Strategies to prevent bacterial biofilms, therefore,
represent a powerful approach to the prevention of biofouling.
Biofouling communities on surfaces of marine plants and
animals can have detrimental effects on the host organism.
Physical damage to the host results from the production of
toxins, digestive enzymes, and waste products by the microbial
community. However, the extent of biofouling on marine or-
ganisms is markedly less than the extent of biofouling on in-
animate structures. Often the later stages of biofouling, such as
the attachment of algae and barnacles, do not occur (2). In
contrast to inanimate surfaces, which are colonized in a rapid
and predictable manner by a diverse assemblage of marine
microbes (62), biotic surfaces frequently harbor species-spe-
cific microbial communities (22, 61, 62). These communities
can be variable and distinct from those found in the surround-
ing environment. Thus, many algal and invertebrate species are
able to regulate the bacterial colonization of their surfaces (29,
32, 62).
Microalgal surfaces are typically covered by bacteria at den-
* Corresponding author. Mailing address: School of Biotechnology
and Biomolecular Sciences and Centre for Marine Biofouling and
Bio-innovation, Biological Sciences Building, University of New South
Wales, Sydney, NSW 2052, Australia. Phone: 61 (2) 9385 2102. Fax: 61
(2) 9385 1779. E-mail: s.kjelleberg@unsw.edu.au.
† Present address: School of Biological Sciences, University of
Southampton, Southampton SO16 7PX, United Kingdom.
sities of approximately 107 bacteria cm⫺2 (3). Studies of the
epiphytic microbial communities present on macroalgae have
emphasized the spatial distribution of bacteria, with specific
parts of the thallus hosting specific bacterial populations (9). In
some cases the bacterial populations change with the season or
the age of the host (55, 56). Associations between algae and
bacteria are common, and studies have generally focused on
the benefits provided to the bacteria, such as support of bac-
terial growth by dissolved organic carbon released by algal cells
(30, 34, 49).
Although some of the bacterium-alga interactions have been
characterized, the ecological significance of most naturally oc-
curring epiphytic bacterial communities is unclear, and in
many cases the bacterial species involved have not been iden-
tified (17). It has been demonstrated that bacterial biofilms are
present on the surface of Ulva australis (formerly known as
Ulva lactuca) (54), and it has been speculated that the seaweed
uses microbial defense to protect against fouling (24, 27). U.
australis has no known physical or chemical defense systems
against fouling organisms, and it has been suggested that the
host may manipulate the bacterial community on its surface,
which in turn protects the host by interfering with the devel-
opment of a mature biofouling community. Such interactions
are not uncommon in the marine environment (for reviews see
references 4, 7, 15, 19, 26, and 44).
One important species of marine bacteria that is found in
association with U. australis is Pseudoalteromonas tunicata.
This bacterium produces a diverse range of biologically active
compounds that specifically target marine fouling organisms
(25). One of these compounds is a 190-kDa multisubunit an-

5547
5548 RAO ET AL.
tibacterial protein designated AlpP which is effective against
both gram-negative and gram-positive bacteria from a range of
environments (28, 37). Recent work has demonstrated that
AlpP can provide a competitive advantage to P. tunicata during
biofilm growth in laboratory biofilm experiments (48).
Roseobacter gallaeciensis is also frequently isolated from the
surface of U. australis. Roseobacter spp. are cosmopolitan and
have been isolated, for example, from green seaweeds (53),
marine snow particles (22), and dinoflagellates (1, 33, 39).
Roseobacter spp. are able to metabolize dimethylsulfoniopro-
pionate (DMSP), and their presence and activity on algal sur-
faces are significantly correlated with DMSP-producing algae,
including dinoflagellates and prymnesiophytes (20).
The extensive, highly diverse microbial community associ-
ated with U. australis makes it an interesting study organism for
addressing questions of surface colonization and host associa-
tion. The dynamics of surface colonization in natural systems,
particularly during the early stages of biofilm establishment,
are poorly understood for marine bacteria. For example, an
assessment of the ability of distinct bacteria to colonize algal
surfaces would be useful for identifying bacterial traits that
contribute to epiphytic fitness. We recently described compet-
itive biofilm interactions of P. tunicata and other marine strains
in a laboratory glass flow cell system (48). However, little is
known about the ecology of colonization and competition on a
living surface, including whether P. tunicata is a dominant
competitor in ecologically relevant settings.
In this study we aimed to investigate, for the first time, the
colonization biology of marine bacteria on the surface of a
marine plant, in this case U. australis. We investigated the
hypothesis that P. tunicata and R. gallaeciensis are effective
colonizers of U. australis and are able to compete with and
dominate other marine bacterial isolates during biofilm forma-
tion on the plant surface. We found that P. tunicata requires
the presence of a natural seawater community to colonize
effectively, whereas R. gallaeciensis is an aggressive colonizer
under all conditions tested. Below we highlight the differences
in colonization strategies exhibited by the epiphytic strains of
P. tunicata and R. gallaeciensis and demonstrate that antibac-
terial compounds have a role in the colonization of U. australis.
This study demonstrated that microbial colonization of the
plant surfaces is a dynamic process in which differences in
attachment, colonization, and competitive biofilm formation
can markedly affect the establishment and organization of
epiphytic microbial communities.
MATERIALS AND METHODS
Collection of plants and generation of axenic U. australis. The common marine
alga U. australis was collected from the rocky intertidal zone (latitude, 151.2572;
longitude, ⫺33.9121) near Sydney, Australia. The method used to make U.
australis axenic was modified from the method described by Scheffel (51). Briefly,
plants were rinsed in 50 ml autoclaved seawater and cut into 5-cm-long frag-
ments. These fragments were swabbed with cotton buds, and 0.6-cm disks were
punched out of the thallus using a cork borer. The disks were rinsed in auto-
claved seawater and incubated in 0.012% NaOCl for 5 min. The disks were
allowed to recover in sterile seawater for 1 h before they were incubated in an
antibiotic cocktail (300 mg liter⫺1 ampicillin, 30 mg liter⫺1 polymyxin B, and 60
mg literl⫺1 gentamicin dissolved in sterile seawater) for 18 h in 24-well tissue
culture plates (Sarstedt). Disks were then incubated in 0.008% NaOCl for 5 min.
These disks were allowed to recover in sterile seawater for at least 3 h to remove
traces of oxidants, suspended in 2 ml of sterile seawater, and agitated at room
temperature at 60 rpm.
APPL. ENVIRON. MICROBIOL.
Plant tissue viability was examined by staining treated U. australis disks with
0.25% (wt/vol) Evans blue (60). This blue dye penetrates into dead and damaged
plant cells, while intact cells are able to exclude the dye. All colonization and
competition experiments were done in triplicate, and three to five individual
disks were randomly sampled at each sampling time.
Colonization experiments. Bacterial strains were isolated from the surface of
U. lactuca as described previously (48). Cultures were stored at ⫺80°C in 50%
(vol/vol) glycerol in VNSS medium (38) and maintained on VNSS medium
plates. P. tunicata and R. gallaeciensis were labeled with a green fluorescent
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protein (GFP) color tag as described previously (48). Bacteria were cultured for
24 h at 25°C in VNSS broth for preparation of inocula. Cells were harvested by
spinning down the culture and resuspending the pellet in seawater. The cell
concentration was estimated by counting cells by epifluorescence microscopy
using a hemocytometer and was adjusted to the desired level by dilution with
seawater. Experiments were conducted in Sarstedt 24-well plates with axenic
disks, and the bacteria were applied by immersing the disks in a suspension of P.
tunicata and R. gallaeciensis for 3 h. The disks were then rinsed twice in filtered
seawater and transferred to fresh Sarstedt 24-well plates containing 2 ml of
filtered seawater. The plates were incubated on a shaker at 60 rpm and 25°C for
16 h in the presence of light.
Factors affecting colonization. In order to investigate some of the factors that
were essential for colonization of P. tunicata, we tested a range of conditions.
(i) Density of P. tunicata required for attachment. P. tunicata was inoculated
onto axenic U. australis disks in 24-well tissue culture plates at a range of
concentrations (104 to 108 cells ml⫺1). The disks were incubated for 3 h, rinsed
twice in sterile seawater, and then incubated at 25°C and 60 rpm for the duration
of the experiment.
(ii) Axenic versus nonaxenic plant surfaces. The attachment of P. tunicata
cells which had been inoculated at a concentration of 107 cells ml⫺1 was com-
pared on the both axenic and nonaxenic surfaces of U. australis disks.
(iii) U. australis disks versus whole plants. Attachment was tested on axenic
disks in 24-well plates, as well as on axenic whole plants (which had been treated
in the same way as the disks). The plants were oriented upright in beakers and
immersed in filtered seawater.
(iv) Dark versus light. Preparations were inoculated in the dark at a concen-
tration of 107 cells ml⫺1 and then incubated in the dark for 3 h. This procedure
was compared to inoculation at the same density and incubation for 3 h in
daylight.
(v) Carbon source. The standard culture conditions for marine strains included
VNSS medium containing glucose. However, we also grew cells for 48 h in
minimal medium (45) with cellobiose as the sole carbon source in a still culture.
The cells were inoculated onto U. australis disks, and attachment was compared
to the attachment of cells grown in minimal medium with trehalose and glucose
as carbon sources.
(vi) Incubation time. Disks were inoculated with 107 cells ml⫺1 and incubated
for 1, 3, 12, and 24 h.
(vii) Effect of multispecies consortia on colonization. Axenic algal disks were
inoculated with overnight cultures of GFP-labeled P. tunicata and 17 unlabeled
strains isolated from the surface of U. australis (48) in filtered seawater. P.
tunicata was also inoculated into a natural seawater community. The resulting
mixed-species biofilm was visualized by staining with acridine orange. Attach-
ment and colonization of GFP-labeled P. tunicata within the mixed biofilm were
visualized by observing the unstained biofilm with a confocal microscope.
To test all factors, a density of 107 cells ml⫺1 was used unless otherwise stated,
and P. tunicata cells were suspended in filtered seawater. Attachment, biofilm
formation, and microcolony development were monitored by epifluorescence
microscopy. The times when preparations were examined were immediately after
inoculation and 1, 3, and 7 days following inoculation. At each sampling time,
three disks were randomly selected, and a total of 12 random fields of view were
counted for each disk. Samples were viewed by epifluorescence microscopy with
an Axiophot microscope equipped with a 10⫻ 1.30-numerical-aperture objective
(Leica).
Although preliminary experiments indicated that R. gallaeciensis was an ag-
gressive colonizer of U. australis, we also tested attachment and colonization of
this organism under all the experimental conditions used for P. tunicata.
Competition in dual-species biofilms on U. australis. Other marine organisms
used in dual-species competition experiments were Pseudalteromonas gracilis,
Alteromonas sp., and Cellulophaga fucicola, all of which were isolated from U.
australis as described previously (48). The strains were labeled with red fluores-
cent protein (DsRed) and GFP as described previously (48). The growth rates of
wild-type marine isolates and their labeled derivatives on the surface of axeni-
cally treated U. australis did not differ significantly (data not shown).
Overnight cultures of bacteria were inoculated onto U. australis at a density of
VOL. 72, 2006 MICROBIAL COLONIZATION ON U. AUSTRALIS 5549

used to make the plants axenic. Rather, other factors appeared

to play a role in the bleaching of plant material after treatment.
This was shown by (i) Evans blue staining of plant tissue, which
revealed that the tissue was viable and healthy after treatment,
and (ii) the fact that treated plant disks that were reinoculated
with bacteria in the course of the experiments did not bleach,
suggesting that the microbial community itself may contribute
to the fitness of the host.

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Factors influencing attachment and colonization. Prelimi-
nary experiments showed that P. tunicata colonized the surface
FIG. 1. Effect of axenic treatment on the U. australis epiphytic of U. australis poorly, which led us to test attachment under
community. (A) Nonaxenic U. australis with the natural community different conditions. Our data provide quantitative information
present. (B) Axenic U. australis with very few bacteria remaining. Only
concerning the attachment of P. tunicata on the surface of U.
plant cells are visible. Specimens were stained with 0.01% acridine
orange. Scale bars ⫽ 50 ␮m. australis under a range of different environmental conditions
(Table 1). P. tunicata harboring a GFP reporter gene was
visualized by epifluorescence microscopy to differentiate P.
tunicata cells from indigenous bacteria on the algal surface. We
108 cells ml⫺1 in filtered and natural seawater and incubated for 3 h without
shaking at room temperature. Disks were rinsed three times with filtered sea- observed cells after a 3-h attachment period and found that
water, transferred to sterile seawater in Sarstedt 24-well plates, and incubated at they were not randomly scattered across the U. australis thallus
25°C at 60 rpm for the duration of the experiment. Unlabeled biofilms were surface. Rather, cells were distributed in patches on the plant
visualized on the surface of the plant by staining with acridine orange. Biofilms surface. Observations after 24 h of growth showed that some of
were grown in filtered seawater and visualized with a confocal laser scanning
microscope (Olympus) using fluorescein isothiocyanate and tetramethyl rhoda-
the attached cells had divided and formed microcolonies,
mine isocyanate optical filters for green and red fluorescent proteins, respec- which occurred in a wide range of sizes (5 to 50 ␮m). Obser-
tively. Labeled P. tunicata was inoculated onto the surface with competing ma- vations after 3 days of incubation revealed that vast areas of U.
rine strains, including R. gallaeciensis. To investigate whether the antibacterial australis thallus tissue remained uncolonized and that micro-
protein produced by P. tunicata improved the competitiveness of this organism
colonies were restricted to certain regions.
on the surface of U. australis, competition studies were also performed with the
AlpP mutant of P. tunicata (37). On the other hand, R. gallaeciensis formed microcolonies
To cultivate mixed biofilms, disks were inoculated with a 500-␮l suspension of which were dispersed evenly over the surface of the thallus
stationary-phase cultures of P. tunicata and the competitive marine strain in tissue. At 24 h, R. gallaeciensis had formed cell chains, which by
filtered seawater. In order to ensure that the initial ratio of attached cells of the day 3 had developed into small microcolonies (diameter, 10 to
two competing strains was 1:1, we first monitored the initial attachment of the
mixed culture after a 3-h adhesion period. In most cases, the initial levels of
20 ␮m). By day 7, these microcolonies had merged to form a
attachment to the plant surface were deemed to be approximately equal, as
determined by counting the numbers of red- and green-labeled cells by epiflu-
orescence microscopy.
To investigate whether P. tunicata and R. gallaeciensis were able to invade and TABLE 1. Factors influencing attachment and colonization
colonize an established biofilm, marine strains (P. tunicata, C. fucicola, and of P. tunicata on U. australisa
Alteromonas sp.) were allowed to preestablish a biofilm for 48 h on U. australis.
No. of cells attached (cells cm⫺2)
The preformed biofilm was inoculated with ⬃108 cells of wild-type P. tunicata or (mean ⫾ SE) at:
R. gallaeciensis, incubated for 3 h without shaking, and then incubated at 60 rpm Factor
for the duration of the experiment. After the resumption of shaking, the biofilm 24 h 3 days 7 days
was examined for red and green fluorescence. Experiments were repeated in 4 ⫺1
three separate rounds with three independent 24-well plates run in parallel. The Density, 10 cells ml 0 0 0
viability of bacterial cells was determined using a Live-Dead staining kit (Mo- Density, 105 cells ml⫺1 0 0 0
lecular Probes Inc., Eugene, Oreg.). Density, 106 cells ml⫺1 52 ⫾ 3.0 13 ⫾ 2.5 0
Density, 107 cells ml⫺1 224 ⫾ 4.9 92 ⫾ 4.9 0
Density, 108 cells ml⫺1 1,880 ⫾ 58b 848 ⫾ 43 0
RESULTS Axenic disks 290 ⫾ 11.4 125 ⫾ 3.9 0
Nonaxenic disks 270 ⫾ 10 131 ⫾ 9.7 0
Obtaining axenic plant tissue. In order to conduct the at- Carbon source, trehalose 250 ⫾ 6.9 112 ⫾ 3.6 0
tachment and colonization experiments, it was necessary to Carbon source, cellobiose 460 ⫾ 7.7b 276 ⫾ 9.9 0
Incubation time, 1 h 184 ⫾ 6.5 72 ⫾ 4.7 0
remove the majority of bacteria on the U. australis thallus Incubation time, 3 h 302 ⫾ 9.3b 118 ⫾ 3.9 0
surface. Observation of untreated algal tissue revealed that Incubation time, 12 h 289 ⫾ 11.4 125 ⫾ 3.9 0
there was a complex community of epiphytic bacteria (approx- Incubation time, 24 h 263 ⫾ 9.2 99 ⫾ 4.4 0
imately 460 cells cm⫺2) (Fig. 1A). However, in treated axenic Inoculation in the light 250 ⫾ 6.8 79 ⫾ 7.2 0
Inoculation in the dark 374 ⫾ 12.6b 171 ⫾ 7.2 0
tissue there were negligible numbers (⬍50 cells cm⫺2) of at- P. tunicata in autoclaved 250 ⫾ 6.6 92 ⫾ 5.2 0
tached solitary cells on the plant surface (Fig. 1B). Antibiotic seawater
treatment resulted in removal of approximately 90% of the P. tunicata in filtered 263 ⫾ 5.8 92 ⫾ 5.8 0
epiphytic bacteria, while there was minimal damage to the U. seawater
australis tissue (as determined by the Evans blue test for plant P. tunicata in natural 539 ⫾ 15.1b 934 ⫾ 26.2 1,880 ⫾ 74b
seawater
viability) (data not shown). Axenic disks that were not inocu-
lated with bacteria in our experiments bleached rapidly and
a
To test all factors apart from density, a density of 107 cells ml⫺1 was used.
The counts are the average values for 12 fields of view for each disk, which were
died within 1 week. However, our data suggest that plant death converted to number of cells cm⫺2.
was probably not the direct result of the chemical treatment b
Significant value.
5550 RAO ET AL.
FIG. 2. Comparison of attachment and colonization by GFP-labeled P. tunicata grown in cellobiose (A) and glucose (B). Magnification, ⫻600.
Scale bars ⫽ 50 ␮m.
relatively flat, mat-like biofilm, which covered a large propor-
tion of the algal surface. Also in contrast to P. tunicata, R.
gallaeciensis cells attached and colonized effectively under all
conditions tested. At cell densities greater than 106 cells ml⫺1,
colonization of U. australis by R. gallaeciensis appeared to be
similar in all situations (data not shown). Furthermore, at-
tached cells persisted indefinitely on algal disks. Compared to
the analysis of P. tunicata, the smaller cell size and the high
rates of cell attachment of R. gallaeciensis made counting dif-
ficult; hence, cell attachment was not quantified.
(i) Effects of inoculum cell density on colonization of U.
australis. Direct in situ observation of epiphytic bacteria re-
vealed that the density of cells in the inoculum affected micro-
colony formation. After an initial attachment period (3 h), we
incubated disks for 24 h and then examined the plant surface
for bacterial cells and microcolony formation. Surprisingly, no
attachment took place when P. tunicata was inoculated at low
densities (104 or 105 cells ml⫺1) (Table 1). At a density of 106
cells ml⫺1 we observed that few cells attached (52 cells cm⫺2).
These cells were mostly solitary; no microcolonies formed, and
cells usually did not persist for more than 3 days. At a density
of 107 cells ml⫺1 some of the attached cells were clustered
together and formed small microcolonies consisting of about
15 to 20 cells after 24 h. At a density of 108 cells ml⫺1, micro-
colonies were considerably larger (up to 100 ␮m across) after
the same growth period (24 h). In spite of the large sizes of
microcolonies obtained with high cell densities, P. tunicata did
not persist for more than 5 days. Although R. gallaeciensis was
able to colonize at lower densities (104 and 105 cells ml⫺1), the
persistence was much greater at higher densities (106 to
108cells ml⫺1). At higher densities R. gallaeciensis rapidly
formed microcolonies that survived and persisted indefinitely
on the plant, compared to the colonies that formed at a low
density, which did not remain for more than 5 or 6 days. Thus,
APPL. ENVIRON. MICROBIOL.
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both organisms exhibited density-dependent colonization and
enhanced microcolony formation at high cell densities.
(ii) Colonization in the dark. On the basis of our observation
of surprisingly low levels of attachment of P. tunicata on the
plant surface, we investigated the possibility that P. tunicata
attachment may be affected by the presence of DMSP on the
plant surface. It is known that DMSP is released in senescent
algae or when algae undergo oxidative stress, particularly un-
der high light intensities (31). We therefore examined coloni-
zation of U. australis by P. tunicata in the dark. We observed
that attachment of P. tunicata improved when cells were inoc-
ulated in the dark, and the number of cells that attached
increased from 250 cells cm⫺2 to 374 cells cm⫺2 (Table 1).
However, the cells did not persist and form biofilms. Most of
the microcolonies formed were small, although the number
was greater (about 16 to 20 microcolonies per cm⫺2 with 12 to
25 cells in each microcolony, compared to about 3 to 6 micro-
colonies per cm⫺2 with 50 to 90 cells in each microcolony for
plant tissue incubated in the presence of light). We repeated
these sets of experiments using the Live-Dead stain to establish
whether cells in smaller microcolonies died or whether they
simply failed to attach. However, the results were inconclusive
as it was difficult to visualize the dead cells against the red
autofluorescence of the algae. In contrast, R. gallaeciensis was
able to attach and colonize at a density of 106 cells ml⫺1
regardless of whether the preparations were inoculated in the
light or in the dark.
(iii) Carbon source. We found that P. tunicata cells pregrown in
media containing cellobiose as a sole source of carbon were able
to attach better on the surface of U. australis. As shown in Table
1 and Fig. 2A, the number of P. tunicata cells that were able to
attach to the surface of the algae was much higher for cells grown
in cellobiose than for cells grown in glucose or trehalose as a
carbon source (Fig. 2B). However, similar to glucose- or treha-
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FIG. 3. Comparison of biofilm formation by mixed-species biofilms containing P. tunicata cells suspended sterile seawater, in natural seawater,
or a mixture of 17 epiphytic strains. P. tunicata inoculated into filtered seawater did not persist for more than 7 days (A). P. tunicata inoculated
into natural seawater was able to attach to U. australis and form biofilms that persisted for up to 3 weeks (B). P. tunicata inoculated with a mixture
of 17 strains isolated from U. australis formed a complex biofilm, which was also able to persist for up to 3 weeks (C). GFP-labeled P. tunicata cells
were enumerated under an epifluorescence microscopy (Table 1) before being stained with 0.01% acridine orange as shown here. Scale bars ⫽
50 ␮m.

lose-grown cells, the cellobiose-grown cells did not persist for
more than 5 days on the plant surface.
(iv) Inoculation in natural seawater and mixed-species bio-
film development. We compared colonization of U. australis by
P. tunicata cells suspended in sterile filtered seawater, auto-
claved seawater, and natural seawater. We observed that in
sterile filtered water, P. tunicata cells did not persist for more
than 5 days (Fig. 3A and Table 1). We also observed that
colonization was most effective in natural seawater. Not only
was there a higher level of attachment in this system, but cells
also became established and persisted indefinitely on the sur-
face of the algal thallus. Together, the bacteria formed a mixed
biofilm, which was stable and was up to 20 ␮m thick (Fig. 3B).
When P. tunicata was inoculated in combination with a mixture
of 17 uncharacterized marine strains isolated from U. australis,
it also formed mixed biofilms and persisted on the surface
indefinitely (Fig. 3C). These biofilms were more stable than
single-species or dual-species biofilms. In contrast to P. tuni-
cata, R. gallaeciensis was able to colonize the surface of the alga
regardless of whether the cells were inoculated in sterile or
natural seawater.
Competitive biofilm development on U. australis. We exam-
ined competitive interactions in mixed-species biofilms during
cocolonization of the surface of U. australis in vivo. After initial
attachment to the plant surface, P. tunicata totally outcom-
peted C. fucicola, Alteromonas sp., and P. gracilis, and no cells
of the latter organisms were visible after 24 h of incubation. In
contrast, P. tunicata was outcompeted by R. gallaeciensis in
these experiments. When C. fucicola, Alteromonas sp., and P.
gracilis were each allowed to preestablish a biofilm on U. aus-
tralis for 48 h before they were challenged with P. tunicata, they
were able to coexist with P. tunicata for the duration of the
experiment. Under these conditions, each of the competing
strains persisted on the plant surface in discrete monospecies
microcolonies, and there was no mixing of the competing or-
ganisms (Fig. 4A). Generally, microcolonies appeared to be
protective structures because, once they were established, re-
moval of microcolonies by competing strains was rarely ob-
served under the conditions used in our experiments. An ex-
ception to this finding occurred in competition experiments
involving R. gallaeciensis; this organism was able to colonize
and to outcompete a preestablished biofilm as effectively as it
colonized the axenic surface of U. australis.
To investigate the role of the antibacterial protein AlpP of P.
tunicata in competitive biofilm development on U. australis, we
repeated the mixed-inoculum biofilm experiments described
above, using the ⌬alpP strain instead of the wild type. When
competing strains were inoculated simultaneously, no differ-
ence in the outcome of the biofilm competition experiments
with either the wild-type or ⌬alpP strain was observed (data
not shown). However, we found that AlpP can play an impor-
tant role in the colonization of preestablished biofilms of other
marine strains on U. australis. The ⌬alpP strain had a greatly
reduced ability to colonize and to establish a biofilm in 48-h-
old biofilms of P. gracilis, C. fucicola, and Alteromonas sp. For
example, the ⌬alpP strain was unable to colonize and invade a
5552 RAO ET AL.
FIG. 4. Competitive biofilm development on the surface of U. australis in preestablished biofilms containing P. tunicata. In competitive biofilm
development P. tunicata (green) was able to dominate in certain situations, but more commonly it was able to coexist with P. gracilis (red) (A).
P. tunicata (green) was, however, outcompeted by R. gallaeciensis (red) (B). The P. tunicata AlpP mutant (green) was less competitive than the
wild-type P. tunicata strain, as shown by competitive biofilm development with P. gracilis (red) (C). Scale bars ⫽ 50 ␮m.
preestablished biofilm of P. gracilis (Fig. 4C). This mutant was
unable to form microcolonies, and the few attached cells had
little or no impact on the proliferation of competing strains.
DISCUSSION
Little is known about the establishment and ecological role
of microbial communities on the surfaces of marine algae.
However, the abundance of bacteria that produce extracellular
inhibitory compounds on the surface of the marine alga U.
australis has prompted speculation that these organisms may
protect the alga against fouling (14, 26). In this study we ex-
plored some of the factors that influence attachment to and
colonization of U. australis by epiphytic bacteria, as well as the
competitive interactions that occur between bacterial strains
on the plant surface.
Effect of cell density on attachment. One of the most im-
portant factors that influenced the attachment of P. tunicata
suspended in filtered seawater was the density of cells in the
inocula. We found, not surprisingly, that the number of cells
attached to the surface was higher when inocula with higher
concentrations of bacteria were used. However, we also ob-
served that surprisingly high densities of cells were necessary
for P. tunicata to become established and grow in microcolo-
nies. Although R. gallaeciensis was able to attach at lower
densities, biofilm formation and colonization were much im-
proved at higher densities in a manner similar to the manner
observed for P. tunicata, perhaps also because high-density
inocula allowed the formation of larger numbers of microcolo-
nies by this organism. The dependence on high cell densities
APPL. ENVIRON. MICROBIOL.
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for microcolony formation has been reported previously for
biofilms on leaf surfaces (41, 42, 65). In these studies, there was
evidence that cells in aggregates had a much greater ability to
survive periodic desiccation stress, and increased tolerance was
correlated with the size of aggregates (41). Presumably, cells in
larger microcolonies are more tolerant of oxidative stress than
cells located in smaller microcolonies are (10), and they may
also be more capable of concentrating nutrients from dilute
sources, which can have important consequences in a nutrient-
poor environment.
The mechanism by which high cell densities allow enhanced
microcolony formation is unclear, but quorum sensing may
have a role in the colonization of U. australis. Quorum sensing
is known to play a role in the formation of multicellular struc-
tures within biofilms (12, 23). To date, there is no evidence that
P. tunicata produces signaling molecules (24), but recent stud-
ies in our laboratory have shown that R. gallaeciensis produces
N-acyl-homoserine lactones, suggesting that it is capable of
quorum sensing (R. Case, A. Low, and S. Kjelleberg, unpub-
lished data). In some epiphytic bacteria, quorum sensing has
previously been shown to play a role in colonization of the
plant, and studies with Pseudomonas syringae have suggested
that N-acyl-homoserine lactone production makes an impor-
tant contribution to epiphytic fitness in the early stages of
colonization of bean leaves (47).
Attachment in the dark. Inoculation in the dark clearly en-
hanced attachment of P. tunicata cells suspended in filtered
seawater, indicating that this organism may be sensitive to
DMSP or reactive oxygen species present on the surface of the
VOL. 72, 2006
algae. DMSP occurs in many diverse species of algae, including
U. australis (14), and is released in senescent algae or under
oxidative stress conditions, particularly under high light inten-
sities (31, 59). There is evidence that DMSP provides protec-
tion against photooxidation for algae (59), but its effects on
epiphytic bacterial communities on Ulva spp. are not fully
understood (46). We observed that attachment of P. tunicata in
the light resulted in fewer but larger microcolonies, whereas
microcolonies established from cells inoculated in the dark
were smaller and more numerous. One possibility is that cells
in larger more resistant microcolonies survived exposure to
oxidative stress, which increased with higher light intensities.
R. gallaeciensis was able to attach and colonize effectively re-
gardless of the light conditions, suggesting that it may be able
to tolerate and/or metabolize DMSP (59). In studies of the
diversity of dimethyl sulfide-producing bacteria from oceanic
and estuarine waters it was found that all the isolates belonging
to the Roseobacter group tested were dimethyl sulfide produc-
ers and therefore could utilize DMSP (21). Another proposed
ecological role for DMSP is that it acts as a precursor of cues
for chemosensory attraction between algae and certain specific
bacteria (57). Thus, DMSP may be one of the chemical signals
released by U. australis which is recognized by R. gallaeciensis.
Furthermore, it has been proposed that the substrate for the
sulfur-based inhibitory compounds produced by R. gallaeciensis
(thiotropocin and tropodithietic acid) is DMSP (8).
Effect of cellobiose on attachment. It is evident that P. tuni-
cata attachment improved considerably when cells were grown
in cellobiose and then suspended in filtered seawater, but the
cells did not persist on the surface of U. australis. Both U.
australis and Ciona intestinalis, from which P. tunicata is most
commonly isolated, contain accessible cellulose polymers in
their cell walls (5, 13). A cellulose binding protein with a high
binding affinity for microcrystalline cellulose was discovered in
P. tunicata (11). Clearly, cellobiose plays a role in attachment,
but its effects may be restricted to the early stages of coloni-
zation, in which it has been suggested to function as an an-
chorage or cue for P. tunicata attachment (58). Recent studies
have found that cellulose stimulated the production of pigment
in this organism, which is known to be coregulated with ex-
pression of AlpP (58), and also induced the expression of
mannose-sensitive hemagglutinin (type IV-like) pili in P. tuni-
cata (11). Thus, cellulose appears to play an important role in
the colonization and responses of P. tunicata on the surface of
U. australis.
Synergistic biofilm formation. P. tunicata persisted on the
surface of the algae only if it was inoculated in seawater that
contained a natural seawater community or if it was inoculated
in filtered seawater that contained a mixture of epiphytic
strains isolated from U. australis. This suggests that P. tunicata
requires the presence of diverse bacteria in the inoculum for
effective colonization, which may allow succession and/or co-
operative colonization of the surface. Cocolonizing bacteria
could modify the habitat and create a “microenvironment” that
encourages the attachment and growth of other colonizing
microorganisms. We tested whether a preexisting biofilm en-
hances the attachment and persistence of P. tunicata by allow-
ing it to colonize nonaxenic U. australis disks with the intact
natural community on the surface. However, P. tunicata was
unable to colonize under these conditions, indicating that an
MICROBIAL COLONIZATION ON U. AUSTRALIS 5553
established biofilm of other bacteria does not facilitate the
colonization. Another possibility is that cooperative interac-
tions during the process of colonization (for example, come-
tabolism or coaggregation) can occur between P. tunicata and
other cocolonizing marine strains. The phenomenon of coop-
erative biofilm formation in mixed consortia has recently been
described for other bacteria (16, 52). Of the 17 strains, it is not
clear which strains or mixtures of strains are responsible for
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allowing P. tunicata to colonize. The underlying mechanisms of
cooperative biofilm formation are the basis of ongoing exper-
iments in our laboratory.
Attachment and colonization of U. australis by P. tunicata
appears to be influenced by many factors. Attachment and
colonization are particularly poor at low cell densities. The com-
bination of environmental factors that appear to enhance col-
onization at a concentration of 107 cells ml⫺1 does not seem to
be effective at lower cell densities (104 to 105 cells ml⫺1). It
appears that colonization is limited by nutrient availability, but
it is also possible that unknown defensive chemicals produced
by the plant play a role.
Competition in biofilms on U. australis. In coinoculation
competition studies, we observed that P. tunicata suspended in
filtered seawater outcompeted all of the other marine isolates
tested except R. gallaeciensis. The results are similar to our
previous findings of competition on glass surfaces in a labora-
tory flow cell model. In that study, we demonstrated that com-
petition was largely controlled by the production of inhibitory
compounds and the relative sensitivity of competing strains to
the inhibitors (48).
However, we did observe some important differences be-
tween bacterial competition on the surface of the marine plant
and the competition in laboratory systems in the preestab-
lished biofilm studies. Unlike laboratory flow cells, which allow
a continuous flow of nutrients, the surface of algae likely pre-
sents nutrient-limited conditions. Our studies demonstrated
that exogenous application of nutrients resulted in a rapid
increase in the microcolony size of P. tunicata (data not
shown), suggesting that microcolonies on the plant surface are
indeed nutrient limited. Furthermore, our observation of the
patchy distribution of microcolonies might reflect the spatial
heterogeneity of nutrients available on algal surfaces. Several
studies conducted with higher terrestrial plants suggested that
most areas of a leaf harbor only small amounts of nutrients (35,
63), and on bean plants inoculated with P. syringae strain
B728A aggregates of bacteria were distributed nonrandomly in
a wide range of cluster sizes, which roughly corresponded to
nutrient availability (36). Our observation that P. tunicata col-
onized a preestablished biofilm poorly suggest that the initial
colonizers could deplete a large percentage of carbon sources
and make it more difficult for the invading bacteria to seques-
ter themselves in the existing biofilm community. This has been
described for epiphytic bacteria on bean leaves, on which a
preestablished biofilm can deplete resources, leading to pre-
emptive exclusion (64).
In competition studies in which P. tunicata was allowed to
invade a preestablished biofilm, it did not outcompete the
other strains and coexisted with the competing strain for the
duration of the experiment. When invading a preestablished
biofilm, P. tunicata tends to colonize and establish microcolo-
nies in areas which remain free from colonization by a prees-
5554 RAO ET AL.
tablished biofilm. This results in spatially segregated micro-
colonies of competing strains with limited interactions and
leads to coexistence of strains. Our observations of competitive
interactions on the surface of U. australis are supported by
recent reports on spatial segregation of epiphytic bacteria on
leaf surfaces (40, 43). The observations differ from the results
of flow cell experiments in which P. tunicata was able to dom-
inate and eventually remove certain competing strains (48).
The evidence of a reduced ability to colonize a preestablished
biofilm was even more pronounced for the AlpP mutant. The
ability of the mutant to form microcolonies in a preestablished
biofilm was impaired, and the mutant had minimal impact on
the competing strain, which remained dominant. Thus, our
results indicate that AlpP enhances colonization of a preestab-
lished biofilm by P. tunicata.
In contrast, R. gallaeciensis did not seem to be constrained by
nutrient limitations and formed a biofilm which eventually
covered most of the surface of the alga. An ability to utilize a
wide range of carbon sources (50) and an ability to metabolize
DMSP may enable this organism to colonize regions of the
plant that are inhospitable to other bacteria. Even when colo-
nizing a preestablished biofilm, R. gallaeciensis might be able to
access nutrients that are inaccessible to other epiphytic bacte-
ria, and this ability might contribute to its success and epiphytic
fitness (6).
Conclusions. P. tunicata and R. gallaeciensis were found to
have different colonization and competition behaviors during
colonization of U. australis. While R. gallaeciensis is capable of
colonizing U. australis under a range of conditions, coloniza-
tion by P. tunicata is enhanced by high cell densities, the pres-
ence of cellobiose in the preculture, inoculation in the dark,
and interactions with a natural seawater community to attach
and persist on the surface of the algae. The epiphytic fitness of
R. gallaeciensis may be attributed to several factors, including
its versatility in utilizing a number of carbon sources (particu-
larly those not available to competing strains) and the produc-
tion of antibacterial compounds and signaling molecules.
Competition in which a preestablished biofilm is challenged
with P. tunicata results in the coexistence of competitors. This
may be due in part to the protective nature of microcolonies,
which may resist invasion. There is a diffusion gradient in
microcolonies, so that metabolically active cells at the outer
edge of the microcolony may perish while cells in the deeper
regions are likely to be protected from the antibacterial pro-
tein. Limited nutrients on the surface of U. australis may lead
strains to inhabit distinct niches on the plant, and this may also
result in the coexistence of competing strains. Microcolonies of
competing strains are spatially separated, which may limit mi-
crobial interactions and provide refuges for competitors. R.
gallaeciensis does not seem to be constrained by low-nutrient
conditions and is able to invade and disperse competing
strains, suggesting that its antibacterial protein is able to dif-
fuse through microcolonies.
The environmental factors that influence nutrient accumu-
lation on the surface of the seaweed and in turn microbial
colonization and competitive interactions are complex. We
propose that these factors may have a profound impact on the
composition and activity of the epiphytic community.
APPL. ENVIRON. MICROBIOL.
ACKNOWLEDGMENTS
This work was supported by a USP-AusAID scholarship, by the
Australian Research Council, by the Leverhulme Trust, United King-
dom, and by the Centre for Marine Biofouling and Bio-innovation,
University of New South Wales, Sydney, Australia.
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