abc pseudomonas
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Molecular Microbiology (2003) 49(4), 905918 doi:10.1046/j.1365-2958.2003.03615.x
2003 Blackwell Publishing Ltd
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 2003494905918Original ArticleP.fluorescens biofilm formationS.M. Hinsa, M. Espinosa-Urgel, J. L. Ramos and G. A. OToole
Accepted 7 May, 2003. *For correspondence. E-mail
[email protected]; Tel. (+1) 603 650 1248; Fax (+1)603 650 1318.
Transition from reversible to irreversible attachmentduring biofilm formation by Pseudomonas fluorescens
WCS365 requires an ABC transporter and a large
secreted protein
Shannon M. Hinsa,
1
Manuel Espinosa-Urgel,
2
Juan L. Ramos
2
and George A. OToole
1
*
1
Department of Microbiology and Immunology, Dartmouth
Medical School, Hanover, NH, USA.
2
Department of Plant Biochemistry and Molecular and
Cell Biology, Estacion Experimental del Zaidin. CSIC.,
Profesor Albareda, 1., 18008 Granada, Spain.
SummaryWe report the identification of an ATP-binding cas-
sette (ABC) transporter and an associated large cell-
surface protein that are required for biofilm formation
by Pseudomonas fluorescens
WCS365. The genes
coding for these proteins are designated lap
for l
arge
a
dhesion p
rotein. The LapA protein, with a predicted
molecular weight of ~~~~
900 kDa, is found to be loosely
associated with the cell surface and present in the
culture supernatant. The LapB, LapC and LapE pro-
teins are predicted to be the cytoplasmic membrane-
localized ATPase, membrane fusion protein and outer
membrane protein component, respectively, of anABC transporter. Consistent with this prediction,
LapE, like other members of this family, is localized
to the outer membrane. We propose that the lapEBC
-
encoded ABC transporter participates in the secre-
tion of LapA, as strains with mutations in the lapEBC
genes do not have detectable LapA associated with
the cell surface or in the supernatant. The lap
genes
are conserved among environmental pseudomonads
such as P. putida
KT2440, P. fluorescens
PfO1 and P.
fluorescens
WCS365, but are absent from pathogenic
pseudomonads such as P. aeruginosa
and P. syrin-
gae
. The wild-type strain of P. fluorescens
WCS365
and its lap
mutant derivatives were assessed for their
biofilm forming ability in static and flow systems. The
lap
mutant strains are impaired in an early step in
biofilm formation and are unable to develop the
mature biofilm structure seen for the wild-type bacte-
rium. Time-lapse microscopy studies determined that
the lap
mutants are unable to progress from revers-
ible (or transient) attachment to the irreversible
attachment stage of biofilm development. The lap
mutants were also found to be defective in attachment
to quartz sand, an abiotic surface these organisms
likely encounter in the environment.
Introduction
In natural settings, bacteria are most often found associ-ated with surfaces in communities known as biofilms, and
not in the planktonic state (Costerton et al
., 1995; Davey
and OToole, 2000). The formation of biofilms by
pseudomonads has been proposed to occur as a series
of regulated steps (OToole et al
., 2000a). First, flagellar-
mediated motility may be required for a bacterium to swim
toward a surface and to initiate reversible (or transient)
attachment (Korber et al
., 1994; OToole and Kolter,
1998a,b). A subpopulation of transiently attached bacteria
become irreversibly attached to the surface to first form a
monolayer, which is followed by the formation of small
microcolonies (Zobell, 1943; Marshall et al
., 1971; vanLoosdrecht et al
., 1990; Jensen et al
., 1992; Fletcher,
1996). The microcolonies develop into a mature biofilm
with an architecture that is typically characterized by mac-
rocolonies separated by fluid-filled channels (Tolker-
Nielsen et al
., 2000). It is believed that these channels
transport nutrients and oxygen to the bacteria and aid in
waste removal (Costerton et al
., 1995; Davey and OToole
2000). Other characteristics of a mature biofilm include
production of an exopolysaccharide matrix and increased
antimicrobial resistance (Costerton et al
., 1995; Mah and
OToole, 2001).
Pseudomonas fluorescens
WCS365, a natural soil iso-
late that is employed as a biological control agent against
plant pathogenic fungi (Geels and Schippers, 1983;
Simons et al
., 1996), has been used to study the molec-
ular genetic basis of biofilm formation. Previous work has
shown that a site-specific recombinase, a two component
regulatory system, the synthesis of certain amino acids,
the O-antigen of lipopolysaccharide, and type IV pili are
important for P. fluorescens
WCS365 to colonize tomato
roots (Simons et al
., 1997; Dekkers et al
., 1998a,b,c;
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S. M. Hinsa, M. Espinosa-Urgel, J. L. Ramos and G. A. OToole
2003 Blackwell Publishing Ltd, Molecular Microbiology
, 49
, 905918
Camacho-Carbajal, 2001). However, in their natural envi-
ronment, bacteria are also likely to adhere to abiotic sur-
faces such as soil particles.
Transposon generated mutations that render P. fluore-
scens
WCS365 defective for attachment to a variety of
abiotic surfaces, both hydrophobic (plastic) and hydro-
philic (glass) were identified previously (OToole and
Kolter, 1998b). Here we report the characterization of one
class of these biofilm-defective mutants. We have
identified an ATP-binding cassette transporter and a large,
cell-surface associated protein that are required for P.
fluorescens
biofilm formation on abiotic surfaces in both
static and flow cell systems. These genes are also
required for robust biofilm formation on quartz sand, which
serves as a model for surfaces typically encountered by
soil pseudomonads. Our analyses suggest that the genes
encoding this ABC transporter are conserved among
sequenced soil pseudomonads, but are absent from
pathogenic Pseudomonas
strains. We discuss possible
roles for this ABC transporter and the cell-surface asso-
ciated protein in biofilm development.
Results
Initial molecular characterization of mutants defective in
biofilm formation
Previous experiments had identified a set of transposon
mutations in P. fluorescens
that render these strains
unable to form a biofilm (OToole and Kolter, 1998b). The
biofilm-defective mutant strains fell into two broad classes
based on their ability to be rescued by changing growth
conditions. Class I mutants could be rescued by growthin medium supplemented with certain amino acids,
organic acids, and/or exogenous iron. Class II mutants
were unable to make a biofilm under any growth condition
tested (OToole and Kolter, 1998b). The studies here focus
on this second class of mutants.
We identified the genes disrupted by the transposon
insertion in Class II mutant strains by determining the
DNA sequence flanking the transposon, either through
arbitrary-primed PCR or by sequencing the region adja-
cent to a cloned transposon fragment (OToole et al
.,
1999). These DNA sequences were then compared with
sequence from the P. putida
KT2440 and P. fluorescens
PfO1 genome projects. Because the P. putida
KT2440
genome is annotated (Nelson et al
., 2002) it was used to
predict open reading frames as well as to assign putative
functions to the genes disrupted in the P. fluorescens
WCS365 mutants.
Eight transposon mutants were analysed, and based on
extensive sequencing of the DNA flanking the transposon
insertions (Fig. 1A and data not shown), all transposons
were found to map to genes located in close proximity to
each other on the chromosome. Four independent trans-
poson insertions mapped to an open reading frame des-
ignated lapA
. This open reading frame had been initially
identified in strain mus-24
, a transposon mutant of P.
putida
KT2440 defective in adhesion to corn seeds
(Espinosa-Urgel et al
., 2000). The four transposon inser-
tions mapping to lapA
in P. fluorescens
WCS365 (
lapA18,
51, 53
and 62
) are located close to the 5
end of the gene
(corresponding to Domain 2 of the protein, Fig. 1B),
whereas the insertion in P. putida
mutant mus-24
is close
to the 3
end of the gene (corresponding to Domain 4 of
the protein, Fig. 1B). The remaining four mutations anal-
ysed mapped to an adjacent gene cluster we have desig-
nated lapEBC
. The lapB
gene is defined by one
transposon insertion just 5
of the start codon (
lapB84
)
and a second transposon located in the middle of the
gene (
lapB52
). The lapC
gene (
lapC87
) and the lapE
gene
(
lapE83
) are each defined by one transposon insertion,
the lapC
insertion is just 5
of the start codon of lapC
,
while the lapE
insertion is in the middle of the gene. The
gene order as shown in Fig. 1A was confirmed in P. fluo-rescens
WCS365 by either sequencing across the junc-
tions of genes or using PCR with primers whose design
was based on the P. fluorescens
WCS365 DNA sequence
(data not shown).
The lap
genes are required for biofilm formation
To further define the role of the lap
genes in biofilm for-
mation, and to assess the defects in the various lap
mutant alleles, we carried out the detailed analysis of
biofilm formation.
To characterize the kinetics of biofilm formation weperformed a time-course study. The extent of biofilm
formation was determined by measuring crystal violet
(CV)-stained biomass accumulating on the walls of the
microtitre dish over 24 h (OToole et al
., 1999). The wild-
type strain reaches maximum biofilm formation in this
assay by 10 h after which the extent of biofilm formed
decreases, then remains steady, until the end of the assay
period at 24 h (Fig. 2A). The lapB52
and lapE83
mutants
were deficient in attachment over the entire 24 h period
(Fig. 3A); similar biofilm results were seen for the lapA51
and lapC87
mutants (data not shown). These data dem-
onstrated that the lap
mutant strains were not simply
delayed in the initiation of biofilm formation. The plank-
tonic growth of all these lap
mutants was identical to the
wild type (not shown). These results are consistent with
those for the P. putida lapA (mus-24
) mutant that is also
unable to form a biofilm on plastic or glass, and has no
planktonic growth defect (Espinosa-Urgel et al
., 2000).
Several approaches were utilized to demonstrate that
the mutations identified above caused the defects in bio-
film formation. First, independent mutations in the lap
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2003 Blackwell Publishing Ltd, Molecular Microbiology
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genes conferred identical phenotypes in several assays,
providing strong genetic evidence that these mutations
were responsible for the observed biofilm defects. Sec-
ond, generalized transduction was utilized to mobilize one
or more alleles in each lap
gene into a wild-type genetic
background. Of the over 40 transductants assayed, all
conferred the documented antibiotic resistance and bio-
film phenotypes of the parental strains, demonstrating
100% linkage between the transposon insertions and the
observed phenotypes. Finally, numerous attempts were
made to clone the lapEBC
region into various vectors
(pGEM, pUCP18, pSMC32 and pME6000), however, we
were unable to successfully clone this locus. These data
suggest that providing the lapEBC
genes in multiple cop-
ies may be toxic to the cell. Despite the inability to perform
complementation assays, the genetic data presented here
demonstrate that the lap
genes are required for biofilm
formation by P. fluorescens
WCS365.
Monitoring early attachment in a static system with
phase-contrast microscopy
Microscopic analysis of plastic tabs confirmed the results
of the CV assay presented above. To visualize the attach-
ment during the early stages of biofilm formation, the wild-
type and mutant strains lapA51
and lapB84were allowed
Fig. 1. Analysis of the lapgenes, the lapchromosomal region and flanking genes.A. Organization of the lapchromosomal region. Shown are the lapgenes and, where known, the predicted flanking genes. The purple and greenarrows represent genes coding for probable regulators, yellow and brown arrows represent genes coding for hypothetical proteins, and the aquaarrows represent genes coding for a putative deoxygenase. The vertical broken line indicates a gene not adjacent to the lapregion on thechromosome. The organization of the lapregions in P. fluorescensWCS365 and P. fluorescensPfO1 is similar. The lapregion of both P. fluorescens
strains is similar to that of P. putidaKT2440, except the P. putida lapgenes are inverted in relation to the flanking ORFs and lapEis separatedfrom the rest of the lapgenes.B. The LapA protein. The structure of LapA, its four domains, and significant features are shown. The yellow arrows represent lapAmutants inP. fluorescensWCS365, whereas the red arrow represents the lapA(mus-24) mutant in P. putidaKT2440 (Espinosa-Urgel et al., 2000). Thecircles in the fourth domain represent putative calcium-binding domains.
C. Consensus sequence of repeats domains. Shown at the top of this panel is the consensus sequence of the 100 amino acid repeats of Domain2. In blue are the positions that vary in one of the repeats, the red residues correspond to amino acids that vary in two to four of the repeats,and all the other residues (black) are identical in all repeats. The consensus sequence for Domain 3 is shown in the lower portion of this panel.The black residues are conserved in 85% of the repeats, the blue in 6585%, and red indicates the amino acid shown is found in 3065% of the
repeats at that position.
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to attach to plastic tabs for up to 5 h (described in the
Experimental procedures) then examined by phase-
contrast microscopy. At early time points (less than 1 h)
there was no difference in attachment between the wildand the lap mutants. For example, in a representative
experiment assessing attachment at 40 min, an average
of 134 (40) wild-type cells were attached per field viewed
by phase-contrast microscopy compared to 135 (34)
lapCmutant bacteria per field (eight fields were analysed
for each strain). However, by 5 h postinoculation, there
was a clear difference in biofilm formation between the
wild-type and lapmutants. The wild type attached to the
surface and formed organized microcolonies comprised
of hundreds of cells by 5 h (Fig. 2B). In contrast, the
lapA51and lapB84strains formed only small clusters of
bacteria (520 cells), but did not establish the larger
microcolonies typical of the wild type. Similar results were
obtained for the lapE83 and lapC87 mutants (data not
shown).
To determine if lapmutants that attached to the tabs at
later time-points were due to a secondary mutation that
rescued the lapmutant defect, these cells were removed
from the tab by sonication and tested for biofilm formation.
Upon retesting, all the lapmutant cells removed from the
tabs still had a biofilm formation deficiency identical to that
of the original strains tested (data not shown). Therefore,
the residual adherence displayed by the lapmutants was
unlikely to be caused by the accumulation of a second,
compensatory mutation.
Analysis of biofilm formation in a flow cell
To analyse development of a mature biofilm, we grew the
wild type and the mutants in a flow cell system. Flow cells
provide a constant influx of fresh nutrients, thus sustaining
the continued development of the biofilm over many days.
Biofilms grown in the flow cell form the characteristic
architecture comprised of large macrocolonies sur-
rounded by fluid-filled channels after 12 days of
incubation.
We examined biofilm formation at 1, 2 and 3 days. As
shown in Fig. 3A, the wild type bacteria established a
dense monolayer of cells by day 1, and then began devel-
oping microcolonies and eventually macrocolonies by
days 2 and 3. The macrocolonies formed by the wild-type
strain were visible by the naked eye throughout the flowchamber by day 3 (Fig. 3C). In contrast, the lapB52,
lapA51,and lapE83mutants showed a severe defect in
attachment to the surface on day 1 (Fig. 3A). The surface
of the flow cell was only sparsely covered at this time
point. By day 2, the mutant bacteria had formed some
small microcolonies, along the edge of the flow cell, which
is generally subjected to slower medium flow. The few
microcolonies formed on day 2 continue to develop and
by day 3 formed some small macrocolonies. Similar
results to those observed for the lap mutants shown in
Fig. 3 were also observed for lapC87 (data not shown).
The lapA (mus-24) mutant of P. putida KT2440 is alsodefective in biofilm formation in a flow cell system with an
architecture similar to the P. fluorescens WCS365 lap
mutants (data not shown).
Quantitative analysis of biofilm structure
The images in Fig. 3A show a striking difference in the
architecture of the biofilms formed by the wild-type strain
and the lap mutants. To quantify the biofilm formed by
each strain we utilized the COMSTAT program (Heydorn
et al., 2000a). COMSTATconverts the digital information of
images (i.e. shown in Fig. 3) into quantitative parameters
representing various aspects of biofilm architecture.
The information required for calculating the quantitative
parameters of the biofilm is acquired by obtaining optical
sections in the z-plane of the biofilms, followed by decon-
volution of these images with the OpenLab software
package. Biofilms grown for two days in the flow cell were
chosen in order to capture the initial stages of biofilm
maturation. Twelve image series in the z-plane (a z-
series) for each strain effectively captured the heteroge-
Fig. 2. Monitoring early biofilm formation.A. The kinetics of biofilm formation. This figure illustrates formation
of a biofilm at the airmedium interface over a 24 h period. Thesurface attached cells were stained with crystal violet, the stain sol-ubilized in ethanol and the absorbance at 550 nm determined (Y-
axis). Legend: wild type, open squares; lapB52, open circles; lapE83,filled diamonds.B. Monitoring early biofilm formation by phase-contrast microscopy.To visualize the bacterial attachment to plastic, bacteria were incu-
bated in the presence of a plastic tab (~3 3 mm) for 5 h at roomtemperature, the tabs washed, and attachment observed usingphase-contrast microscopy at 1400magnification.
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neous structure of each biofilm. Each z-series involved
capturing an image every 0.5 mm starting at the attach-
ment substratum and moving up to a final height of 50 mm
above the surface. The results of the COMSTAT analysis
are displayed in Table 1. The average thickness of the
biofilm formed by the wild type (10.8 mm) was ~ 30-fold
greater than the biofilm formed by the mutant (0.37 mm).
The bio-volume analysis also demonstrated a similar
trend with the average volume of wild type at 9.93 mm3
m-1m2and the mutant registering 0.33 mm3m-1 m2. Further-
more, the wild type covered approximately 34% of the
substratum while the mutant occupied only 5%. The sur-
face to volume ratio was calculated as a measure of the
amount of biomass exposed to media. The wild type aver-
aged 0.17 mm2 m-1m3 and the mutant 0.64 mm2m-1m3.
Large structured biofilms like those formed by the wild
type tend to produce a lower surface to volume ratio than
do individual cells. These quantitative data are consistent
with the biofilm formation phenotypes revealed by the
microscopic images of initial attachment and later biofilm
development for wild-type and mutant strains.
Fig. 3.Monitoring biofilm development in flowcells.A. Flow cell-grown biofilms. The biofilm formedby wild type, lapA51, lapB52and lapE83strainsat days 1, 2 and 3 is shown. Day 1 shows top-
down, phase-contrast images at 1400magni-fication. The day 2 and 3 images are top-down,epifluorescent micrographs at 230magnification.
B. Enlarged view of initial attachment. Phase-
contrast images captured every 3 s (at 4 h postinoculation) show individual cells attaching tothe surface for the lapC87mutant strain and forthe wild type. The blue arrow points to a bacte-
rium that is standing on end, and viewed fromend-on appears as a dot. This bacterium rep-resents a cell that is in the initial reversibleattachment phase. The red arrows point to a
bacterium that moves back and forth laterallywhile one end remains attached. In contrast,the wild-type bacteria are irreversibly attachedto the surface and thus remain fixed in place.C. Macroscopic macrocolonies. Shown is a
picture of the flow cell chamber at day 3. Thewild-type strain has filled the chamber withmacrocolonies that are visible to the naked eye.Few or no macrocolonies are visible in the
channels of the flow cell containing mutantstrains.
Table 1.COMSTAT: Quantitative analysis of biofilm structure.
Parameter measured Wild type lapC87
Average thickness (mm) 10.8 (8.50) 0.37 (0.33)Bio-volume (mm3m-1m2) 9.93 (7.54) 0.33 (0.2)Substratum coverage (%) 33.6 (17.6) 4.9 (3.4)Surface to volume ratio (mm2m-1m3) 0.17 (0.10) 0.64 (0.41)
The standard deviation for each value is shown in parentheses.
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The lapmutants are defective for the transition from
reversible to irreversible attachment during biofilm
development
Microscopy observations from the static and flow cell
experiments suggested that the lapmutants were defec-
tive in an early stage of biofilm development. To further
characterize the biofilm formation defect of the lap
mutants we compared the bacterial attachment of the
lapCmutant to the wild type over the first 8 h of biofilm
development in a flow cell using time-lapse, phase-
contrast microscopy. Images were acquired every 3 s over
a period of 5 min. A series of representative images illus-
trating attachment of the wild type and the lapCmutant
are shown in Fig. 3B, and the time-lapse movies have
been posted on the web (http://www.dartmouth.edu/
~gotoole/hinsamovies/hinsamovies.html).
Both the lapC mutant and the wild type are able to
anchor one pole of the cell to the surface (polar attach-
ment). Bacteria attached in this fashion are known as
reversibly attached because they can readily detach fromthe surface. The wild-type bacteria eventually become
firmly anchored to the surface along the long axis of the
cell in a process referred to as irreversible attachment
(Zobell, 1943; Marshall et al., 1971; van Loosdrecht et al.,
1990; Jensen et al., 1992; Fletcher, 1996). The bottom
panel of Fig. 3B shows a field of approximately 10 wild-
type bacteria which have adhered and remain unmoving
over the course of the 12 s in which these images were
captured. Time-lapse microscopy over a period of 5 min
shows that the wild type is capable of prolonged stable
interactions with the surface (see web site above). These
data suggest that the wild-type strain is able to makestable interactions with the surface even under conditions
of flow. Furthermore, a majority of the wild-type bacteria
are attached to the surface across the long axis of their
cell body (which serves here as the functional definition
of irreversible attachment).
In contrast to the development observed for the wild-
type strain, the majority of lapCmutant bacteria appear
unable to progress to the irreversible attachment phase of
biofilm formation. As shown in Fig. 3B and the web sup-
plement, many of the lapC mutant bacteria are still
anchored by their pole at this time-point and can still be
observed moving, spinning rapidly, and/or frequently
detaching from the cell surface. After extended incubation
in the flow cell, the lapC mutant is able to form small
microcolonies, possibly at sites where a cell was occa-
sionally able to tightly attach during initial colonization.
Similar results were observed for the other lapmutants
(data not shown).
We also quantified the extent of reversible vs. irrevers-
ible attachment for the wild type and the lapCmutant. By
8 h postinoculation 94% (4.5%) of the wild-type bacteria
were irreversibly attached to the surface. That is, these
bacteria were attached by the long axis of the cell body
and did not move over the course of the 5 min time period
in which the images for the time-lapse movies were
acquired. Only 6% (4.5%) of wild-type cells were
attached by one pole and continued to move during this
period. In contrast, for the lapCmutant, only 12% (4%)
of were irreversibly attached, whereas 88% (4%) were
attached by one pole and continued to move for at least
some period during this 5 min interval.
Sequence analysis of the lapgenes
To begin to elucidate the mechanism underlying the bio-
film defect of the lapmutants, we performed a detailed
analysis of the predicted proteins encoded by these
genes. The complete predicted lapA gene, as deduced
from the genome sequence of P. putidaKT2440, is 26 kb
long and would correspond to one of the largest bacterial
proteins (8682 amino acids) with an estimated molecular
weight of ~888 kDa and a predicted pI of 4.1. Thus, wehave named this gene lapA, for large adhesion protein
(lapa is also the Spanish name for limpet, a mollusk that
lives on seashore rocks and sticks firmly to the rock sur-
face when disturbed). At this stage of the sequencing
project of P. fluorescens it has not been possible to
assemble the complete lapAgene, however, as detailed
below, the size and structure of the protein appears to be
very similar to its P. putidacounterpart.
Four domains can be clearly distinguished in LapA, two
of them being composed of long multiple repeats, which
constitute more than three-quarters of the total length of
the protein (Fig. 1C). Because no significantly similar pro-teins of known function could be found in the databases
when LapA was compared as a whole we analysed each
domain separately. Domain 1, encompassing the first 277
amino acids, contains a predicted non-cleavable N-
terminal signal sequence and a transmembrane region
(PSORTprogram, http://psort.ims.u-tokyo.ac.jp). The func-
tion of Domain 1 is unknown, but shows some limited
sequence similarity (29% identity) with the N-terminal part
of the RTX toxin of the fish pathogen Aeromonas salmo-
nicida(GenBank accession #AF218037). The role of the
N-terminal domain of this RTX toxin has not been eluci-
dated. Domain 2, from amino acids 2781178, comprises
nine quasi-perfect repeats of 100 aa stretches (Fig. 1C).
Sequence similarity was found with the surface protein
Bap, from Staphylococcus aureus(Cucarella et al., 2001),
a protein that contains 13 nearly identical repeats of 86
amino acids and is involved in biofilm formation by S.
aureus. Domain 2 also shows structural similarity with the
outer surface protein A of Borrelia burgdorferi(predicted
with 123D+, http://123d.ncifcrf.gov/; (Alexandrov et al.,
1995). Separated from Domain 2 by 18 aa is Domain 3,
http://www.dartmouth.edu/http://psort.ims.u-tokyo.ac.jp/http://123d.ncifcrf.gov/http://123d.ncifcrf.gov/http://psort.ims.u-tokyo.ac.jp/http://www.dartmouth.edu/ -
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which is also a large repetitive region, spanning 6400 aa,
organized in 29 imperfect repeats of 218225 aa
(Fig. 1C). Some sequence similarity was found with the
surface-associated adhesin CshA of the Gram-positive
oral bacterium Streptococcus gordonii (McNab et al.,
1994). CshA also shows a repetitive structure (13 repeats
of 101 amino acids) and is an essential element for oral
cavity colonization, participating in co-aggregation of S.
gordonii with another oral microorganism, Actinomyces
naeslundii (McNab et al., 1994; 1999). Domain 4 (1087
aa) contains several Ca2+-binding motifs similar to those
identified in haemolysins and other secreted proteins
known to participate in bacterialeukaryotic interactions
(Economou et al., 1990). This sequence analysis sug-
gests that LapA may be a cell surface protein working as
a multifunctional adhesin.
Based on sequence analysis, we hypothesize that the
lapEBC genes code for an ABC transporter (Fath and
Kolter, 1993; Young and Holland, 1999; Dassa and
Bouige, 2001). ABC transporters involved in export gen-
erally are composed of three separate components aninner membrane anchored ATPase, a membrane fusion
protein and an outer membrane protein (Dassa and
Bouige, 2001). LapB is the predicted inner membrane
protein of 74 kDa, with several predicted transmembrane
regions and a C-terminal ATPase domain containing the
canonical Walker box motifs characteristic of this family.
LapC is predicted to be 50 kDa, contains a single pre-
dicted transmembrane domain, shows similarity to toxin
secretion proteins of the HlyD family, and is therefore
proposed to be the membrane fusion protein. LapE is a
48 kDa protein predicted to localize to the outer mem-
brane and is similar to AggA (50% identity and 71% sim-ilarity over the 750 bp we have sequenced), a protein that
was described as a factor involved in agglutination and
adherence in Pseudomonas putidastrain Corvallis (Buell
and Anderson, 1992). Furthermore, LapE contains a
domain that is predicted to function as either an outer
membrane efflux protein domain or a TolC-like domain
(NCBI Conserved Domain Search). This TolC domain is
characteristic of the PRT-HLY family of ABC transporters
that are involved in protein export in prokaryotes (Dassa
and Bouige, 2001). Most proteins exported by this sub-
family of ABC transporters contain a series of glycine-rich
repeats that forms a calcium-binding site (Baumann et al.,
1993; Young and Holland, 1999; Dassa and Bouige,
2001). This so-called repeats in toxin or RTX calcium-
binding domain has been identified in LapA. Members of
the PRT-HLY family of proteins can be further distin-
guished based on their C-terminal sequence. LapA is
likely a member of the HLY subfamily, because it lacks the
signature extreme C-terminal motif DXXV (where X is a
hydrophobic residue) of the PRT subfamily members (Let-
offe and Wandersman, 1992; Ghigo and Wandersman,
1994; Duong et al., 1996). Thus, we predicted that LapB
is the ATP-binding element, LapC is the membrane fusion
component and LapE is the outer membrane component
of an ABC transporter responsible for the export of LapA.
Localization of LapE and LapA
The sequence analyses presented above allowed us to
develop a model in which the LapA protein serves as an
adhesin to firmly anchor these bacteria to a surface (i.e.
promote irreversible attachment). Furthermore, the iden-
tification of the LapE, LapB and LapC proteins as compo-
nents of an ABC transporter suggested a mechanism by
which LapA could be transported out of the cell.
To gain insight into the role of the LapA and LapE
proteins in irreversible attachment, we examined the local-
ization of these proteins. The LapA protein was predicted
to be localized to the outer membrane or cell-surface
based on its sequence similarity to known proteins. Frac-
tionation of P. fluorescens inner and outer membranes,
followed by Western blot analysis, did not detect LapA ineither of these fractions (Fig. 4A, lanes labelled IM and
OM). We next tested if LapA was secreted from the cell
and/or was loosely associated with the cell surface. To test
for a loose association of the protein to the surface of the
cell, bacteria were grown as described in the Experimen-
tal proceduresand 20 ml of the culture was centrifuged,
then resuspended in a small volume of buffer resulting in
a 50-fold concentration of the bacteria. These concen-
trated cells were vortexed for five seconds, the suspension
re-centrifuged, and an aliquot of the resulting supernatant
(designated S2) was analysed by Western blot with anti-
LapA antibodies. As shown in Fig. 4A, lane S2, a largemolecular weight band was detected for the wild-type
strain that is absent from the lapAmutant. The exact size
of the band is difficult to estimate as it runs significantly
larger than the largest size marker which runs at
~190 kDa. An identically sized band was also detected in
10-fold concentrated spent supernatant from the wild type
but was absent from the lapAmutant strain supernatant.
Therefore, LapA appears to be found both in the cell
supernatant and in a loose association with the bacterial
cell surface, but not in the outer membrane.
To ensure that the centrifugation and vortexing proce-
dures were not rupturing the cell membrane, we also
probed these same fractions with antibody to LapE, a
predicted outer membrane protein (Fig. 4B, top panel).
LapE was not detected in either supernatant fraction, nor
was it detected associated with the IM fraction (Fig. 4B).
A band corresponding to the molecular weight of LapE
(48 kDa) was detected in the OM fraction, but was absent
from the lapE83 mutant OM fraction. A weakly cross-
reacting band that runs at a molecular weight slightly
greater than LapE was present in all samples.
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Sequence analysis of the LapE, LapB and LapC pro-
teins showed similarity with ABC transporter components,
and therefore suggested a possible role for this putative
ABC transporter system in the secretion of the LapA pro-
tein. To address this hypothesis, we tested for the pres-
ence of cell surface-associated LapA (the S2 fraction) in
the wild type and the lapEBCmutant strains. As shown in
Fig. 4C (top panel) LapA was detected in the preparation
from the wild type, but could not be detected in the S2
supernatant fraction of any of the lapEBC mutants. Fur-
thermore, no LapA was detected in the supernatant of the
lapEBC mutants (data not shown). These observations
suggest the ABC transporter may be responsible for the
delivery of LapA to the exterior of the cell. As expected,
no LapA was detected in the lapA51mutant. In contrast,
LapE was present in the OM fractions of all of the lap
mutant strains except for the lapE83 mutant (Fig. 4C,
lower panel).
Conservation of the lap genes among pseudomonads
Comparisons were performed between the lapAchromo-
somal region of P. putida KT2440 and the equivalent
regions in the incomplete genome sequences of P.
fluorescens PfO1 (http://www.Jgi.doe.gov/JGI_microbial/
html/pseudomonas/pseudo_homepage.html) and P. syrin-
gae (http://www.tigr.org), the finished genome sequence
of P. aeruginosa(http://www. Pseudomonas.com), and the
DNA sequence we obtained from strain P. fluorescens
WCS365. The results of these analyses are shown in
Fig. 1 and Table 2.
The organization of the lapAEBCregion is very similar
in P. fluorescens PfO1 and P. fluorescens WCS365
(Fig. 1A). In P. putidaKT2440, lapE (aggA) is located on
a different part of the chromosome from the rest of the lap
genes and is associated with a different ABC transporter
(not shown). The lapAand lapBCgenes are organized in
a similar fashion to P. fluorescens PfO1, however, they
appear in an inverted orientation relative to the flankinggenes. In spite of these differences in the chromosomal
arrangement of the genes, LapA seems to play a similar
role in P. fluorescens and P. putida. The lapA (mus-24)
mutant of P. putida KT2440 is also defective in biofilm
formation in static conditions (Espinosa-Urgel et al., 2000)
and in a flow cell system, with a biofilm architecture similar
to the P. fluorescens WCS365 lapA mutants (data not
shown). LapA is not present in P. aeruginosaor P. syrin-
gaebut proteins with some similarity to those encoded by
the lapEBCgenes are present, although at different chro-
mosomal locations. It is worth noting that even though
lapA is not present in P. aeruginosa, the gene clustershowing the highest degree of similarity lapEBCgenes is
also associated with a putative large outer membrane
protein of ~2500 amino acids (not shown).
The lapgenes are required for the colonization of
quartz sand
It has been previously reported that the lapmutants of P.
fluorescensare deficient for attachment to polyvinylchlo-
Fig. 4. Protein localization studies.A. Localization of LapA. A Western blot developed with antibody to
LapA was performed on the following fractions: (i) the supernatant ofthe 50-fold concentrated, resuspended and vortexed cells (S2); (ii)the TCA-precipitated, 10-fold concentrated, cell-free supernatant(S1); (iii) the inner membrane fraction (IM), and (iv) the outer mem-brane fraction (OM) of these cells. The results of the Western analysis
on fractions from the wild type (top panel) and lapA51mutant (lowerpanel) are shown.B. Localization of LapE. A Western blot using the fractions describedabove was developed with the LapE antibody. The fractions from the
wild type (top panel) and lapE83mutant (lower panel) are shown.C. Detection of LapA and LapE in the lap mutant strains. The S2supernatants of the wild type and lapmutant strains were analysedfor the presence of LapA (top panel). The OM fractions of the wildtype and lapmutant strains were analysed for the presence of LapE
(lower panel). In all experiments, cells from a ~16 h culture grown inminimal, citrate supplemented medium were used to prepare eachfraction and proteins were resolved on gradient polyacrylamide gels(415%).
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ride, polypropylene, polystyrene and borosilicate glass
(OToole and Kolter, 1998). However, P. fluorescens is
primarily a soil microorganism, therefore its natural sub-
strate is most likely a variety of sands and soils. Therefore,
we tested the wild type and the lapmutant strains for their
ability to attach to a surface this bacterium may encounter
in its natural soil environment, namely, quartz sand. We
implemented assays to follow sand colonization visually
and quantitatively.
To visualize bacteria attached to sand, GFP-labelled
bacteria were allowed to attach to the sand for 5 h and
then the sand was washed and the attached bacteria
visualized by epifluorescent microscopy. Figure 5A illus-
trates the wild type and the lapB52mutant attached to a
Table 2. Sequence similarities of Lap proteins.
P. putidaKT2440 P. fluorescensWCS365 P. fluorescens PfO1 P. aeruginosaPAO1 P. syringae
LapA (PP0168) 39%/52% 41%/53%b
8682 aaa (491 aa) (4785 aa)c
LapB (PP0167) PA1876d
ATPase 86%/93% 85%/92% 37%/58% 30%/52%718 aa (322 aa) 722 aa 723 aa 613 aa
LapC (PP0166) PA1877
MFP 76%/88% 80%/90% 39%/57% 26%/47%452 aa (140 aa) 455 aa 395 aa 526 aa
LapE (PP4519)e PA1875OM protein 50%/71% 53%/71% 22%/42% 24%/42%452 aa (163 aa) 451 aa 425 aa 479 aa
a.The number of amino acids (aa) known from the genome sequence of P. putidaKT4220. The gene designation assigned in the P. putidagenome project are given in parentheses (http://www.tigr.org).
b.Per cent identity and percentage similarity was determined by comparing each complete or partial ORF to the predicted aa sequence of theputative homologue determined from the complete P. putidaKT4220 genome sequence.
c.Per cent identity/similarity corresponds to alignments with partial (in parentheses) or complete ORFs obtained from each respective genomeproject.
d.The PA designations refer to ORF numbers from the P. aeruginosagenome project (http://www.Pseudomonas.com).e.LapE corresponds to the previously identified AggA protein (see text), but is annotated as TolC in the P. putidagenome.
Abbreviations: OM, outer membrane; MFP, membrane fusion protein; ATPase, cytoplasmic membrane-localized ATPase
Fig. 5.Sand attachment.A. Visualizing bacterial attachment to sand. The
wild type and lapB52strains were allowed to
attach to quartz sand for 5 h. The sand waswashed to remove unattached bacteria and epi-fluorescent microscopy used to visualize
attached bacteria at 1400magnification.B. Quantifying attachment of individual strainsto sand. The colony forming units (CFU) pergram of sand (Y-axis) is plotted for the wild typeand lapB52mutant. The initial inoculum of the
wild type and mutant was identical at ~1 109CFU ml-1.C. Quantifying competitive attachment to sand.Equal numbers of wild type and lapB52mutant
bacteria (a total of ~1 109CFU ml-1) weremixed and inoculated onto quartz sand. The percent of each strain attached to the sand particle(Y-axis) was determined as described in theExperimental procedures.
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grain of sand. The wild-type strain efficiently colonizes the
sand particle, whereas the lapB52strain is much reduced
in its ability to colonize. Similar results were seen for the
lapC87and lapA62mutants (data not shown).
To quantify the number of cells attached to the sand
particles, ~1 109 bacteria were allowed to adhere to
sand as described above. The number of surface-
associated bacteria was determined by vortexing and
sonicating the sand to remove attached cells, and the
number of bacteria attached to the sand was deter-
mined by dilution plating (see Experimental procedures
for details). As shown in Fig. 5B, there is a 10-fold differ-
ence in attachment between wild type and the lapB52
mutant. Similar results were seen with the lapA51,
lapC87and lapE83mutants (data not shown). To deter-
mine if competition for attachment or complementation
would affect this outcome, we mixed equal amounts of
wild-type and mutant bacteria (a total of ~1 109 cells)
and allowed them to attach to the sand. After rinsing
and vortexing/sonication to remove the attached bacte-
ria, we found that approximately 80% of the bacteriaattached to the sand were the wild-type strain (Fig. 5C).
Similar results were seen for lapA51, lapC87 and
lapE83 (data not shown). These data also indicate that
the wild-type strain is unable to rescue the biofilm for-
mation defect(s) of the lapmutants.
Discussion
Cell-to-surface interaction events mark the early steps in
the development of a mature biofilm. It has long been
proposed that early attachment events first involve
reversible attachment to a surface, marked by the tran-sient interactions of one pole of the bacterium with a
substratum (Zobell, 1943; Marshall et al., 1971; van
Loosdrecht et al., 1990; Jensen et al., 1992; Fletcher,
1996). Following up on these previous studies, Sauer
et al. (2002) made detailed observations of early biofilm
development, and also observed reversible and subse-
quent irreversible attachment steps by P. aeruginosa. In
the studies presented here, microscopic analysis of
early attachment events in P. fluorescensWCS365 dem-
onstrated that this pseudomonad also undergoes the
same two step early attachment pathway. In the wild-
type strain, cells undergo transient polar attachment fol-
lowed by subsequent undefined events that lead to a
so-called irreversible attachment. The lapmutant strains
are capable of initial attachment to a degree that is
indistinguishable from the wild-type strain, thus they
appear to have no defects in reversible attachment.
However, the lap mutants appear to be unable to
progress normally to the irreversible attachment step of
biofilm development. This phenotype is most clearly
observed in time-lapse movies obtained from flow cell
studies of the wild type and lap mutant strains. Our
studies show that LapA and the lap-encoded ABC trans-
porter, which is required for LapA to be exported from
the cell, are required for irreversible attachment. To our
knowledge, this is the first report of genetic determi-
nants that are necessary for, and define, the transition
from reversible to irreversible attachment.
Despite the apparent defect in irreversible attachment,
the lapmutants can eventually form small clusters of bac-
teria on the surface, however, these mutant bacteria are
unable to develop the architecture observed for the wild-
type bacteria. We demonstrated that the attachment of
these few lapmutant cells to the surface was not due to
a secondary mutation, therefore the lapmutants may use
an undefined pathway to irreversibly attach to a surface
after prolonged incubation. Microcolonies may eventually
form as a consequence of cell-to-cell adherence (suggest-
ing that the Lapgene products may not be required for
these interactions) and/or cell division.
What role does the putative lapEBC-encoded ABC
transporter and the associated LapA protein play in biofilmdevelopment? One possible role for LapE, a predicted
outer membrane protein with sequence similarity to the
AggA adhesin (Buell and Anderson, 1992), may be as an
attachment factor required for early biofilm development.
However, fractionation of the lap mutants and Western
analysis revealed that the LapE protein is localized to the
outer membrane in all strains but the lapEmutant, sug-
gesting that proper localization of this outer membrane
protein is not sufficient for biofilm formation. In contrast,
any mutation within the lapEBCcluster resulted in the loss
of any detectable LapA associated with the cell surface or
in the cell supernatant. These data are consistent with amodel in which the lapEBC-encoded ABC transporter is
required for export of LapA outside of the cell. Particularly
intriguing is the identification of a putative signal sequence
at the N-terminus of LapA, which is not typical of proteins
transported by ABC systems (Young and Holland, 1999;
Dassa and Bouige, 2001). One possibility is that this
secretion signal is cryptic and not typically utilized for
transport of LapA. Another possibility is that LapA is trans-
ported into the periplasm by the Sec-dependent transport
system, and then delivered outside of the cell by the ABC
transporter. The detailed analysis of the mechanism by
which LapA exits the cell awaits future studies. Taken
together, these data are consistent with a hypothesis
wherein LapA, alone or in association with LapE, serves
to promote stable adhesion of P. fluorescensWCS365 to
a surface early in biofilm development.
A role for ABC transporters in cell-to-surface and/or
cell-to-cell interactions and biofilm development has been
proposed in other organisms. An ABC transporter is
important for attachment and virulence of Agrobacterium
tumefacienson carrot cells (Matthysse et al., 1996). In a
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recent report Sauer and Camper (2001) showed that in
another soil pseudomonad, P. putida, the potB gene,
which codes for an ABC transporter component, is
upregulated in the early stages of biofilm formation on
silicone. This observation suggests a possible role for the
PotB ABC transporter in early biofilm development, but
this has not been demonstrated experimentally. In the
oral microbe Streptococcus gordonii, an ABC transporter
was shown to be important for self co-aggregation (cell-
to-cell interactions) in vitro (Kolenbrander et al., 1994).
Taken together, these data indicate that a role for ABC
transporters in biofilm development may be conserved
across Gram-positive and Gram-negative organisms,
perhaps for the purpose of secreting cell surface
adhesins.
All of the lapmutants of P. fluorescensWCS365 and
the lapAmutant of P. putidawere shown to be defective
for attachment to a number of plastics (polyvinylchloride,
polypropylene, polycarbonate and polystyrene) as well as
borosilicate glass (OToole and Kolter, 1998b; Espinosa-
Urgel et al., 2000). Here we report that the lapmutantsare also defective for attaching to quartz sand, a sub-
strate they are very likely to encounter in the environ-
ment. The P. putida mus-24 (lapA) mutant was isolated
as defective for adhesion to corn seeds, and is also
impaired in biofilm formation and competitive root coloni-
zation (M. Espinosa-Urgel, unpubl. obs.). These broad
phenotypic effects on attachment to biotic and abiotic
surfaces caused by mutations in LapA could be
explained in two ways. The interactions mediated by this
protein might be somewhat non-specific, therefore LapA
may act as a general purpose adhesin. Alternatively, the
multiple domains may in fact be different bindingdomains, each promoting adherence to a set of sub-
strates. The repeats in LapA (Domains 2 and 3) are rem-
iniscent of those found in adhesion proteins of Gram-
positive bacteria involved in biofilm formation or in cell
cell interactions, whereas Domain 4 shows similarities
with calcium-binding proteins and haemolysins, that play
a role in cellhost interactions during pathogenesis.
The presence of the lap genes in three different soil
isolates, and their absence from two pathogenic
Pseudomonasstrains, suggests that these genes may be
specifically necessary for biofilm development by only a
subset of pseudomonads. Current studies are exploring
the extent of conservation of the lap genes and their
organization in a wide variety of soil pseudomonads. To
date, no ABC transporter required for biofilm formation by
P. aeruginosahas been identified. This finding, along with
the data presented here, suggest that although both
pathogenic and non-pathogenic pseudomonads make
biofilms, they may utilize distinct mechanisms to transition
from reversible to irreversible attachment during early bio-
film development.
Experimental procedures
Bacterial strains, plasmids, and culture conditions
Pseudomonas fluorescenswas grown in LuriaBertani (LB)
or in minimal media, as specified, at 30C. The minimal salts
medium used was M63 (Pardee et al., 1959) supplemented
with MgSO4 (10 mM) and either glucose (0.2%) or citrate
(0.4%), or AB10 media without trace minerals (Tolker-Nielsen
et al., 2000). Pseudomonas putida was grown in LB andAB10 at 30C and E. coliwas grown in LB at 37C. Antibiotics
were added at the following concentrations: (i) E. coli: gen-
tamycin (Gm), 10 mg ml-1; chloramphenicol (Cm), 30 mg ml-1;
(ii) P. fluorescens: Gm, 50 mg ml-1; kanamycin (Kn),
250 mg ml-1. Generalized transductions were performed as
described (Jensen et al., 1998). Plasmid pSMC21 is derived
from pSMC2 (Bloemberg et al., 1997), expresses the green
fluorescent protein (GFP) under a constitutive promoter, and
carries both Ap and Kn resistance markers. Plasmids pSU21
(Martinez et al., 1988), pME6000 (Itoh et al., 1988), pSMC32
(OToole et al., 2000b), pUCP18 (Schweizer, 1991) and
pGEM (Promega) were utilized for cloning experiments.
Molecular techniques
Sequence of the DNA flanking transposon insertions was
determined by arbitrary primed PCR (OToole et al., 1999).
Selected transposon insertions were cloned to determine
additional DNA sequence flanking the element. Chromo-
somal DNA was prepared as described (Pitcher et al., 1989),
digested with EcoRI and ligated into pSU21 previously
digested with EcoRI. The ligations were electroporated into
E. coliJM109 electrocompetent cells, plated on LB supple-
mented with Cm, then replica printed onto LB supplemented
with Cm and Gm. The CmrGmrcolonies were purified, plas-
mid DNA was prepared, and the plasmids were sequenced
with the Tn5Ext primer (OToole et al., 1999). Polymerase
chain reaction using primers to sequence derived from P.fluorescens WCS365 was performed to confirm the gene
order inferred from sequence analysis (Fig. 1A).
Biofilm assays
Initial biofilm formation was measured using the microtiter
dish assay system performed as described previously
(OToole and Kolter, 1998a,b; OToole et al., 1999) using min-
imal M63 medium with glucose (0.2%) or citrate (0.4%) as
the growth substrate. The visualization of bacterial cells
attached to PVC was performed as previously reported
(Bloemberg et al., 1997) except cultures were incubated at
room temperature for up to 5 h before analysis. The once-
flow through continuous culture flow cell system was assem-
bled as described (Christensen et al., 1999) and modified
AB10, as described above, was utilized as the growth
medium.
To address whether secondary mutations were responsible
for allowing the lapstrains to form microcolonies at later time-
periods, the attached lapAmutant cells were tested for the
ability to make biofilms after re-culture under planktonic con-
ditions. The lap mutant bacteria were allowed to attach to
PVC tabs for 24 h, after which the tabs were rinsed and
placed into an eppendorf tube containing LB. The bacteria
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adhered to the tab were removed by two alternating series
of vortexing (10 s) and sonication (10 s, Tabletop Ultrasonic
Cleaner, FS-60, Fischer Scientific) followed by a final 10 s
vortex (as described Gardener and de Bruijn, 1998). The LB
and any bacteria removed from the tab were used to inocu-
late an LB culture that was outgrown for 24 h, and then a
standard biofilm assay was performed using these cultures
as an inoculum.
Sand attachment
Bacteria were grown overnight in LB and subcultured into
M63 plus citrate (0.4%) at a 1:5 dilution. Sand was placed in
the bottom of the well in a 24-well plate and covered with the
bacterial suspension. The plates were placed on a shaker at
room temperature for four hours. A sample of sand was
removed from each well, placed in an eppendorf tube, and
washed five times with 500 ml of M63. A sand sample could
be removed at this point for visualization of bacter ia attached
to sand. Epifluorescent microscopy indicated that wild-type
bacteria formed a monolayer of cells on the sand particles at
this time under the growth conditions described (see Fig. 5).
Quantification of bacteria attached to sand was performed asfollows: 50 ml of M63 was added to the sand containing tube,
and the bacteria were removed from the sand by two alter-
nating series of vortexing (10 s) and sonication (10 s, Table-
top Ultrasonic Cleaner, FS-60, Fischer Scientific) followed by
a final 10 s vortex (as described by Gardener and de Bruijn,
1998). Ten microlitres of suspension was removed and used
for dilution plating. Bacterial counts are normalized to grams
of sand assayed.
To determine the efficacy of the vortex/sonication regimen,
the percentage of bacteria removed from the sand was deter-
mined. One sample of sand was treated as described above,
whereas a second sample did not receive the vortexing and
sonication treatment. Both treated and untreated samples
were washed an additional three times with 500 ml of M63.Fifty microlitres of M63 was added to the sand post treatment,
the samples were incubated for 2 h and dilution plating per-
formed to determine the number of bacteria present (e.g.
those bacteria shed from the sand and growing planktoni-
cally). These control experiments demonstrated >99.9% of
the bacteria attached to the sand are removed during the
vortexing and sonication steps.
Imaging
Epifluorescent and phase-contrast microscopy were per-
formed with a Model DM IRBE microscope (Leica Microsys-
tems) equipped with an Orca Model C4742-5 CCD camera
(Hamamatsu). Images were acquired and processed on aMacintosh G4 loaded with OpenLab 3.1 software (Improvi-
sion). COMSTATanalysis was performed as described (Hey-
dorn et al., 2000a, b).
Protein localization and Western analysis
Samples for Western analysis were prepared as follows.
Twenty millilitres of minimal M63 medium supplemented with
citrate and MgSO4 were grown shaking at 30C for ~16 h.
The cultures were centrifuged for 10 min at 6000 gand 1 ml
of the supernatant was removed and TCA precipitated as
described (Kunitz, 1952). The precipitated protein pellet was
resuspended in 100 ml of resuspension buffer (Tris-HCl,
20 mM, pH 8 plus 10 mM MgCl2) this sample is designated
S1. The bacterial pellet from the 20 ml culture was resus-
pended in 400 ml of resuspension buffer and vortexed for 5 s.
The samples were centrifuged for 5 min at 12 200 gand the
supernatant was collected (the supernatant of this sample is
designated S2). The inner and outer membranes were sep-
arated as previously described (Lohia et al., 1984) with some
modifications. The cells were grown as described above, then
centrifuged for 10 min at 6000 gand the pellet resuspended
in 1.5 ml of PBS. A Thermo Spectronic French press mini-
cell was used to lyse the cells by processing twice at 20 000
p.s.i. Next, the samples were spun at 12 200 g to pellet
unbroken cells. The supernatant was removed and spun at
100 000 gfor 60 min at 4C. The pellet was resuspended in
100 ml of Hepes buffer (10 mM, pH 5.7) and incubated with
100 mg ml-1 DNAse and RNAse for 20 min at 25C. Nine
hundred microlitres of urea (4 M) was added to the samples
to solubilize the inner membrane. The samples were spun at
100 000 g for 60 min at 4C. The supernatant fraction con-
taining the inner membrane was removed and the pellet(outer membrane) was washed with cold H2O and centrifuged
for another 60 min at 4C at 100 000 g. SDS loading buffer
was mixed with each sample, followed by heat denaturation
at 75C for 10 min. The samples were resolved on a gradient
polyacrylamide gel (415%) at 20 mA. The protein was trans-
ferred in a to a nitrocellulose membrane in transblot buffer as
described (Towbin et al., 1979). Western blots were devel-
oped with ECL Western detection reagents (Amersham).
Acknowledgements
We thank Christian Weinel for providing us with P. putida
sequence data prior to publication. We thank referee #3 forsuggesting the suppressor analysis experiment. This work
was supported by grants from the NSF (CAREER 9984521)
and The Pew Charitable Trusts to G.A.O. G.A.O. is a Pew
Scholar in the Biomedical Sciences. We also acknowledge
grant BMC2001-0576 from the Plan Nacional de I +D +I to
M.E.U. M.E.U. is the recipient of a grant from the Ramn y
Cajal Program (MCYT).
Supplementary material
The following material is available from
http://www.blackwellpublishing.com/products/journals/
suppmat/mmi/mmi3615/mmi3615sm.htm
Time-lapse movies of biofilm-grown bacteria were made
from images captured every 3 s over a period of 5 min. In
this experiment, the biofilm was 8 h old and the flow direc-
tion was from the bottom to the top of the image. Most of
the wild-type bacteria are attached to the surface along the
long length of the cell and do not move during the course of
the 5 min movie. In contrast, most of the lapCmutant bacte-
ria are attached by one pole and can be seen spinning
rapidly or moving with the flow of the medium through the
flow cell.
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