ABC Pseudomonas

8/12/2019 ABC Pseudomonas

1/14

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;


2/14

906

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


3/14

P. fluorescens biofilm formation

907

2003 Blackwell Publishing Ltd, Molecular Microbiology

, 49

, 905918

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.


4/14

908 S. M. Hinsa, M. Espinosa-Urgel, J. L. Ramos and G. A. OToole

2003 Blackwell Publishing Ltd, Molecular Microbiology, 49, 905918

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.


5/14

P. fluorescens biofilm formation 909


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.


6/14



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/


7/14



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.


8/14



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%).
http://www.jgi.doe.gov/JGI_microbial/http://www.tigr.org/http://www/http://www/http://www/http://www.tigr.org/http://www.jgi.doe.gov/JGI_microbial/


9/14



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.
http://www.tigr.org/http://www.pseudomonas.com/http://www.pseudomonas.com/http://www.tigr.org/


10/14



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


11/14



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


12/14



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.
http://www.blackwellpublishing.com/products/journals/http://www.blackwellpublishing.com/products/journals/


13/14



References

Alexandrov, N.N., Nussinov, R., and Zimmer, R.M. (1995)

Fast Protein Fold Recognition Via Sequence to Structure

Alignment and Contact Capacity Potentials.Pacific Sym-

posium on Biocomputing 96. L. Hunter and T. E. Klein.

Singapore: World Scientific Publishing, pp. 5372.

Baumann, U., Wu, S., Flaherty, K.M., and McKay, D.B.

(1993) Three-dimensional structure of the alkaline pro-

tease of Pseudomonas aeruginosa: a two-domain proteinwith a calcium binding parallel beta roll motif. EMBO J12:

33573364.

Bloemberg, G.V., OToole, G.A., Lugtenberg, B.J.J., and

Kolter, R. (1997) Green fluorescent protein as a marker for

Pseudomonasspp. Appl Environ Microbiol63: 45434551.

Buell, C.R., and Anderson, A.J. (1992) Genetic analysis of

the aggAlocus involved in agglutination and adherence of

Pseudomonas putida, a beneficial fluorescent

pseudomonad. Mol Plant-Microb Interact5: 154162.

Camacho-Carbajal, M.M. (2001) Molecular characterization

of type 4 pili, NDHI and PyrR in rhizosphere colonization

of Pseudomonas fluorescensWCS365. PhD Thesis, Insti-

tute of Molecular Plant Sciences, Leiden University:

Leiden, The Netherlands.Christensen, B.B., Sternberg, C., Andersen, J.B., Palmer,

R.J. Jr, Nielsen, A.T., Givskov, M., and Molin, S. (1999)

Molecular tools for study of biofilm physiology. Methods

Enzymol310: 2042.

Costerton, J.W., Lewandowski, Z., Caldwell, D.E., Korber,

D.R., and Lappin-Scott, H.M. (1995) Microbial Biofilms. In

Annual Review Microbiology. Ornston, L.N., Balows, A.,

and Greenberg, E.P. (eds). Palo Alto: Annual Reviews, pp.

711745.

Cucarella, C., Solano, C., Valle, J., Amorena, B., Lasa, I., and

Penades, J.R. (2001) Bap, a Staphylococcus aureussur-

face protein involved in biofilm formation. J Bacteriol183:

28882896.

Dassa, E., and Bouige, P. (2001) The ABC of ABCS: aphylogenetic and functional classification of ABC systems

in living organisms. Res Microbiol152: 211229.

Davey, M.E., and OToole, G.O. (2000) Microbial biofilms:

from ecology to molecular genetics. Microbiol Mol Biol

Revs64: 847867.

Dekkers, L.C., Bloemendaal, C.J.P., de Weger, L.A., Wijffel-

man, C.A., Spaink, H.P., and Lugtenberg, B.J.J. (1998a)

A two-component system plays an important role in the

root-colonizing ability of Pseudomonas fluorescensstrain

WCS365. Mol Plant-Microbe Interact11: 4556.

Dekkers, L.C., Phoelich, C.C., van der Fits, L., and Lugten-

berg, B.J.J. (1998b) A site-specific recombinase is required

for competitive root colonization by Pseudomonas fluore-

scensWCS365. Proc Natl Acad Sci USA95: 70517056.Dekkers, L.C., van der Bij, A.J., Mulders, I.H.M., Phoelich,

C.C., Wentwoord, R.A.R., Glandorf, D.C.M., et al.(1998c)

Role of the O-antigen of lipopolysaccharide, and possible

roles of growth rate and of NADH: ubiquinone oxidoreduc-

tase (nuo) in competetive tomato root-tip colonization by

Pseudomonas fluorescens WCS365. Mol Plant-Microbe

Interact11: 763771.

Duong, F., Lazdunski, A., and Murgier, M. (1996) Protein

secretion by heterologous bacterial ABC-transporters: the

C-terminus secretion signal of the secreted protein confers

high recognition specificity. Mol Microbiol21: 459470.

Economou, A., Hamilton, W.D.O., Johnston, A.W.B., and

Downie, J.A. (1990) The Rhizobiumnodulation gene nodO

encodes a Ca2+-binding protein that is exported without N-

terminal cleavage and is homologous to haemolysin and

related proteins. EMBO J9: 349354.

Espinosa-Urgel, M., Salido, A., and Ramos, J.L. (2000)

Genetic analysis of functions involved in adhesion of

Pseudomonas putida to seeds. J Bacteriol 182: 2363

2369.

Fath, M.J., and Kolter, R. (1993) ABC transporters: bacterial

exporters. Microbiol Rev57: 9951017.

Fletcher, M. (1996) Bacterial attachment in aquatic environ-

ments: a diversity of surfaces and adhesion strategies. In

Bacterial Adhesion: Molecular and Ecological Diversity.

Fletcher, M. (ed.). New York: John Wiley & Sons, pp. 124.

Gardener, B.B.M., and de Bruijn, F.J. (1998) Detection and

isolation of novel rhizopine-catabolizing bacteria from the

environment. Appl Environ Microbiol64: 49444949.

Geels, F.P., and Schippers, B. (1983) Reduction of yield

depressions in high frequency potato cropping soil after

seed tuber treatments with antagonistic fluorescent

Pseudomonasspp. Phytopathol Z108: 207214.Ghigo, J.M., and Wandersman, C. (1994) A carboxyl-

terminal four-amino acid motif is required for secretion of

the metalloprotease PrtG through the Erwinia chrysan-

themi protease secretion pathway. J Biol Chem 269:

89798985.

Heydorn, A., Nielsen, A.T., Hentzer, M., Sternberg, C.,

Givskov, M., Ersboll, B.K., and Molin, S. (2000a) Quantifi-

cation of biofilm structures by the novel computer program

COMSTAT. Microbiology146: 23952407.

Heydorn, A., Ersboll, B.K., Hentzer, M., Parsek, M.R.,

Givskov, M., and Molin, S. (2000b) Experimental reproduc-

ibility in flow-chamber biofilms. Microbiology 146: 2409

2415.

Itoh, Y., Soldati, L., Leisinger, T., and Haas, D. (1988) Low-and intermediate-copy-number cloning vectors based on

the Pseudomonasplasmid pVS1. Antonie Van Leeuwen-

hoek54: 567573.

Jensen, E.T., Kharazmi, A., Hoiby, N., and Costerton, J.W.

(1992) Some bacterial parameters influencing the neutro-

phil oxidative burst response to Pseudomonas aeruginosa

biofilms. Apmis100: 727733.

Jensen, E.C., Schrader, H.S., Rieland, B., Thompson, T.L.,

Lee, K.W., Nickerson, K.W., and Kokjohn, T.A. (1998)

Prevalence of broad-host-range lytic bacteriophages of

Sphaerotilus natans, Escherichia coli, and Pseudomonas

aeruginosa. Appl Environ Microbiol64: 575580.

Kolenbrander, P.E., Anderson, R.N., and Ganeshkumar, N.

(1994) Nucleotide sequence of the Streptococcus gordoniiPK488 coaggregation adhesin gene, scaA, and ATP-

binding cassette. Infect Immun62: 44694480.

Korber, D.R., Lawrence, J.R., and Caldwell, D.E. (1994)

Effect of motility on surface colonization and reproductive

success of Pseudomonas fluorescens. dual-dilution contin-

uous culture and batch culture systems. Appl Environ

Microbiol60: 14211429.

Kunitz, M.J. (1952) Crystalline inorganic pyrophosphatase

isolated from bakers yeast. J Gen Physiol35: 423450.


14/14



Letoffe, S., and Wandersman, C. (1992) Secretion of CyaA-

PrtB and HlyA-PrtB fusion proteins in Escherichia coli:

involvement of the glycine-rich repeat domain of Erwinia

chrysanthemiprotease B. J Bacteriol174: 49204927.

Lohia, A., Chaterjee, A.N., and Das, J. (1984) Lysis of Vibrio

choleraecells: Direct isolation of the outer membrane from

whole cells by treatment with urea. J Gen Microbiol130:

20272033.

van Loosdrecht, M.C., Lyklema, J., Norde, W., and Zehnder,

A.J.B. (1990) Influence of interfaces on microbial activity.

Microbiol Rev54: 7587.

Mah, T.F., and OToole, G.A. (2001) Mechanisms of biofilm

resistance to antimicrobial agents. Trends Microbiol9: 34

39.

Marshall, K.C., Stout, R., and Mitchell, R. (1971) Mechanism

of the initial events in the sorbtion of marine bacteria to

surfaces. J Gen Microbiol68: 337348.

Martinez, E., Bartolome, B., and de la Cruz, F. (1988)

pACYC184-derived cloning vectors containing the multiple

cloning site and lacZa reporter gene of pUC8/9 and

pUC18/19. Gene68: 159162.

Matthysse, A.G., Yarnall, H.A., and Young, N. (1996)

Requirement for genes with homology to ABC transport

systems for attachment and virulence of Agrobacteriumtumefaciens. J Bacteriol178: 53025308.

McNab, R., Forbes, H., Handley, P.S., Loach, D.M., Tannock,

G.W., and Jekinson, H.F. (1999) Cell wall-anchored CshA

polypeptide in Streptococcus gordoniiforms surface fibrils

that confer hydrophobic and adhesive properties. J Bacte-

riol181: 30873095.

McNab, R., Jenkinson, H.F., Loach, D.M., and Tannock, G.W.

(1994) Cell-surface-associated polypeptides CshA and

CshB of high molecular mass are colonization determi-

nants in the oral bacterium Streptococcus gordonii. Mol

Microbiol14: 743754.

Nelson, K.E., Weinel, C., Paulsen, I.T., Dodson, R.J., Hilbert,

H., Martins dos Santos, V.A.P., et al. (2002) Complete

genome sequence and comparative analysis of the meta-bolically versatile Pseudomonas putida KT2440. Environ

Microbiol4: 799808.

OToole, G.A., and Kolter, R. (1998a) Flagellar and twitching

motility are necessary for Pseudomonas aeruginosabio-

film development. Mol Microbiol30: 295304.

OToole, G.A., and Kolter, R. (1998b) Initiation of biofilm

formation in Pseudomonas fluorescensWCS365 proceeds

via multiple, convergent signalling pathways: a genetic

analysis. Mol Microbiol28: 449461.

OToole, G.A., Pratt, L.A., Watnick, P.I., Newman, D.K.,

Weaver, V.B., and Kolter, R. (1999) Genetic approaches

to the study of biofilms. In Methods in Enzymology.Doyle,

R.J. (ed). San Diego, CA: Academic Press, 310:91109.

OToole, G.A., Kaplan, H., and Kolter, R. (2000a) Biofilm

formation as microbial development. Annu Rev Microbiol

54: 4979.

OToole, G.A., Gibbs, K.A., Hager, P.W., Phibbs,P.V. Jr and

Kolter, R. (2000b) The global carbon metabolism regulator

Crc is a component of a signal transduction pathway

required for biofilm development by Pseudomonas aerug-

inosa. J Bacteriol182: 425431.

Pardee, A.B., Jacob, F., and Monod, J. (1959) The genetic

control and cytoplasmic expression of inducibility in the

synthesis of b-galactosidase in E. coli. J Mol Biol1: 165

178.

Pitcher, D.G., Saunders, N.A., and Owen, R.J. (1989) Rapid

extraction of bacterial genomic DNA with guanidium thio-

cyanate. Lett Appl Microbiol8: 151156.

Sauer, K., and Camper, A.K. (2001) Characterization of phe-

notypic changes in Pseudomonas putida. response to

surface-associated growth. J Bacteriol183: 65796589.

Sauer, K., Camper, A.K., Ehrlich, G.D., Costerton, J.W., and

Davies, D.G. (2002) Pseudomonas aeruginosa displays

multiple phenotypes during development as a biofilm. J

Bacteriol184: 11401154.Schweizer, H.P. (1991) Escherichia-Pseudomonas shuttle

vectors derived from pUC18/19. Gene103: 109112.

Simons, M., van der Bij, A.J., Brand, I., de Weger, L.A.,

Wijffelman, C.A., and Lugtenberg, B.J.J. (1996) Gnotobi-

otic system for studying rhizosphere colonization by plant

growth-promoting Pseudomonas bacteria. Mol Plant-

Microbe Inter9: 600607.

Simons, M., Permentier, H.P., de Weger, L.A., Wijffelman,

C.A., and Lugtenberg, B.J.J. (1997) Amino acid synthesis

is necessary for tomato root colonization by Pseudomonas

fluorescensstrain WCS365. Mol Plant-Microbe Interact10:

102106.

Tolker-Nielsen, T., Brinch, U.C., Ragas, P.C., Andersen, J.B.,

Jacobsen, C.S., and Molin, S. (2000) Development anddynamics of Pseudomonas sp. biofilms. J Bacteriol 182:

64826489.

Towbin, H., Staehelin, T., and Gordon, J. (1979) Electro-

phoretic transfer of proteins from polyacrylamide gels to

nitrocellulose sheets: procedure and some applications.

Proc Natl Acad Sci USA76: 43504354.

Young, J., and Holland, I.B. (1999) ABC transporters: bacte-

rial exporters-revisited five years on. Biochim Biophys Acta

1461: 177200.

Zobell, C.E. (1943) The effects of solid surfaces upon bacte-

rial activity. J Bacteriol46: 3956.

ABC Pseudomonas

Documents

Transcript of ABC Pseudomonas