APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2009, p. 6783–6791
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 21
Conjugative Plasmid Transfer and Adhesion Dynamics in an
Escherichia coli Biofilm?†
Cheryl-Lynn Y. Ong, Scott A. Beatson, Alastair G. McEwan, and Mark A. Schembri*
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia
Received 29 April 2009/Accepted 22 August 2009
A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027
was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and
is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes
two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently
found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation
by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system,
however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow
as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a
mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important
mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-
related infections in nosocomial settings.
Bacterial biofilms are complex communities of bacterial cells
living in close association with a surface (17). Bacterial cells in
these protected environments are often resistant to multiple
factors, including antimicrobials, changes in the pH, oxygen
radicals, and host immune defenses (19, 38). Biofilm formation
is a property of many bacterial species, and a range of molec-
ular mechanisms that facilitate this process have been de-
scribed (2, 3, 11, 14, 16, 29, 33, 34). Often, the ability to form
a biofilm is dependent on the production of adhesins on the
bacterial cell surface. In Escherichia coli, biofilm formation is
enhanced by the production of certain types of fimbriae (e.g.,
type 1 fimbriae, type 3 fimbriae, F1C, F9, curli, and conjugative
pili) (14, 23, 25, 29, 33, 39, 46), cell surface adhesins (e.g.,
autotransporter proteins such as antigen 43, AidA, TibA,
EhaA, and UpaG) (21, 34, 35, 40, 43), and flagella (22, 45).
The close proximity of bacterial cells in biofilms creates an
environment conducive for the exchange of genetic material.
Indeed, plasmid-mediated conjugation in monospecific and
mixed E. coli biofilms has been demonstrated (6, 18, 24, 31).
The F plasmid represents the best-characterized conjugative
system for biofilm formation by E. coli. The F pilus mediates
adhesion to abiotic surfaces and stabilizes the biofilm structure
through cell-cell interactions (16, 30). Many other conjugative
plasmids also contribute directly to biofilm formation upon
derepression of the conjugative function (16).
One example of a conjugative system employed by gram-
negative Enterobacteriaceae is the type 4 secretion (T4S) sys-
tem. The T4S system is a multisubunit structure that spans the
cell envelope and contains a secretion channel often linked to
a pilus or other surface filament or protein (8). The Agrobac-
terium tumefaciens VirB-VirD4 system is the archetypical T4S
system and is encoded by 11 genes in the virB operon and one
gene (virD4) in the virD operon (7, 8). Genes with strong
homology to genes in the virB operon have also been identified
on other conjugative plasmids. For example, the pilX1 to
pilX11 genes on the E. coli R6K IncX plasmid and the virB1 to
virB11 genes are highly conserved at the nucleotide level (28).
We recently described identification and characterization
of the mrk genes encoding type 3 fimbriae in a uropatho-
genic strain of E. coli isolated from a patient with a noso-
comial catheter-associated urinary tract infection (CAUTI)
(29). The mrk genes were located on a conjugative plasmid
(pMAS2027) and were strongly associated with biofilm for-
mation. In this study we determined the entire sequence of
plasmid pMAS2027 and revealed the presence of conjugative
transfer genes homologous to the pilX1 to pilX11 genes of E.
coli R6K (in addition to the mrk genes). We show here that
biofilm formation is driven primarily by type 3 fimbriae and
that the T4S apparatus is unable to mediate biofilm growth in
the absence of the mrk genes. Finally, we demonstrate that
conjugative transfer of pMAS2027 within a mixed biofilm con-
fers biofilm formation properties on recipient cells due to
acquisition of the type 3 fimbria-encoding mrk genes.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The strains and plasmids
used in this study are described in Table 1. Cells were routinely grown at 37°C
using solid or liquid Luria-Bertani (LB) medium supplemented with appropriate
antibiotics, unless otherwise stated. M9 minimal medium supplemented with
0.2% glucose and synthetic urine were prepared as previously described (26, 32).
DNA manipulations and genetic techniques. Plasmid DNA was isolated using
QIAprep Spin Miniprep or Midiprep kits (Qiagen, Australia). Restriction en-
donucleases were used according to the manufacturer’s specifications (New En-
gland Biolabs). PCR was performed using Taq polymerase according to the
manufacturer’s instructions (New England Biolabs). DNA sequencing was per-
formed by the Australian Genome Research Facility. E. coli strain MS2027 was
genetically marked by insertion of gfpmut3b* into the chromosomal attachment
site of bacteriophage ? (attB) as previously described (34), which resulted in E.
coli MS2091. Mutation of pMAS2027 was performed by performing ?-red-me-
* Corresponding author. Mailing address: School of Chemistry and
Molecular Biosciences, University of Queensland, Brisbane, QLD
4072, Australia. Phone: 61 7 3365 3306. Fax: 61 7 3365 4699. E-mail:
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 28 August 2009.
diated homologous recombination as previously described (12), except that the
gfp-kan mutants were constructed using pCO13 as the template DNA. Primers
used in this study are described in Table 2. All deletion mutants were confirmed
by PCR and subsequent DNA sequencing.
Sequencing and annotation of pMAS2027. Both strands of plasmid pMAS2027
were sequenced by employing a primer walking strategy. Primers were designed
so that they read progressively outward from the mrkA and mrkF genes, until a
complete, overlapping sequence was obtained. The DNA sequence was assem-
bled using Contig Express (Invitrogen), and annotation was performed by using
Vector NTi (Invitrogen) and Artemis (version 10) (5). BLASTn and BLASTp
searches were performed using the National Center for Biotechnology Informa-
tion website (1).
Plasmid mobilization study. Plasmid mobility was monitored by using filter
paper bacterial conjugation as previously described (44). An overnight culture of
the donor strain was concentrated 10-fold and left to stand at 37°C to allow
growth of the sex pili. The donor and recipient (MS661) were then mixed at a
ratio of 1:10 and then incubated on filter paper for 3 to 4 h. The filter paper
mixture was then resuspended in LB medium, and dilutions were plated on LB
agar containing tetracycline and kanamycin to select for transconjugants. Plates
were incubated overnight at 37°C, and the plasmid conjugation efficiency was
calculated by determining the ratio of transconjugant colonies to donor colonies.
Biofilm study. Biofilm formation on polyvinyl chloride (PVC) surfaces was
monitored by using 96-well microtiter plates (Falcon) essentially as previously
described (33). Briefly, cells were grown for 24 h in urine or M9 minimal medium
(containing 0.2% glucose) at either 28°C or 37°C (statically or with shaking),
washed to remove unbound cells, and stained with crystal violet. Quantification
of bound cells was performed by addition of acetone-ethanol (20:80) and mea-
TABLE 1. Bacterial strains and plasmids used in this study
E. coli strains
Commercial laboratory E. coli cloning
E. coli CAUTI isolate
MS661 harboring pAR163
Kanamycin resistance gene inserted into
Deletion mutant template plasmid (cam)
Deletion mutant template plasmid (kan)
mrk-containing conjugative plasmid
isolated from MS2027
pGEM-T Easy with modified kan
cassette without HindIII
TABLE 2. Primers used in this study
1293 50-bp overhang mrk knockout forward primer TCTTCTCTCTGCAGCAATGGCAACCGCGTTTTTTGGCATGACTGC
1294 50-bp overhang mrk knockout reverse primer
Screening forward primer (mrk)
Screening reverse primer (mrk)
50-bp overhang pilX knockout forward primer
129850-bp overhang pilX knockout reverse primer
Screening forward primer (pilX)
Screening reverse primer (pilX)
50-bp overhang Cmrtag forward primer
1302 50-bp overhang Cmrtag reverse primer
Screening forward primer (tagging)
Screening reverse primer (tagging)
GFP-kan mrk knockout forward primer
1306 GFP-kan mrk knockout reverse primer
1307GFP-kan pilX knockout forward primer
1308 GFP-kan pilX knockout reverse primer
1309GFP-kan tagging forward primer
1310 GFP-kan tagging reverse primer
pCO_F pCO13 internal screening forward primer
pCO13 internal screening reverse primer
pCO13 external screening forward primer
pCO13 external screening reverse primer
aGFP, green fluorescent protein.
6784ONG ET AL.APPL. ENVIRON. MICROBIOL.
surement of the dissolved crystal violet using the optical density at 595 nm. Flow
chamber biofilm experiments were performed as previously described (21).
Briefly, biofilms were allowed to form on glass surfaces in a multichannel flow
system that permitted online monitoring of community structures. Flow cells
were inoculated with overnight cultures that were standardized using the optical
density at 600 nm and were prepared in M9 medium. Biofilm development was
monitored by confocal scanning laser microscopy at 16 to 24 h after inoculation.
All experiments were performed in triplicate. In the flow chamber experiments
with mixed biofilms, the donor and recipient strains were incubated for 6 h
before they were subjected to the medium flow. Biofilm development was mon-
itored by confocal scanning laser microscopy at 16 and 40 h after inoculation. All
experiments were performed in triplicate. Both single-strain biofilm and mixed-
biofilm flow chamber experiments were done using M9 minimal medium sup-
plemented with 100 ?g/?l ampicillin.
SEM. Scanning electron microscopy (SEM) was performed essentially as pre-
viously described (29). Cells were grown as described above for the biofilm study
on PVC surfaces, except that the experiment was performed using a 12-well
microtiter plate (Griener bio-one) with a polystyrene disk placed at the bottom.
The disk was fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer and
postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer. The sample was
then infiltrated with glycerol and frozen in liquid nitrogen. The sample was freeze
substituted in 100% ethanol containing a molecular sieve and incubated at
?80°C for 10 h; then the temperature was increased from ?80°C to ?20°C over
a 10-h period, and the sample was critical point dried. The sample was then
mounted on carbon tabs and sputter coated with platinum at 15 mA for 120 s.
Statistical analysis. Differences in plasmid conjugation efficiency between
pMAS2027mrk::kan and pMAS2027orf24::cam were determined by using a t test
(with two samples assuming unequal variances). Differences in biofilm formation
were analyzed using the analysis of variance single-factor test, and values were
compared with values obtained for E. coli MS2027 (wild type) or E. coli MS2362
(MS2027::cam) (Minitab 15 statistical software).
Nucleotide sequence accession number. The pMAS2027 plasmid sequence has
been deposited in the GenBank database under accession number FJ666132.
FIG. 1. Circular diagram of plasmid pMAS2027. The outer circle indicates (to scale) the genetic organization of ORFs within the plasmid. The
direction of transcription of each ORF is indicated. The middle circle indicates the G?C content, and the inner circle indicates the GC skew. Genes
are color coded as indicated. The image was constructed using cgview (37). CDS, coding sequences.
VOL. 75, 2009 DYNAMICS OF AN EVOLVING E. COLI BIOFILM6785
Complete nucleotide sequence of pMAS2027. Plasmid
pMAS2027 was determined to be a 42,644-bp circular plasmid
with a G?C content of 43.43% (Fig. 1). Bioinformatic analysis
predicted the presence of 58 open reading frames (ORFs),
which represent 88.83% of the total plasmid DNA. Plasmid
pMAS2027 is most closely related to the IncX group of plas-
mids, a narrow-host-range family of conjugative plasmids com-
monly found in the family Enterobacteriaceae (13). The back-
bone structure of pMAS2027 contains genes associated with
plasmid stability and partitioning, as well as a 14,628-kb region
comprising genes predicted to encode a T4S system (Fig. 1).
This region includes the pilX1 to pilX11 genes analogous to the
genes described for R6K (28) and most likely involved in
conjugative DNA transfer. pMAS2027 also contains the
mrkABCDF genes (encoding type 3 fimbriae). The mrk
genes are located on a 5,536-bp segment and have an overall
G?C content of 56.6%. Genes predicted to encode proteins
associated with transposition are located both upstream
(tnpR and tnpA) and downstream (insA, insB, and tnpA) of
the mrk cluster, suggesting that the mrk genes are located on
a mobile genetic element. A summary of each ORF of
pMAS2027, including the predicted function of each en-
coded protein, is shown in Table 1S in the supplemental
material. No genes were predicted to encode proteins asso-
ciated with antibiotic resistance. The remainder of our study
focused on the relative contributions of the mrk and pilX1 to
pilX11 genes to biofilm formation and conjugative transfer.
Bioinformatic analysis of the conjugative transfer locus of
plasmid pMAS2027. The pilX1 to pilX11 genes of pMAS2027
are 43% identical at the nucleotide sequence level to the virB1
to virB11 genes of the archetypical transferred DNA transfer
system of the plant pathogen A. tumefaciens (42). These genes
have also been identified on conjugative plasmids isolated from
other Enterobacteriaceae, including Salmonella enterica serovar
Enteritidis, S. enterica serovar Dublin, Proteus mirabilis, and
other E. coli strains (Fig. 2). The pilX1 to pilX11 regions of
three plasmids (pSE34 from Salmonella serovar Enteritidis,
pOU1115 from Salmonella serovar Dublin, and p2ESCUM
from E. coli) share the strongest overall sequence identity with
the pilX1 to pilX11 genes of pMAS2027 (Table 3). The se-
quences of two other plasmids (pOLA52 from E. coli and
pOU1114 from Salmonella serovar Dublin) were also highly
conserved in this region, except for the eex and pilX6 genes.
The nucleotide sequences of the pilX1 to pilX11 genes of
pMAS2027, R6K (an archetypical E. coli IncX plasmid), and
pHI4320 (P. mirabilis) were more divergent (Table 3). Despite
this, the genetic organization of the pilX1 to pilX11 (and adja-
cent) genes on pMAS2027 and a number of other conjugative
plasmids is highly conserved (Fig. 2).
FIG. 2. Physical map comparing the genes encoding the T4S system (and adjacent genes) in plasmid pMAS2027 to the corresponding regions
in plasmids pSE34 (Salmonella serovar Enteritidis; EU219533), pOU1115 (Salmonella serovar Dublin; DQ115388), p2ESCUM (E. coli;
CU928149), pOLA52 (E. coli; EU370913), pOU1114 (Salmonella serovar Dublin; DQ115387), R6K (E. coli; AJ006342), and pHI4320 (P. mirabilis;
AM942760) and the pathogenicity island (PAI) in E. coli ECOR31 (AY233333). The genes encoding the T4S system are blue and include ddp1
(taxA), taxC, actX, pilX1 to pilX11, and taxB. Adjacent genes (gray) encode hypothetical proteins with unknown functions. No sequence information
is available for genes outside the T4S system gene cluster in E. coli R6K. The direction of transcription of each gene is indicated.
6786 ONG ET AL.APPL. ENVIRON. MICROBIOL.
The pilX locus promotes conjugative transfer of plasmid
pMAS2027. To demonstrate the function of the pilX1 to pilX11
genes, the entire segment was deleted by ?-red-mediated homol-
ogous recombination and replaced by insertion of a kanamycin
resistance gene (to generate plasmid pMAS2027pilX::kan). Plas-
mid pMAS2027pilX::kan could not be mobilized into a recipient
E. coli strain, confirming the function of the pilX1 to pilX11 genes
in conjugative transfer. To examine whether type 3 fimbriae (en-
coded by the mrkABCDF genes on pMAS2027) also contribute to
the efficiency of conjugative transfer, we constructed two addi-
tional plasmids: pMAS2027mrk::kan and pMAS2027orf27::cam.
Plasmid pMAS2027orf27::cam contained a gene encoding resis-
tance to chloramphenicol inserted into orf27 (which encodes a
hypothetical protein of unknown function) and thus served as a
control. The frequency of conjugative transfer, calculated by
determining the number of transconjugants per donor, was
0.0064 for pMAS2027orf27::cam, compared to 0.032 for
pMAS2027mrk::kan (P ? 0.05). Thus, the lack of type 3 fim-
briae resulted in a fivefold increase in conjugative plasmid
Type 3 fimbriae, but not T4S pili, contribute to biofilm forma-
tion. The contributions of type 3 fimbriae and T4S pili to
biofilm growth were examined by using MS2027 harboring
either pMAS2027mrk::kan, pMAS2027pilX::kan, or pMAS2027
orf27::cam and performing two dynamic biofilm assays. First, we
used a PVC microtiter plate assay and showed that type 3 fim-
briae, but not T4S pili, promote biofilm formation after growth in
M9 minimal medium and in urine (Fig. 3). Next, we tested the
ability of the same strains to promote biofilm formation in a
continuous-flow chamber. Consistent with the results of our mi-
crotiter plate assay, MS2072 harboring pMAS2027orf27::cam and
MS2072 harboring pMAS2027pilX::kan produced equivalent,
strong biofilms that were approximately 20 ?m deep, while
MS2072 harboring pMAS2027mrk::kan was unable to form a
biofilm (Fig. 4). An additional plasmid construct with both the
mrk and pilX genes deleted (pMAS2027mrk::kan pilX::cam) was
also examined and did not mediate biofilm growth. Thus, al-
though plasmid pMAS2027 encodes the capacity to produce con-
in these experiments.
Transfer of the mrk genes via conjugation in an evolving
mixed biofilm. A further three deletion mutants were con-
structed by replacing the mrk, pilX, and orf27 genes on
pMAS2027 with gfp and a kanamycin resistance gene, tagging
pMAS2027 with gfp in the process. MS2027 cells harboring
plasmid pMAS2027mrk::gfp-kan, pMAS2027pilX::gfp-kan, or
pMAS2027orf27::gfp-kan were examined for the ability to form
a mixed biofilm in the continuous-flow chamber system with
E. coli K-12 strain MS2199, a non-biofilm-forming strain
tagged with the rfp gene. Biofilm formation was assessed by
performing scanning laser confocal microscopy at 16 h, 30 h,
and 40 h after inoculation. E. coli MS2199 was unable to form a
biofilm at all three time points when it was alone and when it was
grown in a coculture with MS2027(pMAS2027mrk::gfp-kan) or
MS2027(pMAS2027pilX::gfp-kan) (Fig. 5A). However, when
E. coli MS2199 was grown in a coculture with MS2027
MS2027(pMAS2027orf27::gfp-kan) cells (green) and transconju-
gant MS2199 cells (yellow) formed (Fig. 5A). Close examina-
tion of the mixed biofilm (magnification, ?100) revealed re-
gions with a mixture of green and yellow cells, suggesting that
there was conjugative transfer within the evolving biofilm.
Taken together, the data demonstrate that conjugative transfer
of pMAS2027orf27::gfp-kan from MS2027 to MS2199 enabled
MS2199 transconjugant cells to form a strong biofilm due to
production of type 3 fimbriae. No red-tagged MS2199 cells
were observed in the mixed biofilm. When the mixed biofilm
containing MS2199 and MS2027(pMAS2027orf27::gfp-kan)
mixed biofilmconsisting of
TABLE 3. Comparison of the E. coli plasmid pMAS2027 T4S system protein and nucleotide sequences with the corresponding sequences
from other T4S system conjugative plasmids
T4S system protein
% Amino acid identity (% nucleotide sequence conservation)
pSE34 pOU1115p2ESCUM pOLA52 pOU1114 R6K pHI4320
apilX3 and pilX4 and virB3 and virB4 in R5K and pTi are separate genes, but in all other plasmids they are fused in one ORF.
bpTi contains the virB operon, which was compared to the pilX operon of pMAS2027.
cNA, not applicable.
VOL. 75, 2009DYNAMICS OF AN EVOLVING E. COLI BIOFILM 6787
was viewed with an SEM, T4S pilus-like structures linking
cells within the biofilm were observed (Fig. 5B). Thus, it is
possible that the T4S pili encoded on pMAS2027 contribute
to the structural composition of a mixed biofilm both di-
rectly and through their ability to transfer the type 3 fim-
bria-encoding mrk genes to recipient cells.
Biofilm formation by uropathogenic E. coli is mediated by a
range of cell surface factors, including fimbriae, flagella, and
adhesins. Often, the genes encoding these factors are located
on mobile genetic elements, such as plasmids, transposons, and
pathogenicity islands. Here we determined the complete nucle-
otide sequence of a conjugative plasmid isolated from a strain of
uropathogenic E. coli that caused CAUTI and defined the prop-
erties of this plasmid that are associated with biofilm growth.
Plasmid pMAS2027 is most closely related to conjugative
plasmids belonging to the IncX group (36), and its nucleotide
sequence strongly suggested that it belonged to IncX1, a subset
of this group (20). The genetic organization of pMAS2027 is
similar to that previously described for several characterized
virulence plasmids from S. enterica (9, 15, 41). Indeed, of the 58
ORFs identified on pMAS2027, 40 (69%) exhibit the strongest
nucleotide sequence similarity to S. enterica genes. The major
genetic load region of pMAS2027 comprised the mrk genes
(encoding type 3 fimbriae), which appear to be located on a
mobile genetic element. Recently, a large conjugative plasmid
(pOLA52) isolated from swine manure that also contains both
the mrkABCDF and pilX1 to pilX11 genes was described (27).
FIG. 3. Biofilm formation by E. coli MS2027 and derivatives.
Strains were grown in urine for 16 h in PVC microtiter plates under
various conditions, as indicated. Biofilm formation was quantified by staining
adhered cells with 0.1% crystal violet, resuspending them in ethanol-acetate
(80:20), and measuring the absorbance at 595 nm. The results are the aver-
the results for MS2027, MS2362 [MS2027(pMAS2027orf27::cam)], MS2181
[MS2027(pMAS2027mrk::cam)], MS2103 [MS2027(pMAS2027pilX::kan)],
and MS2183 [MS2027(pMAS2027mrk::cam, pilX::kan)].
FIG. 4. Flow chamber biofilm formation for (A) E. coli MS2091 (MS2027 attB::bla-PA1/04/03-gfpmut3b*-T0;Gfp), (B) MS2519 [MS2027
(pMAS2027orf27::gfp-kan)], (C) MS2517 [MS2027(pMAS2027mrk::gfp-kan)], and (D) MS2518 [MS2027(pMAS2027pilX::gfp-kan)]. Biofilm develop-
ment was monitored by confocal scanning laser microscopy 24 h after inoculation. The large micrographs show horizontal sections. To the right of and
above each large image are images of the yz plane and the xz plane, respectively, obtained at the positions indicated by the lines.
6788 ONG ET AL.APPL. ENVIRON. MICROBIOL.
The mrkABCDF genes of pOLA52 are flanked by transposon-
like sequences, and the nucleotide sequences of these genes
are ?94% identical to the nucleotide sequences of the
mrkABCDF genes of pMAS2027. A comparison of the pilX1 to
pilX11 genes of the two plasmids also revealed a high degree of
nucleotide sequence conservation, except for the eex and pilX6
genes. Although pOLA52 is approximately 10 kb larger than
pMAS2027 and contains genes that impart multidrug resis-
FIG. 5. (A) Flow chamber biofilm formation for E. coli MS2199 (Rfp?) and mixed cultures of E. coli MS2199 (Rfp?) and MS2517
[MS2027(pMAS2027mrk::gfp-kan)] (Gfp?), MS2199 (Rfp?) and MS2518 [MS2027(pMAS2027pilX::gfp-kan)] (Gfp?), and MS2199 (Rfp?) and
MS2519 [MS2027(pMAS2027orf27::gfp-kan)] (Gfp?). Magnification, ?40. Biofilm development was monitored by confocal scanning laser mi-
croscopy at 16 h, 30 h, and 40 h after inoculation. The micrographs show horizontal sections. To the right of and above each large panel are images
of the yz plane and the xz plane, respectively, obtained at the positions indicated by the lines. The largest micrograph shows a higher magnification
(?100) of a defined region of the MS2199-MS2519 mixed biofilm. (B) SEM micrograph of a mixed MS2199-MS2519 microtiter plate biofilm.
Putative T4S pilus structures are indicated by arrows.
VOL. 75, 2009 DYNAMICS OF AN EVOLVING E. COLI BIOFILM 6789
tance, the similarity between the backbone sequences of the
two plasmids is striking considering that the plasmids were
identified in strains isolated from two very different environ-
ments (i.e., swine manure and the urine of a patient with
The ability to produce type 3 fimbriae was an absolute re-
quirement for biofilm growth of E. coli MS2027. No biofilm
formation was observed with mutants that lacked the
mrkABCDF genes but retained the ability to produce conjugative
T4S pili. Thus, unlike the F pilus (16, 30), T4S pili do not mediate
binding to abiotic surfaces and do not promote biofilm formation.
Although the role of type 3 fimbriae in biofilm formation was
consistent in previous studies performed with plasmid pOLA52
(4), we found that deletion of mrkABCDF resulted in a fivefold
increase in the conjugation efficiency of pMAS2027. This finding
is in contrast to the results of studies performed with pOLA52,
where mutation of the mrkC gene (which abrogates production of
type 3 fimbriae) caused a dramatic reduction in conjugation effi-
ciency (4). It is possible that this discrepancy is due to differences
in the makeup of other cell surface components that might inter-
fere with this process between the E. coli strains harboring
pMAS2027 and pOLA52.
Mixed-culture flow chamber assays were employed to
examine the contribution of conjugative plasmid transfer
to biofilm development. Maintenance of transconjugant
MS2199(pMAS2027orf27::cam) cells within a biofilm was
dependent on the production of type 3 fimbriae. Thus, the
genetic load of pMAS2027 (mrkABCDF) defined its ability
to spread laterally within the biofilm. Genes encoding type 3
fimbriae have been identified in many gram-negative uro-
pathogens, including Klebsiella spp., E. coli, Enterobacter
spp., P. mirabilis, Serratia spp., Yersinia spp., and Providentia
spp. It is likely that the widespread occurrence of type 3
fimbria-encoding genes in these pathogens is associated with
plasmid transfer within biofilms in the hospital setting.
This work was supported by grant DP666852 from the Australian
Research Council and by grant 455914 from the National Health and
Medical Research Council.
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