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Journal of Bacteriology, October 1999, p. 5993-6002, Vol. 181, No. 19
Laboratoire de Génétique
Moléculaire des Microorganismes et des Interactions Cellulaires,
CNRS UMR 5577, Institut National des Sciences Appliquées de
Lyon, 69621 Villeurbanne, France
Received 10 March 1999/Accepted 23 July 1999
To get further information on bacterial surface sensing and
biofilm-dependent regulation of gene expression in Escherichia coli K-12, random insertion mutagenesis with Mu dX, a mini-Mu carrying the promoterless lacZ gene, was performed with an
ompR234 adherent strain, and a simple screen was developed
to assess changes in gene expression in biofilm cells versus planktonic
cells. This screen revealed that major changes in the pattern
of gene expression occur during biofilm development: the transcription
of 38% of the genes was affected within biofilms. Different
cell functions were more expressed in sessile bacteria: the OmpC porin,
the high-affinity transport system of glycine betaine (encoded by the
proU operon), the colanic acid
exopolysaccharide (wca locus, formerly
called cps), tripeptidase T (pepT), and the
nickel high-affinity transport system (nikA). On the other
hand, the syntheses of flagellin (fliC) and of a putative
protein of 92 amino acids (f92) were both reduced in
biofilms. Such a genetic reprogramming of gene expression in biofilms
seems to result from changes in multiple environmental physicochemical conditions. In this work, we show that bacteria within
biofilms encounter higher-osmolarity conditions, greater oxygen
limitation, and higher cell density than in the liquid phase.
Many bacteria can attach to solid
surfaces and form biofilms, which are defined as matrix-enclosed
microbial populations adherent to each other and to surfaces or
interfaces (11). Biofilm formation is such a common
phenomenon that virtually every material that comes into contact with
naturally occurring fluids, such as blood or seawater, becomes rapidly
covered with bacteria (8, 10). Given the important medical
and economic consequences of this detrimental situation, understanding
the colonization process would help in the design of surface coating
methods able to prevent biofilm formation.
Abundant evidence indicates that cells grown on solid surfaces within
biofilms are in a physiological state different from that of planktonic
cells (reviewed in reference 22). The most difficult
properties of biofilm bacteria are their extreme resistance to
treatment with biocides and detergents and their high tolerance to
prolonged antibiotic therapy in human infections (19, 26). Bacterial structures involved in biofilm formation have been well characterized for some bacteria; AtlE (an autolysin) in
Staphylococcus epidermidis (25), type I pili
(42) and curli (56) in Escherichia coli, and type IV pili in Pseudomonas aeruginosa
(41) have been described as major structures for interaction
with the surface. The importance of flagellar motility in initiation of
biofilm development in Pseudomonas fluorescens, P. aeruginosa, and E. coli was also reported
(40-42).
However, there is still limited information available on bacterial
surface sensing at the gene expression level. Surface-induced expression of the laf genes, which are responsible for the
synthesis of peritrichous lateral flagella, was correlated with a
decreased rotation ability of the unique polar flagellum in
Vibrio parahaemolyticus (6, 35) and Proteus
mirabilis (5). Specific induced expressions of the
P. aeruginosa genes algC (13, 14) and
algD (27) and of the sfaA gene
(encoding S fimbrial adhesins) of a pathogenic strain of E. coli (47) were also observed after contact of the bacteria with a solid surface, but the surface-sensing mechanisms were not investigated. Furthermore, in many bacterial species, regulation of metabolic functions, particularly those relating to
virulence, involves cell-to-cell signalling molecules (such as
N-acylhomoserine lactones in gram-negative bacteria). These signal molecules accumulate in the bacterial environment as a function of cell number. Thus, the high density of bacteria within biofilms led to the hypothesis that cell-to-cell signal mechanisms may
play an important role in the establishment of the biofilm-specific physiological state. Evidence in support of this hypothesis has been
recently provided. Acylhomoserine lactones were detected in biofilms
formed on urethral catheters removed from patients (52) and
on immersed stones from the San Marcos river in Texas (36).
Moreover, Davies and coworkers (15) demonstrated that the
LasI-LasR system (one of the two cell-to-cell signalling systems of
P. aeruginosa) is required for the normal development of biofilms.
The present work was undertaken in the very well characterized E. coli K-12 context in order to identify biofilm-regulated functions
and to gather information on the regulation processes triggered by
bacterial contact with the surface. Using a new, reliable system to
assess changes in gene expression in biofilm cells versus planktonic
cells, we found that 38% of the E. coli genes are
differentially expressed in the biofilm. In addition to cell-to-cell
signalling mechanisms, microenvironmental conditions of osmolarity and
oxygen concentration can be correlated with this major change in gene expression.
Bacterial strains, plasmids, and media.
All of the E. coli K-12 strains and plasmids used in this work are listed in
Table 1. The bacteria were grown in
complete Luria-Bertani (LB) medium (37) or in minimal M63
medium (37) supplemented with mannitol (0.2%) or glucose
(0.2%) as carbon sources. MOPS (morpholinepropanesulfonic acid) medium
supplemented with glycerol (0.4%) was prepared as described previously
(39). Antibiotics were used at the following concentrations:
ampicillin, 50 µg/ml; kanamycin, 50 µg/ml; tetracycline, 10 µg/ml; and chloramphenicol, 20 µg/ml. For all experiments with
derivatives of strain YK3421, histidine (50 µg/ml), thymine (25 µg/ml), uracil (100 µg/ml), and cytosine (25 µg/ml) were added to
the M63 medium.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Abiotic Surface Sensing and Biofilm-Dependent
Regulation of Gene Expression in Escherichia coli
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
E. coli K-12 strains and plasmids used
Genetic methods. Random insertion mutagenesis with phage Mu dX (4) carrying the promoterless lacZ gene was performed by the procedure described by Miller (38). Phage P1 vir was used for transductions, which were carried out as described by Miller (37). The ompR234 mutation was transferred by using its genetic linkage (cotransduction at 50%) with malA or malT followed by screening of adherent transductants in 24-well microtitration plates.
Screening of lacZ fusions with altered expression
within biofilms.
Mutant clones obtained by transposition of Mu dX
were grown in 200 µl of M63-mannitol (0.2%) medium in the wells of a
96-well microtitration plate. After 24 h of incubation at 30°C,
a visible biofilm was present on the wall of each well. The medium
containing the free-living bacteria of each well was removed carefully
and was introduced into the corresponding well of a precooled
microtitration plate, whereas attached bacteria were suspended in 200 µl of cold M63-mannitol medium by vigorous shaking of the plate. The
turbidity (optical density at 630 nm [OD630]) and the
initial absorbance of suspensions at 405 nm were estimated by using a
microplate photometer. Bacteria were then permeabilized by the addition
of 20 µl of a permeabilization reagent (48), and
o-nitrophenyl-
-D-galactoside was added to a
final concentration of 2.7 mM. Quantification of -galactosidase
activity was performed at 405 nm with a microplate photometer after
various periods of incubation at room temperature. The ratio between
the OD405 and OD630 was calculated for the
free-living and biofilm bacteria (44).
Monitoring of lacZ fusion expression within biofilms
developed on inert surfaces.
The wells of 24-well microtitration
plates, petri dishes, or glass tubes containing Plexiglas strips were
filled with minimal M63 medium and inoculated with about 5 × 106 cells of an overnight culture of the clone of interest.
After different incubation times at 30°C, the liquid medium
containing free-living bacteria was carefully removed; biofilms
developed on inert surfaces were washed, and sessile bacteria were
suspended in cold medium by pipetting up and down. Cell biomass was
estimated by OD600 measurements, and -galactosidase
assays were performed as described below.
DNA manipulations. Standard techniques were used for chromosomal DNA preparation, plasmid extraction, gel electrophoresis, and DNA sequencing (45). Restriction endonucleases and T4 DNA ligase were used as recommended by the manufacturers.
Construction of plasmid pCP994. A 697-bp fragment containing the csgD open reading frame (ORF) was amplified by PCR with chromosomal DNA of strain MC4100 as the template and two primers (5'-CCGCCACACTGCAGCGTAAATAACG-3', and 5'-CGGGGTTTCATCATGACTAATGAAG-3') overlapping the csgD ORF and containing PstI and BspHI cutting sites, respectively. The PCR fragment was digested with PstI and BspHI and then purified and cloned in the PstI and NcoI sites of the plasmid cloning vector pKK233-2 (Apr) (2).
Identification of genes affected by Mu dX insertion.
The
chromosomal DNAs of mutants were extracted and digested with
PstI, and fragments of more than 10 kb were cloned in pBR322 or pPH126 (51). Transformants displaying -galactosidase
activity were analyzed. The chromosomal region adjacent to the
lacZ end of the Mu genome was sequenced with a 20-bp
lacZ-targeted oligonucleotide (5'-CAGGCATCAGGATTTGTGGC-3').
-Galactosidase assay.
-Galactosidase specific activity
in toluenized samples was measured by spectrophotometrically monitoring
the hydrolysis of o-nitrophenyl--D-galactoside into
o-nitrophenol at 420 nm and 37°C (37) and was
expressed as units per milligram of protein, where 1 U corresponds to 1 nmol of product liberated per min.
Potassium assay. Cells from 1-ml portions of suspensions of free-living and sessile bacteria grown in M63-mannitol medium in petri dishes, and containing the same number of cells (108), were harvested by centrifugation for 30 s, the supernatants were removed, and the pellets were suspended in 1 ml of deionized water. The suspensions were boiled for 1 min to release potassium from the cells, and the potassium concentrations were determined by flame photometry (55).
Preparation of conditioned medium.
An actively growing
culture of E. coli DH5
was diluted 1,000-fold in LB
medium and grown for 24 h at 37°C to a final OD600 of 3.5. Cells were removed by centrifugation for 15 min at 4°C. The
supernatant was sterilized by filtration through a 0.2-µm-pore-size filter and stored at 
80°C. When conditioned medium was used to support growth, tryptone and yeast extract were added to concentrations of 10 and 5 g/liter, respectively (20). Fifty milliliters of conditioned medium or fresh LB medium was inoculated at a final dilution of 1/100 with a stationary-phase culture of the appropriate strain. The cultures were incubated at 30°C with vigorous shaking. The conditioned medium supported growth to a cell division rate equal
to that of cells grown in fresh LB medium.
Electron microscopy. Cell cultures were grown in M63-mannitol medium at 30°C in gently shaken glass tubes, each containing one plastic strip (attached bacteria) or not containing a strip (free-living bacteria). At different incubation times between 8 and 48 h, suspensions of free-living bacteria and attached bacteria were carefully recovered to prevent breakdown of flagella and were allowed to adhere to carbon-coated 200-mesh grids. After being stained with 1% phosphotungstic acid (Sigma), the grids were examined with a Philips CM120 electron microscope.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A major change in the pattern of gene expression occurs in E. coli biofilms.
Random insertion mutagenesis with Mu dX, a
bacteriophage Mu derivative carrying the promoterless lacZ
reporter gene (4), was performed on the biofilm-forming
E. coli K-12 strain PHL644, and, for 885 clones, the
-galactosidase specific activities of the biofilm population and the
nonattached cells were compared in microtitration plates (see Materials
and Methods). A total of 446 clones were Lac+, and three
classes of lacZ fusions could be distinguished. Ninety-eight fusions belong to the first class, corresponding to fusions
significantly (1.5- to 10-fold) more expressed in the biofilm cells
(up-regulated fusions). In the second class, 73 fusions were
significantly (2- to 10-fold) less expressed in the biofilm
(down-regulated fusions). Most of the fusions (275) belong to the third
class, corresponding to invariant fusions. A total of 38% of the
fusions, therefore, were differently expressed in biofilm. A second
-galactosidase assay was conducted on a representative sample of 36 clones to compare the levels of expression in biofilm and free-living
cells grown in 24-well microtitration plates (see Materials and
Methods). The results supported those of the first analysis.
The higher osmolarity encountered in biofilm triggers major changes in gene expression. As detailed by Goodman and Marshall (22), a bacterium approaching a solid-water interface could encounter a gradient of inorganic ions and organic ionized molecules (attracted to counterbalance the electric charges existing at the solid surface), alterations in surface free energy, a lower pH level (resulting from proton accumulation), modified viscosity and osmolarity, and altered rates of gas exchange. Among these conditions, we chose to focus first on osmolarity.
It is well established that in E. coli, the intracellular content of potassium ions varies proportionally to the external osmotic pressure (12, 18). Intracellular K+ concentrations were measured for attached and planktonic PHL644 bacteria in 20 ml of liquid M63-mannitol medium introduced in petri dishes and incubated at 30°C without agitation (see Materials and Methods). After 10 h of incubation at 30°C, the intracellular potassium content in free-living bacteria was 1.81 ± 0.08 mmol/g of protein, and that in attached bacteria was 2.84 ± 0.28 mmol/g of protein (means ± standard errors for 10 repetitions; P < 0.001 by Student's t test). This corresponds to 223 ± 10 and 349 ± 34 mmol/liter for free-living and attached bacteria, respectively, where the cell volume was estimated to 1.2 µm3 for both by electron microscopy observations (more than 10 independent measurements; this result is in good agreement with data reported previously [17]). These results suggest that attached bacteria indeed encounter higher-osmolarity conditions than planktonic cells from the beginning of the colonization process. To study the effects of osmolarity further, the expression of four genes known to be osmoregulated was compared for attached and free-living bacteria grown in petri dishes, as described in Materials and Methods. The porin ompC gene (46); the proU operon, which encodes a high-affinity glycine betaine transport system (23); and the wcaB gene (formerly called cpsB), which is involved in the synthesis of the capsular exopolysaccharide colanic acid (50) are up-regulated by high osmolarity. On the other hand, the flagellin gene fliC is down-regulated by high salt concentrations (49). For each fusion up-regulated by osmolarity, a clear induction (two- to threefold) was observed in sessile populations during the first 40 h (Fig. 1A, B, and D). The down-regulated fusion fliC is consistently less expressed in biofilm bacteria (Fig. 1C). Similar weak differences in fliC-lacZ expression in the presence of regulatory mutations conferring a flagellum-deficient phenotype have been reported (30). This suggests that flagellum synthesis is reduced in biofilms, even if the level of fliC expression is far from being totally abolished. An electron microscopic study (see Materials and Methods) revealed that biofilm cells of the motile strain PHL628 did not synthesize flagella (and produced a copious amount of curli); no flagella were visualized on sessile bacteria and no broken flagella were released in biofilm cell suspensions (Fig. 2B), whereas flagellated free-living bacteria were easy to visualize (Fig. 2A). Synthesis of curli was lower in these last cells (43).
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-D-galactopyranoside), in
the presence or absence of NaCl (0.3 M). Differences in the intensity
of the blue coloration permitted the isolation of PHL825, a clone
containing a biofilm-down-regulated fusion which is repressed by a high
salt concentration. A kinetic analysis of the expression of this fusion
confirmed its biofilm regulation (Fig.
3A). To determine the precise extent of
osmoregulation, the expression of the fusion in shaken liquid cultures
containing different salt concentrations was monitored (Fig. 3B); a
fourfold-lower expression was observed at high osmolarity (in the
presence of 0.3 M NaCl). To identify the target gene of the Mu dX
insertion, the DNA fragment surrounding the lacZ gene was
cloned on a multicopy plasmid to give pCP832 and sequenced (see
Materials and Methods). A BLAST search identified this gene as the
uncharacterized f92 ORF (ECAE000245; min 33.5), encoding a
putative short protein (92 amino acids).
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Effects of the ompR234 allele.
Since wild-type
E. coli K-12 strains are not able to attach to surfaces, the
experiments reported above were done in the ompR234 genetic
background. EnvZ-OmpR is a two-component signal transduction system
involved in bacterial osmoregulation. The ompR234 allele increases the expression of csgA, the curlin-encoding gene
(the resulting overproduction of curli confers the adherence
properties), and of porin genes ompC and ompF
(56). Although the osmoregulation mediated by EnvZ-OmpR is
still efficient in ompR234 strains (56), the
effect of this allele on the biofilm-regulated fusions had to be
examined. The expressions of f92-lacZ, fliC-lacZ,
wcaB-lacZ, and proU-lacZ in wild-type,
ompR234, and ompR::Tn10
strains in planktonically grown cultures were compared (Table
2). For all of these strains, similar
growth rates were observed (data not shown). The expression of
proU, a gene which is osmoregulated independently of
the EnvZ-OmpR system (32), was similar in the three backgrounds. The same results were observed for fliC,
whereas f92 and the colanic acid gene wcaB were
strongly dependent on the ompR allele. This last result
indicates that these two genes belong to the OmpR regulon.
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Cells in biofilm also encounter limited diffusion of oxygen. Because of the particular physicochemical conditions on uncolonized surfaces and the high cellular density in established biofilms, oxygen diffusion could be an important parameter to explain differential gene expression in attached and free-living bacteria. Furthermore, in all of the experiments reported above, the microtitration and petri dishes were incubated in the absence of agitation. Due to the small total amount present in the liquid medium, the local oxygen availability could rapidly become a discriminating factor between the bacteria colonizing the bottom of the vessel and the cells swimming in the liquid phase. A nitrate assay (28) performed on strain PHL644 cultivated in liquid medium in unshaken petri dishes indicated that microaerophilic conditions were reached after 16 h (5 mM nitrite was detected in the medium). nikA encodes a high-affinity nickel transport system, and the expression of this gene is known to be precisely tuned by the level of oxygen availability (57). The nikA-lacZ fusion of strain HYD723 was introduced in an ompR234 background (to give strain PHL965), and its expressions in biofilm and planktonic bacteria under standard incubation conditions were compared. A fivefold-higher expression was observed in biofilm after 23 and 45 h (Fig. 5A). These differences were not observable in anaerobiosis (Fig. 5B), indicating that the local oxygen concentration is the main parameter responsible for the differential nikA expression under the standard conditions.
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Cell-to-cell signalling.
Since we have shown that osmolarity
is a key factor in biofilm-dependent gene regulation, a possible
osmoregulation of pepT was examined. Similar levels of
expression of the pepT-lacZ fusion were obtained in liquid
culture in MOPS-glycerol medium (126 U/mg of protein) and in
MOPS-glycerol containing 0.3 M NaCl (118 U/mg of protein), indicating
that pepT, in contrast to f92, ompC,
fliC, wcaB, and proU, is not
osmoregulated. In addition, a similar expression was observed in the
ompR+, ompR234, and
ompR::Tn10 backgrounds (Table 2).
Kinetic analysis of the expression of the pepT-lacZ fusion
during growth in liquid M63-mannitol medium gave some clues concerning
its regulation. A sudden increase in -galactosidase activity was
measured at the end of the exponential phase (Fig.
7A), probably resulting from a cell
density or starvation signalling mechanism. As a cell-to-cell signalling mechanism has been shown to be involved in P. aeruginosa biofilm development (15), a quorum-sensing
mechanism was investigated for pepT regulation. Conditioned
medium (i.e., medium containing the suspected signalling factors) was
removed from a stationary-phase culture of strain DH5, as described
previously (3, 20) (see Materials and Methods) and used to
support growth of the pepT-lacZ strain. Overexpression of
the fusion was observed at a low cell density (Fig. 7B), confirming the
emission of some cell-to-cell signalling factors in the conditioned
medium in response to high cell density. As the cellular density is far
higher in our biofilm (1011 cells/ml are reached in a
20-µm-thick biofilm after 24 h) than in the liquid phase
(109 cells/ml), we can suggest that the biofilm-dependent
activation of pepT resulted from a cell-to-cell signalling
mechanism.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In natural environments, bacteria are often found as sessile communities known as biofilms (8, 10). To date, the bacterial structures of adherence (25, 40-42, 56) and the physiological processes involved in bacterial surface colonization (15, 40, 41) are better understood than the genetic responses of bacteria adhering to a surface. By using a library of lacZ fusions and a reliable screen for identifying genes whose expression changes in biofilm versus planktonic cells, the transcription of 38% of the E. coli genes was shown to be modified during the colonization process. Several genes with altered expression in biofilms were identified. Different cellular functions were induced in attached bacteria: the OmpC porin, the high-affinity transport system of glycine betaine, colanic acid production (the E. coli class I exopolysaccharide), tripeptidase T, and synthesis of a nickel high-affinity transport system. On the other hand, the syntheses of flagella and of a putative protein of 92 amino acids were both reduced in biofilms. The induction of colanic acid synthesis at the gene expression level is consistent with the results of other authors, who have described a similar surface activation of exopolysaccharidic alginate genes in P. aeruginosa (13, 14, 27). These results emphasize the major role of exopolysaccharides in biofilm development, as previously reported (1, 9). Another important aspect of the biofilm formation process is triggered by flagella in different organisms, such as P. fluorescens, P. aeruginosa, and some strains of E. coli (40-42). However, in biofilms of E. coli K-12 strains overproducing curli, no flagella are produced (Fig. 2B). Furthermore, nonmotile strains (such as adherent derivatives of MC4100 overproducing curli) form thick biofilms as well as motile strains (such as adherent derivatives of MG1655 overproducing curli) do. It seems that, depending on environmental conditions and biofilm communities, different approaches and cell surface structures such as flagella, pili, S-fimbrial adhesins, autolysins, and curli could be used to initiate biofilm formation (25, 41, 42, 47, 56).
The surprising major change in gene expression observed within E. coli biofilms is consistent with the results of several teams that have shown that new protein synthesis is required for biofilm formation (40) and that patterns of proteins synthesized by attached cells versus planktonic cells are significantly different (11). Our study suggests that such a genetic reprogramming of gene expression in E. coli biofilms results from changes in multiple environmental physicochemical conditions: bacteria within biofilms encounter higher-osmolarity conditions, a lower oxygen supply, and higher cell density. Indeed, the intracellular concentration of potassium ions, which is essentially proportional to the osmolarity of the external medium in the absence of exogenous solutes such as proline or betaine (18), is 1.6-fold higher in attached bacteria than in free-living bacteria (see Results). This magnitude of change is weak but corresponds to that previously published (18; see Fig. 3 of reference 29). According to these studies, our data correspond to external osmolarities of about 220 mosM (corresponding to an intracellular potassium concentration of 223 mM) for free-living bacteria and about 380 mosM (corresponding to an intracellular potassium concentration of 349 mM) for attached cells. The osmolarity of the freshly prepared sterile M63-mannitol medium was 250 mosM (as measured with a Fiske OS/220 osmometer) (data not shown). A bacterium approaching a surface may encounter a gradient of organic and inorganic ions attracted to counterbalance the negative electric charges existing at the solid surface (22). Moreover, the copious amount of appendages present at the cell surface (such as curli [Fig. 2B]) and the various exopolymers excreted by bacteria could concentrate ionic molecules from the bulk phase as the biofilm develops.
Osmoregulated genes, whether they belong to the OmpR regulon (such as ompC, wcaB, and f92) (Table 2) or not (such as fliC, and proU) (Table 2), respond within biofilms to changes in osmolarity. Fusions up-regulated by high osmolarity (such as ompC, wcaB, and proU-lacZ) were more expressed in sessile cells, whereas fusions down-regulated by high osmolarity (such as fliC and f92-lacZ) were more expressed in planktonic cells (Fig. 1 and 3). The effect of ompR234, the particular allele used in this work, on these responses was investigated for one OmpR-independent osmoregulated gene, proU, and for one OmpR-dependent osmoregulated gene, wcaB. For these two genes, the same differential expression of free-living bacteria and attached bacteria was observed in biofilms of wild-type ompR strains overproducing curli through the overexpression of the activator CsgD (Fig. 4). Thus, an ompR234 strain is still able to detect osmotic changes and react efficiently (56); the OmpR234 protein is thought to interact more strongly with the regulatory sites of the target genes or with the RNA polymerase. This hypothesis is in good agreement with the higher repression of the wca promoter observed with OmpR234 than with OmpR (Table 2).
As the biofilm develops, oxygen consumption and emission of
density signals could cause new patterns of gene expression.
Using microelectrodes, Costerton and collaborators have shown that
microaerophilic conditions were encountered by attached bacteria
(11). Here, by using a fusion in nikA, a gene
which is highly expressed in anaerobiosis and belongs to the FNR
regulon (57), limitation of oxygen availability to cells
within biofilms and its impact on gene expression were demonstrated
(Fig. 5). A 100-fold-higher density was encountered in biofilms of
ompR234 E. coli K-12 mutants than in the liquid phase.
Cell-to-cell signals may therefore be accumulated and be involved in
regulation of gene expression. This seems to be the case for
pepT. This gene was shown to be more highly expressed in
biofilm cells than in free-living bacteria and to be highly induced in
the late exponential growth phase, suggesting its probable regulation
by quorum sensing. In E. coli K-12 prototroph strains, such
as MG1655, two types of autoinducers are produced: the first, encoded
by the luxS gene (similar to the AI-2 factor produced by
Vibrio harveyi), is expressed in the mid-exponential phase
and is degraded when bacteria enter the stationary phase, whereas the
second operates in the stationary phase (3). The first one
is not produced by the domesticated laboratory strain DH5, due to
the presence of a frameshift mutation in the luxS gene
(54). Since the pepT gene was induced in the late
exponential phase, only the role of the second type of signal molecules
was investigated: a conditioned medium, prepared from a
stationary-phase culture of DH5 cells, was able to induce the expression of the pepT-lacZ fusion at a low cell density.
pepT could therefore be considered a new cma (for
conditioned medium activated) gene (3). Previously described
cma genes were shown to be involved in amino acid
metabolism; this fact is in good agreement with the function of
tripeptidase T.
Biofilm gene expression patterns appear to be modulated by multiple changing external physicochemical conditions and to involve very complex regulation pathways; several bacterial sensors, many two-component signal transducing systems (such as OmpR-EnvZ), and perhaps some alternative sigma factors (such as AlgU, which is required for alginate gene expression in P. aeruginosa [16, 33]), may be suggested to coordinately regulate genes within biofilms. The analysis of additional biofilm-regulated genes isolated in the screen described in this study is in progress. It will allow us to assess the role of physicochemical factors other than osmolarity, oxygen limitation, and cell density in biofilm gene expression in E. coli K-12. Further studies of bacterial structures involved in surface sensing and biofilm formation may provide ways to move forward in the search for efficient surface coating methods able to prevent biofilm formation or, at least, to interfere with their inconvenient increased resistance to biocides.
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ACKNOWLEDGMENTS |
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We acknowledge Rémy Gourdon and Karim Lounis for helping us with the potassium assay, Valérie de Crécy and Philippe Bertin for gifts of strains, and the members of the Laboratoire de Génétique des Microorganismes for their interest in this work and helpful discussions.
This work was supported by grants from the French Defense Ministry (96-048/DRET) and the Centre National de la Recherche Scientifique (Réseau "Infections Nosocomiales").
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Génétique des Microorganismes, INSA de Lyon, 20 avenue A. Einstein, 69621 Villeurbanne, France. Phone: (33) 4 72 43 87 06. Fax: (33) 4 72 43 87 14. E-mail: lejeune{at}insa.insa-lyon.fr.
Present address: Department of Animal Sciences, University of
Illinois, Urbana, IL 61801.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Prigent-Combaret, C., Brombacher, E., Vidal, O., Ambert, A., Lejeune, P., Landini, P., Dorel, C.
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Rajkumari, K., Gowrishankar, J.
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Becker, P., Hufnagle, W., Peters, G., Herrmann, M.
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