The Two-Component Response Regulator RcsB Regulates Type 1 Piliation in Escherichia coli

Article (PDF Available)inJournal of Bacteriology 189(19):7159-63 · November 2007with11 Reads
DOI: 10.1128/JB.00705-07 · Source: PubMed
The ability of Escherichia coli cells to produce type 1 pili depends upon the orientation of the fimA promoter. The orientation depends upon the ratios of the FimB and FimE recombinases. Here, we report that the two-component response regulator RcsB influences the piliation state by controlling fimB and fimE transcription.
JOURNAL OF BACTERIOLOGY, Oct. 2007, p. 7159–7163 Vol. 189, No. 19
0021-9193/07/$08.000 doi:10.1128/JB.00705-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
The Two-Component Response Regulator RcsB Regulates Type 1
Piliation in Escherichia coli
William R. Schwan,
Satoshi Shibata,
Shin-Ichi Aizawa,
and Alan J. Wolfe
Department of Microbiology, University of Wisconsin—La Crosse, La Crosse, Wisconsin 54601
; Department of Life Sciences,
Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan
; CREST Soft Nano-Machine Project,
Innovation Plaza Hiroshima, 3-10-23 Kagamiyama, Higashi-Hiroshima 739-0046, Japan
; and Department of
Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago,
Maywood, Illinois 60153
Received 3 May 2007/Accepted 12 July 2007
The ability of Escherichia coli cells to produce type 1 pili depends upon the orientation of the fimA promoter.
The orientation depends upon the ratios of the FimB and FimE recombinases. Here, we report that the
two-component response regulator RcsB influences the piliation state by controlling fimB and fimE
Type 1 pili are filamentous proteinaceous appendages pro-
duced by many members of the family Enterobacteriaceae (4)
that play a major role in biofilm development and pathogenesis
during the course of human infections (26). Escherichia coli
cells can switch from a completely piliated state to a completely
nonpiliated state (7). This ability depends on a process called
phase variation (Fig. 1), in which a 314-bp invertible DNA
element switches between two interconvertible orientations
(1). When this element is in the “phase-on” orientation, the
fimA promoter (fimAp) faces the fim operon and thus can drive
its transcription. Since the fim operon includes genes for struc-
tural components and the machinery required for pilus assem-
bly, cells in the “phase-on” orientation elaborate numerous
type 1 pili. When the element is oriented in the “phase-off”
state, the promoter faces the opposite direction, the promoter
cannot drive fimA transcription, and the cells lack type 1 pili
altogether. The invertible nature of the fimAp element de-
pends upon two site-specific recombinases, FimB and FimE.
Whereas FimE recombinase activity favors switching from
“phase-on” to “phase-off,” FimB facilitates switching in both
directions (16, 22, 23).
We previously reported that the fim operon is regulated by
acetyl phosphate (35), a central metabolite that functions as a
global signal (12, 34) by donating its phosphoryl group to a
subset of response regulators (RRs) of the family of two-
component signal transduction (2CST) pathways (8, 15a, 19).
The most fundamental of 2CST pathways consists of an RR
and a sensor kinase (SK). The SK autophosphorylates a con-
served histidinyl residue, using ATP as its phosphoryl donor.
The phospho-SK then serves as the phosphoryl donor to the
RR, which autophosphorylates a conserved aspartyl residue (6,
29, 33). For a subset of RRs, the central metabolite acetyl
phosphate can serve as an alternative phosphoryl donor (re-
viewed in reference 34). As its name implies, the RR is typi-
cally associated with a response domain, often one that permits
binding to DNA. Thus, many RRs function as transcription
factors (6, 29, 33).
The phosphorelay, a more complex version of the 2CST
pathway, contains two additional domains. As in the funda-
mental 2CST pathway, ATP donates a phosphoryl group to the
SK, which then donates it to an RR. In the phosphorelay, a
histidine phosphotransferase transfers the phosphoryl group
from the first RR to a second one (reviewed in references 2, 14,
and 24). The core of the Rcs phosphorelay is composed of
three proteins: RcsC (a hybrid SK-RR), RcsD (a histidine
phosphotransferase also known as YojN), and RcsB (the ter-
minal RR) (reviewed in references 10 and 20). RcsB can bind
DNA either as a homodimer (3) or as a heterodimer in asso-
ciation with the accessory protein RcsA (31, 32). The stability
of RcsA is controlled by the proteases Lon (30) and ClpYQ
(18). Another accessory protein, the outer membrane lipopro-
tein RcsF, serves to activate the kinase activity of RcsC (20).
The Rcs phosphorelay is estimated to regulate some 5% of
the Escherichia coli genome (reviewed in references 21 and
25). Most of these genes encode functions associated with the
cell envelope. For instance, the Rcs phosphorelay activates the
genes required for the biosynthesis of colanic acid, an extra-
cellular polysaccharide required for biofilm development (13).
It also activates the expression of several multiple-stress effec-
tors that localize to the periplasm (3, 5), while repressing genes
required for the biogenesis of flagella (11).
While studying the impact exerted by acetyl phosphate upon
the network of 2CST pathways (12), we obtained electron
microscopic evidence that led us to hypothesize that RcsB
functions as a positive regulator of type 1 pili. Here, we report
attempts to test that hypothesis.
We grew cells at 37°C in tryptone broth (1% [wt/vol] tryp-
tone, 0.5% [wt/vol] NaCl), harvested them during mid-expo-
nential growth and shortly after entry into stationary phase,
and monitored piliation by transmission electron microscopy
as described previously (12). At both stages of growth, about
60% of wild-type (WT) cells (strain AJW678) (17) elaborated
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Stritch School of Medicine, Loyola University
Chicago, Maywood, IL 60153. Phone: (708) 216-5814. Fax: (708) 216-
9574. E-mail:
Published ahead of print on 20 July 2007.
pili (Fig. 2A and C); isogenic rcsC mutant cells (strain
AJW2144) (12) displayed similar behavior (Fig. 2C). In con-
trast, most isogenic rcsB mutant cells (strain AJW2143) (12)
were nonpiliated (Fig. 2B). About 40% of the rcsB mutant cells
elaborated pili after entry into stationary phase, while only
20% displayed pili during exponential growth (Fig. 2C).
The observation that the percentage of piliated cells was
affected rather than the number of type 1 pili per cell or their
length led us to hypothesize that RcsB influenced the orienta-
tion of the 314-bp fimAp invertible element. To test this hy-
pothesis, we initially performed multiplex PCR amplifications
on chromosomal DNA extracted from the rcsB mutant, the
rcsC mutant, or their WT parent, using oligonucleotide prim-
ers specific for the “phase-on” and “phase-off” orientations of
the invertible element (28) or, as a control, the E. coli ftsZ gene
We previously reported that pH values and salt relevant to
murine urine exerted substantial effects on phase variation
(27). Therefore, we grew cells at 37°C to mid-exponential
phase in LB (1% [wt/vol] peptone 140, 0.5% [wt/vol] yeast
extracts, 1% [wt/vol] glycerol, 0.1 M sodium phosphate, 0.5%
[wt/vol] NaCl) at either neutral pH (7.0) or acidic pH (5.5) and
in either the presence of additional NaCl (final concentration,
490 mM [referred to as high salt]) or its absence (final con-
centration, 90 mM [referred to as low salt]).
Overall, the PCR results followed those of our previous
study (27): more WT cells grown at neutral pH (Fig. 3A, lanes
1 and 4, and B) than cells grown at acidic pH (Fig. 3A, lanes 7
and 10, and B) positioned their invertible fimAp element in the
“phase-on” orientation. This analysis also showed that rcsB
mutant cells grown under neutral-pH, low-salt conditions (Fig.
3A, lane 2) positioned their invertible element more in the
“phase-off” orientation than did cells of either their WT parent
(lane 1) or the isogenic rcsC mutant (lane 3) grown under the
same conditions. Under neutral pH, high-salt conditions, the
rcsC mutant (lane 6) appeared to position the fimAp promoter
element less in the “phase-on” orientation than did either its
WT parent (lane 4) or the rcsB mutant (lane 5). At acidic pH,
regardless of salt, all strains produced similar “phase-off” and
“phase-on” distributions (lanes 7 to 12).
To confirm the rcsB finding, we complemented the rcsB
mutant with a plasmid containing a His-tagged WT rcsB gene
(pHRcsB) (5), harvested cells during mid-exponential growth,
and compared the complemented strain to the WT strain and
the rcsB mutant parent (Fig. 3C and D). At the neutral-pH,
low-salt condition, the complemented rcsB mutant (lane 3)
oriented its invertible element more like the WT strain (lane 1)
than the rcsB mutant (lane 2). Similar results were obtained
when cells were harvested following entry into stationary phase
(data not shown).
Taken together, these results support the hypothesis that
RcsB regulates piliation under neutral-pH, low-salt conditions
by influencing the orientation of the fimAp invertible element.
These results also suggest that RcsC may influence the posi-
tioning of the invertible element under high-salt growth con-
ditions. Finally, they show that neither RcsB nor RcsC exerts
much influence at acidic pH and that some other factor must
be involved.
To determine how RcsB may regulate the orientation of the
invertible element, we asked whether RcsB and/or RcsC influ-
ences the transcription of fimB,fimE, or both recombinase genes.
The rcsB mutant, the rcsC mutant, and their WT parent were
transformed with single-copy plasmids pJB5A and pJLE4-3,
which express the transcriptional fusions fimB-lacZYA and fimE-
lacZYA, respectively (27). Because pH and salt have been shown
to influence the transcriptional states of both fimB and fimE (27),
we performed these reporter studies under the growth conditions
used for the PCR analyses.
When cells were grown at acidic pH in the presence of either
the low or high salt, transcription of either fimB or fimE was
largely unaffected by the status of the Rcs phosphorelay (Table
FIG. 1. In E. coli, the abilities of cells to assemble type 1 pili
depend upon the orientation of the fimA promoter. The orientation
depends primarily on the ratio of the activities of FimB and FimE. A
high activity favors the “phase-on” orientation (top); a low activity
favors the “phase-off” orientation (bottom).
FIG. 2. RcsB enhances piliation. Cells were grown aerobically with
250-rpm agitation at 37°C in tryptone broth. Samples were negatively
stained with 2% phosphotungstic acid (pH 7.0) and observed with a
JEM-1200EXII electron microscope (JEOL, Tokyo, Japan). Micro-
graphs were taken at an accelerating voltage of 80 kV. Transmission
electron micrographs of WT cells (strain AJW678) (A) and rcsB mu-
tant cells (strain AJW2143) (B) harvested at an optical density at 600
nm of 0.5 are shown . The thin and thick appendages are type 1 pili and
flagella, respectively. The bar represents 2 m. (C) Histogram showing
percentages of piliated cells harvested at either 0.5 (solid bar) or 1.0
(open bar) optical density units at 600 nm. The sample sizes ranged
from 83 to 178 cells.
1). This is consistent with the lack of any significant effect upon
the populations of invertible elements (Fig. 3). In contrast,
when cells were grown at neutral pH, the states of certain Rcs
phosphorelay components influenced transcription. Further-
more, the critical Rcs component and the promoter affected
depended upon the salt. For example, at the low salt, rcsB
mutant cells transcribed fimB at significantly reduced levels
relative to their WT parent and the rcsC mutant (Table 1). This
effect appears to be specific because expression of the His-
tagged WT rcsB allele from a compatible plasmid (pHRcsB)
restored transcription to WT levels. In contrast, growth in
high-salt medium resulted in a distinctly different pattern. Un-
der this condition, relative to WT cells, both the rcsB and rcsC
mutants exhibited elevated fimE transcription. Here, both the
RcsB and RcsC effects appeared to be specific because expres-
sion from a compatible plasmid either with the His-tagged WT
rcsB allele in the rcsB mutant (Table 1) or with the WT rcsC
allele (pSG980) (9) in the rcsC mutant restored fimE transcrip-
tion to WT levels (data not shown). In contrast, the vector
controls had no effect (data not shown).
The PCR analysis (Fig. 3) and the fim-lacZ fusion data
(Table 1) supported the hypothesis that RcsB can influence the
orientation of the fimAp invertible element by controlling the
transcription of the recombinase genes fimB and fimE. This
RcsB-dependent behavior occurred only at neutral pH and
depended upon RcsC only in the presence of the high salt. On
the basis of these observations, we predicted that type 1 pilus
expression would be affected in a condition-dependent manner
by mutations in rcsB and rcsC. To test this prediction, enzyme
immunoassays were performed according to the procedure of
Hultgren et al. (15). As predicted, at neutral pH with the low
salt, the rcsB mutant strain displayed significantly reduced type
1 pilus expression relative either to its WT parent or to the rcsC
mutant (Fig. 4). At neutral pH with the high salt, however,
both the rcsB and rcsC mutants displayed lower levels of type
FIG. 3. Determination of the invertible element orientation by PCR.
(A) Analysis was performed on chromosomal DNA isolated from WT cells
(strain AJW678), an rcsB mutant (strain AJW2143), and an rcsC mutant
(strain AJW2144). Cells were harvested during mid-exponential phase fol-
lowing aerobic growth with 250-rpm agitation at 37°C in pH 7.0 LB medium
with either no added NaCl (low salt) or 400 mM added NaCl (; high salt)
or in pH 5.5 LB medium with either no added NaCl (low salt) or 400 mM
NaCl (high salt). Multiplex PCRs were set up with INV and FIMA primers
to amplify “phase-on”-oriented DNA (ON; 450-bp product) (28), FIME and
INV primers to amplify “phase-off”-oriented DNA (OFF, 750-bp product)
(28), and EcFtsZ 1 and 2 primers to amplify the ftsZ gene (302-bp product)
(27). Each multiplex was run at least three separate times. The lanes were
loaded as follows: lane 1, AJW678 (pH 7.0, low salt); lane 2, AJW2143 (pH
7.0, low salt); lane 3, AJW2144 (pH 7.0, low salt); lane 4, AJW678 (pH 7.0,
high salt); lane 5, AJW2143 (pH 7.0, high salt); lane 6, AJW2144 (pH 7.0,
high salt); lane 7, AJW678 (pH 5.5, low salt); lane 8, AJW2143 (pH 5.5, low
salt); lane 9, AJW2144 (pH 5.5, low salt); lane 10, AJW678 (pH 5.5, high salt);
lane 11, AJW2143 (pH 5.5, high salt); and lane 12, AJW2144 (pH 5.5, high
salt). (B) Quantification of the data from panel A. Using ImageQuant soft-
ware (Molecular Dynamics), the number of pixels for each band was quan-
tified. For each lane, the intensities of the OFF and ON states were corrected
to the intensity of the ftsZ band. The corrected values for both states were
standardized to the respective WT band (lane 1). Since the resultant values
were plotted as log
numbers, the WT strain for both the OFF and ON states
had a value of zero, while increased and decreased PCR products resulted in
positive and negative values, respectively. Solid bars, strain AJW678 (WT);
open bars, strain AJW2143 (rcsB mutant); hatched bars, strain AJW2144
(rcsC mutant). (C) Analysis was performed on chromosomal DNA isolated
from WT cells (strain AJW678), an rcsB mutant (strain AJW2143), and an
rcsB mutant complemented with the pHRcsB plasmid (strain AJW2307)
without induction. Multiplex PCR amplifications were performed with the
same primer pairs, using DNAs isolated from the strains grown as described
for panel A. Each multiplex was run at least three separate times.
Lane 1, AJW678 (pH 7.0, low salt); lane 2, AJW2143 (pH 7.0, low
salt); lane 3, AJW2307 (pH 7.0, low salt); lane 4, AJW678 (pH 7.0,
high salt); lane 5, AJW2143 (pH 7.0, high salt); lane 6, AJW2307
(pH 7.0, high salt); lane 7, AJW678 (pH 5.5, low salt); lane 8,
AJW2143 (pH 5.5, low salt); lane 9, AJW2307 (pH 5.5, low salt);
lane 10, AJW678 (pH 5.5, high salt); lane 11, AJW2143 (pH 5.5,
high salt); and lane 12, AJW 2307 (pH 5.5, high salt). All PCR
products were subjected to electrophoresis on 1.5% agarose gels.
(D) Quantification of the data from panel C as described for panel
B. Solid bars, strain AJW678 (WT); open bars, strain AJW2143
(rcsB mutant); hatched bars, strain AJW2307 (complemented rcsB
TABLE 1. Effects of pH and salt on fimB-lacZ and fimE-lacZ
fusions in WT E. coli compared to effects on isogenic
rcsB and rcsC mutants
Fusion and strain
Expression level (Miller units) under indicated
growth condition
pH 7.0 pH 7.0 () pH 5.5 pH 5.5 ()
fimB-lacZ fusion
WT 345 30 259 36 271 20 219 23
rcsB 201 19
229 27 274 62 182 48
329 21 315 13 297 11 231 11
rcsC 305 34 235 36 284 51 238 57
fimE-lacZ fusion
WT 335 26 318 54 252 35 237 26
rcsB 332 34 463 68 260 54 221 60
rcsB/pRcsB 339 32 377 70 231 55 245 13
rcsC 365 21 437 53 260 32 253 48
Values indicate fimB and fimE promoter expression in terms of -galacto-
sidase activity and are means standard deviations from at least three indepen-
dent experiments. Cells were grown in LB at pH 7.0 or pH 5.5 with the low or
high () salt and harvested during mid-exponential phase.
Bold denotes that the differential expression is significant (P0.05) as
determined by Student’s ttest.
rcsB/pRcsB is the rcsB mutant transformed with pHRcsB, which encodes a
His-tagged WT rcsB allele under the control of the lac promoter. Induction,
however, was unnecessary.
VOL. 189, 2007 NOTES 7161
1 pili than did their WT parent. In contrast, at acidic pH
(regardless of salt), the status of rcsB or rcsC had, at most, a
minor effect.
At neutral pH, therefore, it appears that RcsB helps mediate
the inversion of the fimAp element either by increasing fimB
transcription (low salt) or by decreasing fimE transcription
(high salt). These results are consistent with our observation
that significantly fewer rcsB mutant cells than cells of their WT
parent expressed pili. Whether RcsB acts directly upon fim
transcription awaits further experimentation; however, inspec-
tion of the sequence upstream of the fimB and fimE open
reading frames reveals several sequences with some similarity
to RcsB and RcsAB boxes (31, 32). Although RcsC appeared
to have no discernible effect under neutral-pH, low-salt growth
conditions, it appeared to influence the orientation of the
invertible element under neutral-pH, high-salt conditions, pre-
sumably by decreasing fimE transcription. These results argue
for the hypothesis that RcsB receives its phosphoryl groups
from RcsC (its cognate SK) under neutral-pH, high-salt con-
ditions but from an alternative donor (e.g., acetyl phosphate or
a noncognate SK) under neutral-pH, low-salt conditions.
Our previous work showed that acidic growth conditions
reduce type 1 pilus expression, while implicating the EnvZ/
OmpR 2CST pathway as a fim inhibitor at least in the presence
of the high salt (27). The current study supports the former
conclusion and shows that it occurs in an RcsB-independent
manner. Whether fim regulation under acidic growth condi-
tions involves the EnvZ/OmpR pathway or some other acid
tolerance gene product is currently under examination.
In summary, we propose that the fim locus is part of the
RcsB regulon and that this global regulator inversely affects
the transcription of fimB and fimE. Furthermore, this inverse
regulation increases the probability of the “phase-on” orienta-
tion of the fimAp element and consequently the elaboration of
type 1 pili. On the basis of the current study and our previous
report (12), we can conclude that RcsB enhances the produc-
tion of type 1 pili as well as the synthesis of the capsule, while
inhibiting the biogenesis of flagella. Since these surface or-
ganelles play critical and/or essential roles in biofilm develop-
ment and urinary tract infections, RcsB should now be consid-
ered a coordinator of these processes.
We thank Sylvia Reimann for strain construction.
This work was supported by NIH grant GM066130, awarded to
A.J.W., grant RAI065432A, awarded to W.R.S., and a grant from the
CREST project of JST to S.-I.A.
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VOL. 189, 2007 NOTES 7163
    • "The Rcs cascade was originally identified as a positive regulator of colanic acid biosynthesis in Escherichia coli [5] and is one of the most thoroughly studied signaling pathways in bacteria. It is known to regulate genes involved in the production of important cell-surface-associated structures, such as flagella [6], exopolysaccharides [7], fimbriae [8] and some proteins with predicted roles in the maintenance and/or modification of the bacterial cell surface [9]. The cascade is composed of three proteins, i.e., RcsC (a hybrid kinase), RcsD (a phosphotransferase) and the response regulator RcsB [10]. "
    [Show abstract] [Hide abstract] ABSTRACT: Edwardsiella tarda, a Gram-negative bacterium of the family Enterobacteriaceae, is the causative agent of the systemic disease edwardsiellosis, which is a major problem in aquaculture industry worldwide. Many virulence-related genes in E. tarda have been investigated, but the Rcs phosphorelay, a two-component pathway, which regulates several cell-surface-associated structures related to invasion and survival in host cells, has not yet been thoroughly studied. In the present study, an rcsB in-frame deletion mutant ΔrcsB was constructed through double-crossover allelic exchange. To complement the rcsB mutation, the ΔrcsB (pACYC184K-rcsB) mutant was constructed by transformation of a low-copy plasmid carrying the intact rcsB into the ΔrcsB mutant of E. tarda. Several virulence-associated characters of the mutants and wild-type strain were tested. Compared with wild-type strain EIB202, biofilm formation decreased significantly in ΔrcsB, while ΔrcsB (pACYC184K-rcsB) recovered the phenotype to some extent. In addition, the capacity for autoagglutination, the percentage of adherence and internalization to Epithelioma papulosum cyprini cells and lethality toward zebrafish embryos significantly increased in ΔrcsB. All these phenomena displayed by mutant ΔrcsB showed a certain degree of recovery, though incomplete, in strain ΔrcsB (pACYC184K-rcsB). Present results indicate that rcsB is involved in regulating the gene expression of virulence factors in E. tarda, as shown in other members of Enterobacteriaceae.
    Article · Apr 2014
    • "According to this scenario, we supposed that both procaine and RfaH activate the pathway and that the cognate regulator of the system inhibits fimB transcription. It has been shown previously that fimB transcription is controlled by the response regulator RcsB that forms part of the Rcs phosphorelay system, a regulatory pathway that is also responsive to envelope stress (41). However, it was found that fimB transcription was unaffected by RcsB under the growth conditions used in this study and that the effect of procaine on the expression of the fimB-lacZ transcriptional fusion remained intact in this mutant background (data not shown). "
    [Show abstract] [Hide abstract] ABSTRACT: The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE, and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.
    Full-text · Article · Oct 2013
    • "This capsule can consist of many different substances, among them the K-capsule polysaccharide that is a contributor to the intracellular lifestyle of uropathogenic E. coli [1] and colanic acid, which has been recognized early as an important factor in forming the three dimensional structures that constitute the bio- film [39] . The phosphorelay system RcsCDB is an activator of colanic acid production [40], while also activating the synthesis of type I fimbriae [25]. These multiple "
    [Show abstract] [Hide abstract] ABSTRACT: Biofilms are communities of bacteria that are characterized by specific phenotypes, including an increased resistance towards anti-microbials and the host immune system. This calls for the development of novel biofilm prevention and treatment options to combat infectious disease. In Escherichia coli, numerous global regulators have been implicated in the control of biofilm associated cell surface organelles. These include the flagellar regulator FlhD/FlhC, the osmoregulator EnvZ/OmpR, and the colanic acid activator RcsCDB. Using flow cell technology and fluorescence microscopy, we determined the temporal expression from flhD::gfp, ompR::gfp, and rcsB::gfp in E. coli biofilm, as well as the impact of the negative regulation of flhD by OmpR and RcsB. Spatial gene expression was investigated from flhD::gfp. The temporal gene expression profile for flhD yielded an early peak at 12 h, a minimum of expression at 35 h, and a second increase in expression towards 51 h of biofilm development. In contrast, the ompR profile showed a peak at 35 h. A mutation in ompR abolished time dependence of flhD expression after the initial growth period of 12 h. Intriguingly, rcsB expression did not correlate inversely with flhD expression, yet a mutation in rcsB abolished time dependence of flhD expression as well. Spatially, expression of flhD was highest in the outermost layer of the biofilm in the parent strain. In ompR and rcsB mutants, flhD was expressed throughout the biofilm. Mutations in both, ompR and rcsB increased flhD expression throughout all temporal and spatial experiments. This increase was paralleled by reductions in biofilm amounts at four tested time points. Our data lead to the conclusion that FlhD/FlhC and its regulation by OmpR and RcsB may be our first target mechanism for the development of novel biofilm prevention and treatment techniques.
    Full-text · Article · Aug 2013
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