Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli

Caroline Blumer1,{dagger}, Alexandra Kleefeld1, Daniela Lehnen1, Margit Heintz1, Ulrich Dobrindt2, Gábor Nagy3, Kai Michaelis2, Levente Emödy3, Tino Polen4, Reinhard Rachel5, Volker F. Wendisch4 and Gottfried Unden1

1 Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg Universität Mainz, Becherweg 15, 55099 Mainz, Germany
2 Institut für Molekulare Infektionsbiologie der Universität Würzburg, 97070 Würzburg, Germany
3 Institute of Medical Microbiology and Immunology, University of Pécs, 7624 Pécs, Hungary
4 Forschungszentrum Jülich, Institut für Biotechnologie I, 52425 Jülich, Germany
5 Institut für Mikrobiologie, Universität Regensburg, 93040 Regensburg, Germany

Correspondence
Gottfried Unden
unden{at}uni-mainz.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF transition of the fim switch. Enhanced type 1 fimbrial expression as a result of lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also.


Abbreviations: Ap, ampicillin; H-NS, histone-like nucleoid-structuring protein; IHF, integration host factor; IRL, left inverted repeat; Km, kanamycin; Lrp, leucine-responsive regulatory protein; Sm, spectinomycin; UPEC, uropathogenic E. coli

{dagger}Present address: Miltenyi Biotec GmbH, 51429 Bergisch-Gladbach, Germany.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The lrhA gene of Escherichia coli encodes a transcriptional regulator of the LysR family (Bongaerts et al., 1995). Recently, the function of LrhA has been elucidated by a microarray-based comparison of the transcriptional profiles of wild-type E. coli and an isogenic lrhA mutant. LrhA has been identified as a regulator of genes involved in flagellation, motility and chemotaxis (Lehnen et al., 2002). The lrhA mutants showed an increased expression of flagellar, motility and chemotaxis genes (e.g. flhDC, fliA, fliC) and a corresponding increased motility and chemotactic response. LrhA acts as transcriptional repressor of the flhDC genes encoding the FlhD2C2 master regulator, and thus of the flhDC regulon. The flhDC regulon contains nearly 50 genes required for flagellum biosynthesis and function, which are arranged in at least 13 operons and whose expression is under hierarchical control (Kalir et al., 2001; Macnab, 1996). LrhA also controls its own expression, but unlike most LysR-type regulators, LrhA is positively autoregulated (Lehnen et al., 2002). In addition, the LrhA protein also controls the stationary-phase sigma factor {sigma}S (RpoS), which regulates gene expression in response to general stress in enteric bacteria (Gibson & Silhavy, 1999; Mukherjee et al., 2000). The concentration of {sigma}S is regulated through degradation by the ClpXP protease. LrhA represses RpoS stability by affecting the function of ClpXP and the response regulator RssB (or SprE), which serves as a {sigma}S recognition factor.

Differential gene-expression studies have indicated that LrhA also represses structural (fimAICDFG) and regulatory (fimE) genes related to the synthesis of type 1 fimbriae (Lehnen et al., 2002). Type 1 fimbriae of E. coli mediate adherence to mannose-containing receptors, which is an important early step in colonization of host cells (Brinton, 1959; Keith et al., 1986; Mulvey et al., 1998). The fimbriae consist of repeating structural subunits (FimA) and several minor components, including an adhesin (FimH) (Jones et al., 1995). The expression and assembly of type 1 fimbriae require at least eight genes that are localized within one gene cluster. The expression of the fim structural subunit genes (fimAH operon) is controlled by an invertible 314 bp promoter element (‘fim switch’). Inversion of the 314 bp switch element is the basis of phase-variable expression of type 1 fimbriae, which leads to a heterogeneous population of fimbriate (phase-ON) and afimbriate (phase-OFF) cells. The fim switch is promoted by the recombinases FimE and FimB. As a result, the fimA promoter can be in the OFF orientation (no transcription) or in the ON orientation, which allows transcription (Klemm, 1986). FimB facilitates inversion from phase-OFF to phase-ON, as well as inversion from phase-ON to phase-OFF. FimE, on the other hand, causes only switching from phase-ON to phase-OFF (Blomfield et al., 1991; Gally et al., 1996). In addition to the fim recombinases, efficient inversion of the fim switch requires the accessory proteins integration host factor (IHF) and leucine-responsive regulatory protein (Lrp) (Blomfield et al., 1993, 1997; Dorman & Higgins, 1987; Gally et al., 1994). The histone-like nucleoid-structuring protein (H-NS) is also involved in the recombination event (Donato et al., 1997; O'Gara and Dorman, 2000; Olsen & Klemm, 1994).

Fimbriae-mediated adherence is important for the virulence of uropathogenic E. coli (UPEC), and the expression of type 1 fimbriae is linked to urinary tract colonization and pathogenesis (Connell et al., 1996; Martinez et al., 2000; Mulvey, 2002). Type 1 fimbriae have also been described as key factors in biofilm establishment on abiotic surfaces (Pratt & Kolter, 1998; Schembri & Klemm, 2001). Under static growth conditions, type 1 fimbriae provide stable interactions between bacteria and different surfaces, including polystyrene, PVC, polycarbonate and borosilicate glass – a prerequisite for biofilm formation. The non-specific attachment to abiotic surfaces and the specific binding to receptor molecules are mannose-inhibitable and are mediated by the fimbrial adhesin FimH. Thus, in addition to other specific outer-membrane proteins, type 1 fimbriae promote the transition from a free-living, planktonic existence to a complex sessile lifestyle.

The aim of this study was to examine the role of LrhA in the regulation of type 1 fimbriae expression at the molecular level and with regard to adherence and biofilm formation.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and media.
The bacterial strains, plasmids and phage used are shown in Table 1. Unless otherwise stated, plasmids were maintained in E. coli JM105 (Yanisch-Perron et al., 1985). For genetic experiments, the bacteria were routinely grown in Luria–Bertani (LB) broth and on LB agar. For expression studies, M9 medium (Miller, 1992) supplemented with acid-hydrolysed Casamino acids (0·1 %), tryptophan (0·005 %) and 20 mM glycerol was used. Cultures were incubated aerobically at 37 °C in flasks with shaking at 180 r.p.m. When required, antibiotics were added as follows: 100 µg ml–1 ampicillin (Ap), 50 µg ml–1 kanamycin (Km), 50 µg ml–1 spectinomycin (Sm), and 20 µg ml–1 tetracycline. Cell densities were measured as the OD at 578 nm.


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Table 1. E. coli strains, plasmids and phage used in this study

 
RNA preparation.
RNA was extracted from bacteria grown aerobically at 37 °C in the modified M9 mineral medium. For DNA array analysis, total RNA was extracted as described by Lehnen et al. (2002) and Wendisch et al. (2001) from cells harvested at OD578 0·3–0·45. For real-time RT-PCR, total RNA was extracted from cultures grown to an OD578 ~0·5 using RNeasyProtect Bacteria Mini kits (Qiagen) as recommended by the manufacturer. Traces of genomic DNA were removed by DNase treatment with DNA-free reagent (Ambion).

DNA microarray and macroarray (‘E. coli Pathoarray’) analysis.
Microarray preparation, probe labelling, hybridization and analysis were carried out as described previously (Lehnen et al., 2002; Wendisch et al., 2001). The DNA macroarray covered 456 pathogenicity island- or virulence-associated genes of UPEC strain 536 and other extraintestinal and intestinal pathogenic E. coli (including Shigella) and has been described previously (Dobrindt et al., 2002, 2003). In order to compare the gene expression of wild-type strain 536 and its isogenic lrhA mutant, ‘E. coli Pathoarrays' were hybridized in five different experiments using independently labelled cDNA probes. Reverse transcription of RNA, radioactive probe labelling and hybridization were performed as described by Dobrindt et al. (2003). The scanned arrays were analysed with ArrayVision software (Imaging Research, St. Catharines, Canada) followed by visual inspection. For identification of genes with statistically significant changes in expression, significance analysis of microarrays (SAM), a statistical technique for finding significant genes in a set of array experiments, was used (Tusher et al., 2001) (http://www-stat.stanford.edu/~tibs/SAM/).

Determination of fim transcript levels by quantitative, real-time PCR following reverse transcription.
Before reverse transcription reactions were performed, the absence of DNA from RNA samples was verified by PCR amplification (40 cycles) of the fimE, fimA, fimI and gapA genes with 0·2 µg RNA as template. cDNA synthesis was performed with 320–550 ng total RNA, isolated from three independent bacterial cultures, and random hexamer primers, using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions.

The resulting cDNA samples with concentrations varying between 1·7 and 2·5 µg µl–1 were diluted 100- to 1000-fold prior to real-time PCR. PCR reactions were done using the iQ SYBR Green Supermix (Bio-Rad) and the following specific primers (Table 2): FimE_for and FimE_rev for amplification of fimE, FimA_for and FimA_rev for amplification of fimA, and FimI_for and FimI_rev for amplification of fimI. As an LrhA-independent internal control, gapA (encoding D-glyceraldehyde-3-phosphate dehydrogenase A) was amplified using primers GapA_for and GapA_rev. The reactions containing 1x iQ SYBR Green Supermix, 100 nM of each primer and 5 µl of the template cDNAs were run on an iCycler iQ system (Bio-Rad) with the following cycling parameters: initial denaturation at 95 °C for 3 min and 45 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s. Each PCR was performed in duplicate. Specificity for all amplicons was confirmed via melting curves and gel analysis.


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Table 2. Primers used in this study

 
The threshold cycle for each real-time PCR was determined by using the software package supplied with the iCycler system, and fimA, fimE and fimI expression was normalized with gapA expression for each sample.

DNA manipulation and cloning procedures.
DNA cloning and preparation of plasmid and chromosomal DNA were performed according to standard methods (Sambrook et al., 1989). The fimE'–'lacZ fusion plasmid (pMW193) was constructed by PCR amplification of the promoter region of fimE (–463 to +79) from genomic DNA of strain MG1655 with primers (Table 2) fimEHindIII and fimEBamHI. The resulting 542 bp fragment comprising the complete intergenic region was cloned into the HindIII and BamHI sites of the protein fusion vector pJL28. The fimB'–'lacZ (–1119 to +68; plasmid pMW251), fimA'–'lacZ (–532 to +22; plasmid pMW194) and fimA2'–'lacZ (–400 to +22; plasmid pMW282) fusions were constructed in the same way with the following primers (Table 2): fimBEcoRI and fimBHindIII, fimABamHI and fimAHindIII, and fimA2BamHI and fimAHindIII, respectively. After sequence verification, translational lacZ fusions were transferred to the genome of E. coli (wild-type and lrhA mutant) with phage {lambda}RZ5 (Ostrow et al., 1986), and monolysogens were identified and used for further work (Bongaerts et al., 1995). Fusion fimA2'–'lacZ on plasmid pMW282 was introduced into receptor strains by chemical transformation. The construction of plasmid pMW132 used for the overexpression and isolation of His6–LrhA has been described previously (Lehnen et al., 2002).

Inactivation of the lrhA gene in E. coli strain 536 by allelic exchange.
The lrhA gene was inactivated by insertion of an {Omega}-Km cassette in the unique StuI site and cloned into pCVD442, a suicide vector that confers sensitivity to sucrose in Gram-negative bacteria (Donnenberg & Kaper, 1991). First, the lrhA gene was amplified with flanking up- and downstream regions by PCR using primers lrhABglII and lrhA3 (Table 2), and cloned in pBluescriptKS cut with BamHI and EcoRI. After insertion of the {Omega}-Km cassette originating from plasmid pGS607, a hybrid plasmid with pCVD442 was constructed by SalI restriction of both plasmids and subsequent ligation. By deletion of the SacI fragment, bla and the origin of pBluescriptKS were eliminated. The resulting plasmid (pMW192) allowed allelic exchange with the chromosomal lrhA gene of E. coli 536, resulting in strain IMW330. Vector pCVD442 and derivatives thereof, which carry a pir-dependent origin of replication, were propagated in the E. coli strain SY327{lambda}pir. Allelic exchange was performed as previously outlined (Nagy et al., 2002). Briefly, plasmid pMW192 was mobilized from the donor strain E. coli SM10{lambda}pir into the recipient strain E. coli 536 (SmR). Isolates that were sucrose-resistant and Ap-sensitive, which is indicative of the loss of vector sequence, as well as Km- and Sm-resistant, were selected for further studies. The allelic exchange was confirmed by PCR amplification of the chromosomal region.

Gel retardation.
For gel-retardation assays, DNA fragments containing fimE (570 bp), fimB (1218 bp) and fimA (554 bp) promoter regions were used. They were obtained by PCR amplification of chromosomal DNA from a colony of MG1655 with primers fimEHindIII and fimEBamHI, fimBEcoRI and fimBHindIII, and fimABamHI and fimAHindIII, respectively. After digestion with the appropriate restriction enzymes, the promoter fragments were purified and labelled with [{alpha}-33P]dATP on both strands, as previously described (Lehnen et al., 2002).

The gel-retardation assays were performed essentially as described by Drapal & Sawers (1995). The His6–LrhA proteins were purified as described earlier (Lehnen et al., 2002) and incubated with labelled DNA (5 nM) in binding buffer (10 mM Tris/HCl, pH 7·5, 10 %, w/v, glycerol, 2·5 mM EDTA, 50 mM KCl, 0·1 mM DTT, 4 mM spermidine, 12·5 µg sonicated calf thymus DNA, 1 µg BSA, in a final volume of 20 µl) for 30 min at room temperature. After incubation, the reaction mix was applied to a non-denaturing polyacrylamide gel (5 %) buffered with Tris/borate/EDTA (Sambrook et al., 1989). The concentration of LrhA required for half-maximal retardation was designated apparent KD.

{beta}-Galactosidase assay.
The strains were grown at 37 °C in supplemented M9 medium with glycerol (20 mM). From exponentially growing bacteria (OD578 0·5–0·6), {beta}-galactosidase specific activities were measured and calculated (Miller, 1992).

Agglutination of yeast cells, haemagglutination and haemolysis.
The capacity of bacterial strains to express a D-mannose-binding phenotype was measured by the ability to agglutinate Saccharomyces cerevisiae cells on glass slides. Aliquots of washed bacterial suspensions of various optical densities were incubated with a yeast suspension (10 mg ml–1 dry weight). Agglutination, which is susceptible to inhibition by D-mannose (2 %), was monitored visually by aggregation and precipitation of the cells. The lowest OD578 of bacterial suspensions at which agglutination was observed was determined.

The presence of P and S fimbriae was demonstrated by haemagglutination of defibrinated human and bovine erythrocytes, respectively. A suspension of human and bovine blood (Elocin-lab, Muelheim) was mixed on a glass slide with a toothpicked colony of E. coli. After incubation for some minutes on ice, agglutination occurred. Expression of S or P fimbriae was also checked by Western blot analysis using polyclonal sera raised against S or P fimbriae of E. coli strain 536, as described by Nagy et al. (2002).

To test their haemolytic activity, cells from E. coli colonies were spread onto sheep blood agar plates (Oxoid) with a toothpick and incubated overnight at 37 °C. Lysis of the blood cells by {alpha}-haemolysin was detected by formation of clear haloes around the colonies after incubation. Haemolytic activities of culture supernatants were determined quantitatively using a method previously described (Nagy et al., 2002).

Ascending urinary tract infection model.
Intravesical infections of 3–4 day old CFLP mice (Gödöllö, Hungary) were performed as previously described (Allison et al., 1994). Six to 26 infant mice were infected with each of the strains in two separate experiments. UPEC strain 536 and the lrhA mutant IMW330 were administered intravesically using an inoculum of 2·5x104 or 2·5x105 cells. The mortality rate was determined after 3 days of infection.

Biofilm formation.
Overnight cultures were diluted 1 : 50 into LB broth and 200 µl aliquots were transferred to the wells of a 96-well polystyrene microtitre plate (Nunc). For growth in the presence of 1 % methyl-{alpha}-D-mannopyranoside ({alpha}MM), 20 µl of a 10 % {alpha}MM solution was added to 180 µl LB broth inoculated with an overnight culture already grown in the presence of 1 % {alpha}MM. After growth of the samples for 24 h at 30 °C, biofilm formation was measured. Medium and unbound cells were discarded, the wells were rinsed with water, and adhered cells were stained by incubation with 200 µl 1 % crystal violet (Merck) for 15 min. Non-bound dye was removed by thorough rinsing. Biofilm formation was subsequently quantified by adding 200 µl ethanol (99 %) twice to the stained microtitre wells. The ethanol was brought up to a volume of 1 ml (with water) and the absorbance of crystal violet was measured at 540 nm (O'Toole & Kolter, 1998).

Electron microscopy of flagella.
Cells were washed with 50 mM potassium phosphate (pH 7·0) and fixed with glutaraldehyde (2 %, v/v, final concentration). The cells were allowed to sediment to carbon-coated grids for 1–2 min. After negative staining with 1 % uranyl acetate, the grids were examined with a Philips CM12 transmission electron microscope (Scharf et al., 2001).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transcriptional regulation of fim genes by LrhA
Preliminary experiments suggested an effect of LrhA on the mRNA levels of E. coli fimbrial genes. For a more detailed analysis, the expression profile of wild-type E. coli MG1655 genes was compared to that of the isogenic lrhA mutant IMW325 by DNA microarray analysis after growth in mineral medium with glycerol. In addition to the genes involved in flagellation, motility and chemotaxis (Lehnen et al., 2002), genes encoding type 1 fimbriae showed increased relative mRNA abundances in the lrhA mutant in three different experiments (Table 3). The identified genes belong to the fimAICDFGH operon, and the mRNA contents were increased by factors between 1·6 and 7·3. The microarray experiments suggested that LrhA is a negative transcriptional regulator of type 1 fimbrial genes. Expression of the fimAICDFGH operon is regulated by the fim switch, which is accomplished by the recombinases FimE and FimB. The relative mRNA level of fimE, encoding FimE (ON-to-OFF switch), was elevated in the lrhA mutant by a factor of 6·9, but only two interpretable microarray experiments were obtained. For FimB, no information on the mRNA levels was obtained due to interference from high background fluorescence levels.


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Table 3. Fimbrial genes with increased mRNA abundances in the lrhA mutant

mRNA from E. coli wild-type (MG1655) and the lrhA derivative IMW325 was used for DNA microarray analysis (Lehnen et al., 2002). The relative mRNA levels are the mean from three different experiments and genes with relative mRNA levels increased by factors >=1·6 are shown. All genes are members of the same operon and are transcribed in the order shown. An asterisk indicates P values of <0·05. The P values were calculated based on a t test distribution (Lehnen et al., 2002). The function of the gene products is given according to http://coli.berkeley.edu/cgi-bin/ecoli/coli_entry.pl.

 
To determine the role of the resolvases FimB and FimE in the LrhA control of type 1 fimbrial gene expression, translational lacZ fusions of the fimA, fimB and fimE genes were constructed and inserted in single copies into the chromosome of wild-type and lrhA mutant strains. The {beta}-galactosidase activities of the fimA'–'lacZ fusion showed a large variation in different experiments, both in the wild-type and the lrhA background, presumably due to effects of promoter inversion (Table 4). Multiple repeated experiments suggested, however, that fimA expression increases by a factor of about two in the lrhA-negative background. The fimA2'–'lacZ fusion lacks the left inverted repeat (IRL) essential for inversion of the fimA promoter region. Therefore, the switch is locked in the phase-ON orientation and any expression changes should reflect only transcription initiation levels at the fimA promoter. In contrast to the ‘unlocked’ fimA'–'lacZ fusion, wild-type and lrhA mutant showed no difference in fimA2'–'lacZ expression. Similarly fimB'–'lacZ expression was not affected by LrhA, whereas the expression of fimE'–'lacZ was decreased about 2·5-fold in the lrhA mutant compared to the wild-type (Table 4). Plasmid-borne lrhA expression complemented the lrhA mutant for fimE'–'lacZ expression, which then reached the same level as that of the wild-type. Expression of lrhA from the plasmid had no significant effect on fimB'–'lacZ expression. These results imply that LrhA has no direct effect on fimA promoter activity, but enhances fimE expression, thereby promoting the ON-to-OFF switch at fimA.


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Table 4. Expression of translational lacZ fusions of type 1 fimbrial genes in wild-type and lrhA mutant strains

ND, Not determined.

 
The positive effect of LrhA on fimE'–'lacZ expression is in conflict with the data obtained from the DNA microarray analysis, which show an increased fimE expression in the absence of LrhA (repression factor of 6·9 in two DNA-chip experiments compared to a factor of 0·40 in the lacZ-expression studies). To resolve this contradiction, the mRNA levels in wild-type and the lrhA mutant were determined by quantitative RT-PCR with RNA isolated from three independent cultures. The mRNA levels of fimE decreased by a factor of 2·9 in the lrhA mutant (Table 5), confirming the results obtained for the fimE'–'lacZ reporter gene fusion rather than the results of the DNA microarray. The mRNA levels of fimA (1·7-fold) and fimI (1·9-fold), on the other hand, increased slightly in the lrhA mutant. The quantitative RT-PCR experiment of fimA (Table 5) was therefore in good agreement with the DNA microarray analysis and the lacZ reporter-gene studies.


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Table 5. Quantification of fimA, fimE and fimI gene expression in E. coli wild-type (MG1655) and lrhA mutant (IMW325) by quantitative RT-PCR

RNA was prepared from three independent cultures grown in M9 medium with glycerol to OD578 0·5. Using primers specific for each of the indicated genes, RNA levels were quantified by RT-PCR relative to the level of the housekeeping gene gapA. The level of the genes in wild-type background was set at 1.

 
The regulation of the flagella, motility and chemotaxis by LrhA is effected via the regulator FlhD2C2, which is under the transcriptional control of LrhA (Lehnen et al., 2002). The requirement of FlhD2C2 for the LrhA-mediated transcriptional regulation of fim genes was tested in a flhDC-negative background. In a similar manner to the flhDC-positive background, inactivation of lrhA in mutant IMW462 resulted in a threefold decrease of fimE'–'lacZ expression (545±17 Miller units) compared to the parental strain IMW461 (170±4 Miller units). Therefore, LrhA-steered control of fim gene expression is independent of FlhD2C2.

Binding of LrhA to the promoters of type 1 fimbrial genes
To demonstrate binding of LrhA to target promoters, gel-retardation experiments were performed. Promoter fragments of fimA, fimE and fimB were radioactively labelled and incubated with purified His6–LrhA protein. When the samples were subjected to native gel electrophoresis, the band of free fimA promoter DNA disappeared at concentrations of 50–100 nM LrhA, and a DNA band with decreased electrophoretic mobility appeared. This band presumably represents a DNA–LrhA complex and was formed with an apparent KD of about 100 nM LrhA (Fig. 1A).



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Fig. 1. Gel-retardation assay with a 554 bp fimA (A) and a 570 bp fimE (B) promoter DNA fragment and purified LrhA protein. The radioactively labelled fragment (5 nM in each lane) was incubated with various concentrations of LrhA in the presence of a 625-fold excess of competitor DNA and subjected to non-denaturing DNA PAGE (5 % acrylamide). Arrows indicate bands of free DNA and of a retarded LrhA–DNA complex.

 
Similarly, gel retardation of fimE (Fig. 1B) and fimB promoter DNA by the addition of LrhA was observed. The concentrations of LrhA required for half-maximal retardation were 20–50 and 200–300 nM for the fimE and fimB promoter fragments, respectively. As for fimA, a large (625-fold) excess of non-specific DNA did not inhibit the formation of LrhA–promoter complexes, suggesting specific interactions between LrhA and the tested promoters. Thus, LrhA seems to bind to the promoter regions of all three genes, with the strongest binding affinity for the fimE promoter, but only fimE expression is directly influenced by LrhA.

Effect of lrhA mutation on agglutination of yeast, and biofilm formation
To verify an altered expression of the fimbrial genes in the lrhA mutant, various fimbriae-associated phenotypes and functions were tested in E. coli wild-type and lrhA mutants. Type 1 fimbriae of E. coli mediate adherence to mannose-containing receptors, allowing colonization of many host surfaces. The classical way to monitor type 1 fimbriae-mediated adhesion is agglutination of yeast (S. cerevisiae) cells. Agglutination was tested with the E. coli K-12 strains and the UPEC strain 536, since adhesion is of particular significance for pathogenic E. coli strains. The capacity of the wild-type strains and the corresponding lrhA mutants to agglutinate S. cerevisiae cells was tested on glass slides. Upon mixing, the agglutination reaction produced by the lrhA derivatives was significantly stronger and occurred at lower cell densities (OD578 0·25) than with the wild-type (OD578 1) (Table 6). Complementation of the lrhA mutant IMW325 with lrhA carried by plasmid pMW213 conferred a weak agglutination phenotype similar to that of the wild-type strains. As expected, the fim mutant strain AAEC185, lacking the fimbrial structural genes, caused no agglutination. Furthermore, the addition of mannose abolished agglutination completely. Thus, agglutination was specific for mannose-containing surface proteins and depended on the type 1 fimbriae, and LrhA repressed the agglutination phenotype.


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Table 6. Effect of lrhA mutation on E. coli agglutination of yeast cells compared to wild-type and fim-negative E. coli strains with and without mannose

Cell suspensions of different optical densities were incubated with yeast cells and agglutination was detected. NA, no agglutination.

 
Flagella, and type 1 and curli fimbriae, are the bacterial structures involved in the non-specific adhesion of E. coli to organic or inorganic surfaces, which represents the first step in biofilm formation (Pratt & Kolter, 1998; Vidal et al., 1998). Attachment and adherence by fimbriae and flagella therefore represent important factors in biofilm formation. Since LrhA represses the synthesis of flagella (Lehnen et al., 2002) and type 1 fimbriae, the effect of LrhA on biofilm formation under static growth conditions was examined. To this end, E. coli was grown in polystyrene microtitre plates for 24 h, and attached bacteria were visualized and subsequently quantified by staining with crystal violet (Fig. 2). The lrhA mutant IMW325 displayed a significantly increased biofilm formation compared to the wild-type strain MG1655, which adhered to the well surface but to a lesser extent (Fig. 2A). When the lrhA mutant was complemented with a plasmid-borne lrhA gene, IMW325 (pMW213), biofilm formation was no longer visible, presumably due to an increased lrhA copy number. No biofilm development was observed for the type 1 fimbriae-negative mutant strain AAEC185, demonstrating the requirement of type I fimbriation to build biofilms. Quantitative analysis of biofilm formation by measuring the dye adsorbed to the attached bacteria (O'Toole & Kolter, 1998) confirmed these phenotypes (Fig. 2B).



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Fig. 2. Effects of lrhA on biofilm formation under static conditions. Cultures of MG1655, its isogenic lrhA mutant IMW325, the complemented strain IMW325 (pMW213) and the fim-negative strain AAEC185 were grown in polystyrene microtitre plates for 24 h. (A) Representative samples stained by crystal violet; (B) quantification of biofilm-associated dye, as described in Methods. In addition (C), the flagella-negative MC4100 (flhDC) and IMW330 (flhDC lrhA) strains were analysed for biofilm-associated dye. (D) Quantification of biofilm formation in the presence of 1 % methyl-{alpha}-D-mannopyranoside ({alpha}MM). Experiments were done in triplicate. Error bars represent standard deviation.

 
Next we tried to dissect the contribution of flagella and type 1 fimbriae to biofilm formation and the role therein of LrhA. E. coli MC4100, an flhDC deletion strain which lacks flagella but not fimbriae synthesis, showed largely decreased biofilm formation (Fig. 2C). In the flhDC lrhA double mutant strain IMW41, biofilm formation was impaired to a similarly low degree. The ability of type 1 fimbriae to bind both specifically to mannose and non-specifically to abiotic surfaces can be abolished by addition of methyl-{alpha}-D-mannopyranoside ({alpha}MM), a non-metabolizable analogue of mannose (Pratt & Kolter, 1998). This feature was exploited to further analyse the role of LrhA and type 1 fimbriae on biofilm formation. In the presence of {alpha}MM, biofilm formation was almost completely eliminated for both the wild-type and the lrhA mutant to similar levels to those of the fim deletion strain, which was not influenced by the addition of {alpha}MM (Fig. 2D). These results confirmed that under static growth conditions, both type 1 fimbriae and flagella are important for biofilm formation, and even suggested a slightly more prominent role for type 1 fimbriae. Since both flagella and type 1 fimbriae are essential for the development of biofilms, the clear effect of LrhA on biofilm formation has to be the result of the repression of both flagellar as well as type 1 fimbrial genes by LrhA.

Flagella in wild-type E. coli and lrhA mutants (electron microscopy)
The influence of lrhA on the number of flagella per cell was studied by electron microscopy of E. coli wild-type MG1655 and lrhA mutant IMW325. On average, the lrhA mutant showed an about threefold increased number of flagella compared to the wild-type (Fig. 3), including the number of attached and broken flagella. It was not possible to quantify the difference in length of the flagella in electron micrographs due to frequent twisting and breakage of the flagella. Even so, these results indicate that the increased motility of an lrhA mutant (Lehnen et al., 2002) can be attributed to an increased number of flagella per cell.



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Fig. 3. Electron micrographs of wild-type E. coli MG1655 (A) and lrhA mutant strain IMW325 (B). The bacteria were grown overnight in LB broth, harvested by centrifugation, fixed and negatively stained with uranyl acetate.

 
Significance of LrhA for virulence of UPEC
Type 1 fimbriae, together with other virulence factors, including several fimbrial adhesins, toxins and iron-uptake systems, contribute to the ability of UPEC to colonize the urinary tract. To test whether additional adhesins and other virulence factors specific for UPEC strains are also under the control of the LrhA protein, we used the ‘E. coli pathoarray’, which carries 456 DNA probes for typical virulence-associated genes of extra-intestinal and intestinal pathogenic E. coli, and Shigella (Dobrindt et al., 2003). The mRNA profile of UPEC strain 536 (O6 : K15 : H31), which expresses virulence factors such as S- and P-related fimbriae, {alpha}-haemolysin, yersiniabactin and capsule (Dobrindt et al., 2003), was compared with that of its isogenic lrhA mutant IMW330. The genes encoding type 1-, S- and P-fimbrial determinants (fim, sfa and prf genes), {alpha}-haemolysin and ORF32 showed moderately increased mRNA levels (about twofold) in the lrhA mutant relative to the wild-type. Other virulence-related genes showed no significant alteration in transcript levels. Physiological tests for S and P fimbriae that measure their ability to bind to sialic-acid-specific receptors on bovine erythrocytes or Gal{alpha}[1-4]Gal{beta} disaccharides on human erythrocytes showed no difference between the strains. Similarly, Western blot analysis with specific antibodies revealed no significant changes in the amount of S and P fimbriae (data not shown). The same applied to haemolytic activities and the overall virulence in an ascending urinary tract infection model in suckling mice, which gave no significant difference for wild-type and the lrhA mutant. It was concluded that type 1 fimbriae, but not other virulence factors, are transcriptionally regulated by LrhA.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
It has previously been established that LrhA is a key repressor of genes associated with motility, chemotaxis and flagella synthesis in E. coli (Lehnen et al., 2002). The present investigation demonstrates that LrhA serves also as a repressor of type 1 fimbriae expression. Due to its effects on flagella and fimbriae synthesis, LrhA is also a regulator of biofilm formation.

Control of fimbrial expression
Gene-expression profiling experiments revealed an upregulation of the genes belonging to the fimAICDFGH operon in the lrhA mutant. The expression of the structural genes for type 1 fimbriae is regulated by the site-specific inversion of the fimA promoter regionby the two recombinases FimE and FimB. Although LrhA binding to the promoter regions of fimA, fimB and fimE was demonstrated, only fimE expression seemed to be directly influenced by LrhA. The expression and mRNA levels of fimE were decreased in the lrhA mutant compared to the wild-type, whereas mRNA abundances of the structural genes for type 1 fimbriae were elevated in the lrhA mutant. These results led us to propose a model (Fig. 4) in which LrhA positively controls the expression of fimE, as demonstrated by lacZ fusion and quantitative RT-PCR. Since FimE exhibits a strong bias for phase switching from the ON to the OFF orientation, expression of the fim operon is reduced in the wild-type. In the absence of LrhA, however, fimE expression is decreased, leading to an elevated fimbrial expression in the lrhA mutant. This model is further supported by the fact that LrhA does not affect fimA promoter activity (in the locked-ON situation), even though LrhA binds to the fimA promoter. The binding of LrhA to the fimE promoter was specific, with an estimated apparent KD value between 20 and 50 nM, whereas a 10-fold-higher LrhA concentration (200–300 nM) was required for half-maximal retardation of the fimB promoter. Other promoters proven to be targeted by LrhA, such as the flhDC and lrhA promoters, were bound by LrhA with a similar KD to that of the fimE promoter (Lehnen et al., 2002). These observations suggest that the binding of the fimE promoter by LrhA is more relevant for the regulation of fimbrial expression, and that the effect on fimB might be too small to be detected, if indeed it is present at all. Whether LrhA directly activates fimE expression, which is in contrast to the function of LrhA as a repressor for other target genes, or whether it alleviates the action of an additional repressor, remains to be elucidated.



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Fig. 4. Proposed model of regulation of type 1 fimbriae synthesis by LrhA. Activation of fimE expression by LrhA leads to repression of expression of the fimA operon.

 
The binding affinity of LrhA for the fimA promoter lies in-between those for the fimE and fimB promoters. Although our model predicts that LrhA controls expression of the fim genes primarily by controlling fimE expression, we cannot exclude that LrhA influences the fim switch by other mechanisms also; in other words, LrhA binding to the fimA promoter might influence site-specific inversion in a similar manner to Lrp, IHF and H-NS (Blomfield et al., 1993, 1997; O'Gara & Dorman, 2000). Lrp, for instance, slightly affects the transcription of both fimB and fimE. Additionally, it also binds with high affinity within the fim switch, and this binding stimulates recombination in both directions (Gally et al., 1994). In the case of H-NS, it has been shown that H-NS is a repressor of the FimB recombinase, but there is evidence that this regulation is not the sole cause of the increased DNA flipping observed in hns mutant backgrounds (Donato et al., 1997). By binding to the fim switch, LrhA might also compete with Lrp and/or IHF for DNA binding and disturb the structure needed for the recombination event. Alternatively, LrhA could steer the phase switching by directly influencing FimE or FimB activity. Such a mechanism has been shown for PapB, a transcriptional regulator of pyelonephritis-associated pili (pap) that has a negative effect on the expression of type 1 fimbriae. In vitro recombination assays have demonstrated that PapB directly inhibits FimB-promoted switching. Moreover, PapB promotes fimE expression to a similar degree to LrhA (Xia et al., 2000).

The expression of fimE is also controlled by the orientation of the fim switch, which operates only when fimE is in cis to the invertible element (Sohanpal et al., 2001). The expression of fimElacZ fusions is not subject to this negative control that is exhibited by the fim switch in the OFF orientation, whereas quantitative RT-PCR of fimE transcripts mirrors also the orientational control. Nevertheless, both approaches revealed a similar reduction of fimE expression in the lrhA mutant. These independent results are more reliable than the fimE expression data obtained by microarray analysis, which implied a regulation in the opposite direction. Transcript profiling with whole-genome microarrays is well suited to monitor the global changes associated with an altered genotype. However, alternative methods should be used to confirm single results.

Future experiments will have to determine whether the repression of type 1 fimbriae synthesis by LrhA can be solely attributed to the LrhA control of fimE expression or whether additional mechanisms influencing the phase variation play a role.

Biofilm formation
Biofilm development was strongly affected by LrhA, with increased biofilm formation in the lrhA mutant and loss of biofilm formation when LrhA was overexpressed. In E. coli, type 1 fimbriae as well as flagellar-mediated motility contribute to the initial stages of biofilm development on abiotic surfaces (Pratt & Kolter, 1998). Therefore, the effects of LrhA on biofilm formation are largely due to changed expression of flagella and type 1 fimbriae. Under the conditions tested, inhibition of fimbrial adhesion by methyl-{alpha}-D-mannopyranoside reduced biofilm formation even more drastically than lack of flagella, demonstrating the crucial role in biofilm formation of type 1 fimbriae and of their regulation by LrhA.

Recently, the RNA-binding global regulatory protein CsrA (carbon storage regulator) has been reported to be a repressor of biofilm formation (Jackson et al., 2002). In contrast to LrhA, CsrA apparently affects biofilm formation essentially through the regulation of glycogen metabolism, and not through alteration of surface-molecule expression. As expected, the CsrA-antagonizing regulatory RNA CsrB stimulated biofilm formation (Jackson et al., 2002). Interestingly, HexA, a close relative of LrhA (>60 % identity) from the plant pathogen Erwinia carotovora, negatively regulates the expression of rsmB, a homologue of E. coli csrB (Mukherjee et al., 2000). One could therefore speculate that LrhA might repress biofilm formation both through the control of flagella and fimbriae synthesis as well as indirectly through carbon balance. Expression of csrB and csrA, however, was not affected by LrhA (unpublished results).

Virulence assessment of an lrhA-defective UPEC strain
Urinary tract infection is the major bacterial infectious disease in industrialized countries. Type 1, S and P fimbriae are typical virulence factors of UPEC. Fimbriae are involved in specific adherence to epithelia of the urogenital tract and promote both bacterial attachment to and/or invasion of host tissues within the urinary tract (Connell et al., 1996; Keith et al., 1986; Martinez et al., 2000; Mulvey et al., 1998). In vivo expression of type 1 fimbriae contributes to virulence in the urinary tract and fim expression is critical for infection of the lower urinary tract (cystitis) (Bahrani-Mougeot et al., 2002; Gunther et al., 2001).

Inactivation of lrhA results in an increased expression of genes encoding flagella and type 1 fimbriae both in the E. coli K-12 laboratory strain MG1655 and the UPEC strain 536. LrhA is closely related to HexA and PecT from plant-pathogenic Erwinia species, which function as repressors both of motility and of multiple virulence factors, such as lytic exoenzymes (Harris et al., 1998; Surgey et al., 1996). However, neither haemolytic activity nor other established virulence factors of the UPEC strain 536, such as S or P fimbriae, appear to be significantly affected by LrhA.

Thus, LrhA has a particular effect on type 1 fimbriae synthesis and hence on biofilm formation, but only a limited effect on the virulence of the UPEC strain as established in a murine model of ascending urinary tract infection. Other forms or models of virulence, in which type 1 fimbriae play a more important role, could depend more directly on LrhA.


   ACKNOWLEDGEMENTS
 
We thank Dr R. E. Streeck (Mainz) for allowing the use of an S2 laboratory. The work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie to G. U., SFB 479 (Würzburg), OTKA grants T037833 and ETT086/2001 to L. E., and from the Swiss National Science Foundation (project 83EU-059835) to C. B.


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Received 7 April 2005; revised 20 June 2005; accepted 29 June 2005.



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