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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Miltenyi Biotec GmbH, 51429 Bergisch-Gladbach, Germany.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 320550 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 µl1 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.
|
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
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 His6LrhA 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 -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
-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
pir. Allelic exchange was performed as previously outlined (Nagy et al., 2002
). Briefly, plasmid pMW192 was mobilized from the donor strain E. coli SM10
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 [-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 His6LrhA 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.
-Galactosidase assay.
The strains were grown at 37 °C in supplemented M9 medium with glycerol (20 mM). From exponentially growing bacteria (OD578 0·50·6), -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 ml1 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 -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 34 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--D-mannopyranoside (
MM), 20 µl of a 10 %
MM solution was added to 180 µl LB broth inoculated with an overnight culture already grown in the presence of 1 %
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 12 min. After negative staining with 1 % uranyl acetate, the grids were examined with a Philips CM12 transmission electron microscope (Scharf et al., 2001).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
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 His6LrhA protein. When the samples were subjected to native gel electrophoresis, the band of free fimA promoter DNA disappeared at concentrations of 50100 nM LrhA, and a DNA band with decreased electrophoretic mobility appeared. This band presumably represents a DNALrhA complex and was formed with an apparent KD of about 100 nM LrhA (Fig. 1A).
|
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.
|
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (200300 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.
|
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-
-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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bahrani-Mougeot, F. K., Buckles, E. L., Lockatell, C. V., Hebel, J. R., Johnson, D. E., Tang, C. M. & Donnenberg, M. S. (2002). Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract. Mol Microbiol 45, 10791093.[CrossRef][Medline]
Berger, H., Hacker, J., Juarez, A., Hughes, C. & Goebel, W. (1982). Cloning of chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli. J Bacteriol 152, 12411247.[Medline]
Blomfield, I. C., McClain, M. S., Princ, J. A., Calie, P. J. & Eisenstein, B. I. (1991). Type 1 fimbriation and fimE mutants of Escherichia coli K-12. J Bacteriol 173, 52985307.[Medline]
Blomfield, I. C., Calie, P. J., Eberhardt, K. J., McClain, M. S. & Eisenstein, B. I. (1993). Lrp stimulates phase variation of type 1 fimbriation in Escherichia coli K-12. J Bacteriol 175, 2736.[Abstract]
Blomfield, I. C., Kulasekara, D. H. & Eisenstein, B. I. (1997). Integration host factor stimulates both FimB- and FimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli. Mol Microbiol 23, 705717.[CrossRef][Medline]
Bongaerts, J., Zoske, S., Weidner, U. & Unden, G. (1995). Transcriptional regulation of the proton translocating NADH dehydrogenase genes (nuoA-N) of Escherichia coli by electron acceptors, electron donors and gene regulators. Mol Microbiol 16, 521534.[CrossRef][Medline]
Brinton, C. C. (1959). Non flagellar appendages of bacteria. Nature 183, 782786.[Medline]
Connell, I., Agace, W., Klemm, P., Schembri, M., Marild, S. & Svanborg, C. (1996). Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract. Proc Natl Acad Sci U S A 93, 98279832.
Dobrindt, U., Blum-Oehler, G., Nagy, G., Schneider, G., Johann, A., Gottschalk, G. & Hacker, J. (2002). Genetic structure and distribution of four pathogenicity islands (PAI I536 to PAI IV536) of uropathogenic Escherichia coli strain 536. Infect Immun 70, 63656372.
Dobrindt, U., Agerer, F., Michaelis, K. & 7 other authors (2003). Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays. J Bacteriol 185, 18311840.
Donato, G. M., Lelivelt, M. J. & Kawula, T. H. (1997). Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS. J Bacteriol 179, 66186625.
Donnenberg, M. S. & Kaper, J. B. (1991). Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59, 43104317.[Medline]
Dorman, C. J. & Higgins, C. F. (1987). Fimbrial phase variation in Escherichia coli: dependence of integration host factor and homologies with other site-specific recombinases. J Bacteriol 169, 38403843.[Medline]
Drapal, N. & Sawers, G. (1995). Promoter 7 of the Escherichia coli pfl operon is a major determinant in the anaerobic regulation of expression by ArcA. J Bacteriol 177, 53385341.
Gally, D. L., Rucker, T. J. & Blomfield, I. C. (1994). The leucine-responsive regulatory protein binds to the fim switch to control phase variation of type 1 fimbrial expression in Escherichia coli K-12. J Bacteriol 176, 56655672.[Abstract]
Gally, D. L., Leathart, J. & Blomfield, I. C. (1996). Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12. Mol Microbiol 21, 725738.[CrossRef][Medline]
Gibson, K. E. & Silhavy, T. J. (1999). The LysR homolog LrhA promotes RpoS degradation by modulating activity of the response regulator sprE. J Bacteriol 181, 563571.
Gunther, N. W., 4th, Lockatell, V., Johnson, D. E. & Mobley, H. L. T. (2001). In vivo dynamics of type 1 fimbria regulation in uropathogenic Escherichia coli during experimental urinary tract infection. Infect Immun 69, 28382846.
Harris, S. J., Shih, Y., Bentley, S. D. & Salmond, P. P. C. (1998). The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants. Mol Microbiol 28, 705717.[CrossRef][Medline]
Jackson, D. W., Suzuki, K., Oakford, L., Simecka, J. W., Hart, M. E. & Romeo, T. (2002). Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli. J Bacteriol 184, 290301.
Jensen, K. F. (1993). The Escherichia coli K-12 wild-types' W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels. J Bacteriol 175, 34013407.[Abstract]
Jones, C. H., Pinkner, J. S., Roth, R., Heuser, J., Nicholes, A. V., Abraham, S. N. & Hultgren, S. J. (1995). FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae. Proc Natl Acad Sci U S A 92, 20812085.
Kalir, S., McClure, J., Pabbaraju, K., Southward, C., Ronen, M., Leibler, S., Surette, M. G. & Alon, U. (2001). Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292, 20802083.
Keith, B. R., Maurer, L., Spears, P. A. & Orndorff, P. E. (1986). Receptor-binding function of type 1 pili affects bladder colonization by a clinical isolate of Escherichia coli. Infect Immun 53, 693696.[Medline]
Klemm, P. (1986). Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J 5, 13891393.[Abstract]
Lehnen, D., Blumer, C., Polen, T., Wackwitz, B., Wendisch, V. F. & Unden, G. (2002). LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli. Mol Microbiol 45, 521532.[CrossRef][Medline]
Macnab, R. M. (1996). Flagella and motility. In Escherichia coli and Salmonella, pp. 123145. Edited by F. C. Neidhardt. Washington, DC: American Society for Microbiology.
Martinez, J. J., Mulvey, M. A., Schilling, J. D., Pinkner, J. S. & Hultgren, S. J. (2000). Type 1 pilus-mediated bacterial invasion of bladder epithelial cells. EMBO J 19, 28032812.
Miller, J. H. (1992). A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Miller, V. L. & Mekalanos, J. J. (1988). A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170, 25752583.[Medline]
Mukherjee, A., Cui, Y., Ma, W., Liu, Y. & Chatterjee, A. K. (2000). hexA of Erwinia carotovora ssp. carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Environ Microbiol 2, 203215.[CrossRef][Medline]
Mulvey, M. A. (2002). Adhesion and entry of uropathogenic Escherichia coli. Cell Microbiol 4, 257271.[CrossRef][Medline]
Mulvey, M. A., Lopez-Boado, Y. S., Wilson, C. L., Roth, R., Parks, W. C., Heuser, J. & Hultgren, S. J. (1998). Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science 282, 14941497.
Nagy, G., Dobrindt, U., Schneider, G., Khan, A. S., Hacker, J. & Emödy, L. (2002). Loss of regulatory protein RfaH attenuates virulence of uropathogenic Escherichia coli. Infect Immun 70, 44064413.
O'Gara, J. P. & Dorman, C. J. (2000). Effects of local transcription and H-NS on inversion of the fim switch of Escherichia coli. Mol Microbiol 36, 457466.[CrossRef][Medline]
Olsen, P. B. & Klemm, P. (1994). Localization of promoters in the fim gene cluster and the effect of H-NS on the transcription of fimB and fimE. FEMS Microbiol Lett 116, 95100.[CrossRef][Medline]
Ostrow, K. S., Silhavy, T. J. & Garrett, S. (1986). cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12. J Bacteriol 168, 11651171.[Medline]
O'Toole, G. A. & Kolter, R. (1998). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28, 449461.[CrossRef][Medline]
Pratt, L. A. & Kolter, R. (1998). Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type 1 pili. Mol Microbiol 30, 285293.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scharf, B., Schuster-Wolff-Buhring, H., Rachel, R. & Schmitt, R. (2001). Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: importance of flagellin A for flagellar filament structure and transcriptional regulation. J Bacteriol 183, 53345342.
Schembri, M. A. & Klemm, P. (2001). Biofilm formation in a hydrodynamic environment by novel fimH variants and ramifications for virulence. Infect Immun 69, 13221328.
Silhavy, T. J., Berman, M. & Enquist, L. W. (1984). Experiments with Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1, 781791.
Six, S., Andrews, S. C., Unden, G. & Guest, J. R. (1994). Escherichia coli possesses two homologous anaerobic C4-dicarboxylate membrane transporters (DcuA and DcuB) distinct from the aerobic dicarboxylate transport system (Dct). J Bacteriol 176, 64706478.[Abstract]
Sohanpal, B. K., Kulasekara, H. D., Bonnen, A. & Blomfield, I. C. (2001). Orientational control of fimE expression in Escherichia coli. Mol Microbiol 42, 483494.[CrossRef][Medline]
Surgey, N., Robert-Baudouy, J. & Condemine, G. (1996). The Erwinia chrysanthemi pecT gene regulates pectinase gene expression. J Bacteriol 178, 15931599.
Tusher, V. G., Tibshirani, R. & Chu, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98, 51165121.
Vidal, O., Longin, R., Prigent-Combaret, C., Dorel, C., Hooreman, M. & Lejeune, P. (1998). Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J Bacteriol 180, 24422449.
Wendisch, V. F., Zimmer, D. P., Khodursky, A. B., Peter, B. J., Cozzarelli, N. & Kustu, S. (2001). Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays. Anal Biochem 290, 205213.[CrossRef][Medline]
Xia, Y., Gally, D., Forsman-Semb, K. & Uhlin, B. E. (2000). Regulatory cross-talk between adhesion operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein. EMBO J 19, 14501457.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103119.[CrossRef][Medline]
Received 7 April 2005;
revised 20 June 2005;
accepted 29 June 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |