Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung (ZMBE), Von Esmarch Strasse 56, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany
Correspondence
M. Alexander Schmidt
infekt{at}uni-muenster.de
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ABSTRACT |
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The EMBL accession number for the sequences of the genes identified in the pix operon is AJ307043.
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INTRODUCTION |
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The genetic organization, the regulation of expression, and the assembly process leading to functional filaments of P pili have been addressed by numerous investigators and have been elucidated in great detail. P pili are encoded by the 11 genes of the pap operon (Hultgren et al., 1993). Expression is regulated by a region directly upstream of the gene of the major pilus subunit PapA encompassing the papI and papB genes. Transcription of the polycistronic mRNA is additionally influenced by global regulators such as the leucine-responsive protein (Lrp), the catabolite activator protein (CAP) and H-NS (Nou et al., 1995
; Weyand et al., 2001
; White-Ziegler et al., 1998
). The different pilus systems show remarkable similarities in the coding sequences as well as in the protein structures of their different subunits (Smyth et al., 1996
).
The transcriptional regulation of pili expression can be modulated by environmental factors, such as temperature, carbon source and growth rate, for example (Uhlin, 1994). In several pilus systems, the regulatory mechanisms involve changes in primary DNA sequence thereby inducing a phase variation due to an on or off state at the transcriptional level (Jonsson et al., 1992
; Swansson & Koomey, 1989
; Willems et al., 1990
). In the pap operon, differential methylation patterns of GATC sites located in the promoter region are responsible for inhibiting and inducing pilus transcription. Two pap operon genes (papB and papI) are involved in this process, acting in concert with global regulators such as Lrp (Nou et al., 1995
; Weyand et al., 2001
).
Here we report on the molecular cloning and characterization of a novel filamentous adhesin derived from the clinical UTI isolate X2194 (O2 : K- : H6). This adhesin has been designated Pix for pilus involved in E. coli X2194 adhesion. The Pix adhesin exhibits genetic and structural similarity to the well-characterized P pilus; however, it lacks a papI equivalent. Instead, a fragment of the R6 gene which had been identified recently in the pathogenicity island (PAI) of the UTI E. coli CFT073 (Guyer et al., 1998; Kao et al., 1997
) was detected upstream of pixB. Initial analysis of transcriptional fusions of this region indicated that the expression of Pix pili is influenced by the R6 gene fragment. Binding and inhibition studies indicated that Pix pili apparently recognize surface antigens which are distinct from the known UTI E. coli adhesin receptors.
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METHODS |
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Haemagglutination assay and inhibition by carbohydrates.
A single colony was grown in 1 ml LB medium, and the overnight culture was pelleted and resuspended in 0·1 % BSA/PBS containing 1 % mannose to inhibit haemagglutination mediated by type 1 pili expression. Fifty microlitres of the bacterial suspension was incubated with 50 µl of a 1 % suspension of human erythrocytes (HRBCs in PBS) for 1 h at 20 °C. HRBCs of blood group A and O were used. For inhibition the following compounds were added (16 mM final concentration): D-arabinose, D-fructose, D-galactose, D-glucose, lactose, D-mannose, D-melibiose, L-rhamnose, D-ribose, D-xylose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminosyl-D-lactose, sucrose, asialofetuin, fetuin, transferrin, TammHorsfall protein, glycophorin AM and L-serine. Agglutination of Gal-(1,4)Gal-coated P-latex beads (Chembiomed) was tested in PBS.
Adhesion assay.
Bacterial adherence to HeLa cells was monitored essentially as described by Cravioto et al. (1979) with modifications. For each assay, 108 bacteria grown overnight at 37 °C with aeration in LB medium were incubated for 5 min in 1 ml PBS containing 0·5 % D-mannose. The bacterial suspension was added to HeLa cell monolayers, which had been cultured overnight on cover-slips, just before reaching confluency. After 1 h incubation at 37 °C, the cells were washed extensively with PBS to remove non-adherent bacteria. The cells were fixed in 70 % methanol, stained with Giemsa (10 % solution in water) for better contrast, and evaluated by light microscopy.
Recombinant DNA techniques.
All DNA manipulations were performed by standard genetic and molecular biology techniques (Sambrook et al., 1989). Plasmid DNA was purified using a Qiagen kit. Restriction and DNA-modifying enzymes were obtained from New England Biolabs and used according to the manufacturer's instructions. Oligonucleotides were synthesized in a Beckman oligonucleotide synthesizer. The sequences of oligonucleotide primers are summarized in Table 2
. E. coli strains were transformed by the CaCl2 method as described by Hanahan (1983)
.
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Cloning of the 5'-regulatory region.
Cloning of sequences upstream of the major pilin gene pixA was performed by a modified chromosome walking method as described by Dominguez & Lopez-Larrea (1994). In brief, a pixA-specific primer (SP1) was used in combination with a non-specific primer (USP1) for PCR (20 pmol of each primer, 2 U Taq polymerase, 2·5 mM MgCl2) in standard PCR buffer (Roche Biochemicals) in a Biometra Trio PCR block. After initial denaturation at 95 °C for 3 min, five cycles were performed with the following settings: 95 °C for 60 s, 25 °C for 120 s, 72 °C for 120 s with a temperature ramping of 0·5 °C s-1 from 25 to 72 °C. An additional 30 cycles were carried out at 95 °C for 60 s, 55 °C for 90 s, and 72 °C for 90 s. Amplified fragments were evaluated by gel electrophoresis (1·0 % agarose). A 1·3 kb fragment could be identified by Southern blotting using a DIG-labelled pixA-specific oligonucleotide probe (Southern, 1975
). This fragment was purified from a 1·0 % agarose gel by using the Qiaex gel extraction kit (Qiagen) according to the manufacturer's instructions, reamplified by PCR and cloned into the pGEM-T vector, resulting in pAL1. A second chromosomal walking step was carried out as described above using a different pair of primers (SP2 and USP2). A positive fragment (950 bp) was identified by digestion with BglII (removal of 252 bp from the 3'-end) and cloned into the pGEM-T vector (pAL4). Subsequently, the complete fragment was reamplified by PCR using the 5'-primer pixIBA and the 3'-primer pixABI (Table 2
). Double-stranded plasmid DNA sequencing of the pix operon was carried out by the dideoxy-chain termination procedure with a T7 sequencing kit (Pharmacia). The software package HUSAR (Heidelberg Unix Sequence Analysis Resources, version 4.0) of the DKFZ Heidelberg was used for sequence analysis.
The sequences of the genes identified in the pix operon have been deposited in the EMBL database (accession no. AJ307043).
Construction of lacZ fusions for the evaluation of transcriptional activity.
For the evaluation and quantification of the putative transcriptional activity of the 5'-flanking sequence of pixB, two fragments were cloned into the promoter test vector pCB192, which harbours a promoterless lacZ gene (Schneider & Beck, 1986).
After digestion of pAL1 with SphI and SalI, the insert was cloned into the vector pBS(+) digested with the same enzymes to generate pAL2. Subsequently, the fragment now flanked by HindIII and PspAI sites was directionally cloned as a 1·3 kb insert into pCB192 (pAL3), generating a pixAlacZ fusion for the investigation of the putative promoter activity residing in the pixBA region. Positive clones were identified by a low activity of lacZ and were additionally characterized by restriction analysis.
For construction of pAL6, chromosomal DNA of E. coli X2194 was amplified using the two primers pixABI and pixIBA (Table 2) in a standard PCR reaction (30 cycles of 95 °C for 60 s, 60 °C for 90 s, 72 °C for 60 s). These primers are flanking the 1923 bp fragment of the pix operon, including the R6 gene fragment, and additionally contain a BamHI restriction site at their 5'-end to allow cloning into pCB192. The orientation of the insert was checked by digestion with EcoRI/SphI. Positive clones showed a high activity of lacZ as detected on X-Gal-containing LB-agar plates.
-Galactosidase activity in bacteria was determined as described by Miller (1972)
.
Influence of growth conditions on pix operon expression (growth phase, temperature, carbon source).
The influence of different culture conditions was investigated by growing the bacteria in M9CA medium containing glucose as carbon source. Cultures were started with the addition of a 1 : 100 dilution of an overnight culture. To investigate the effect of growth phase on Pix pilus expression, samples were taken at different time points and the OD600 and lacZ activity were measured. To analyse the effect of temperature on pixA expression, cultures were grown at 37 and 26 °C, and lacZ activity was monitored at different time points. To assess the influence of different carbon sources, bacteria were grown in M9CA medium containing 0·1 % glucose or 0·1 % glycerol.
Electron microscopy.
Expression of pili in E. coli X2194 and in recombinant clones was evaluated by transmission electron microscopy using uranyl acetate for contrast. Bacteria suspended in PBS were placed on a Formvar- and carbon-coated copper grid. In some experiments, the pili were stabilized by the addition of 1 % BSA/PBS. After applying a 1 % uranyl acetate solution for negative staining, the sample was inspected using a Philips 400 electron microscope set for transmission electron microscopy or in the scanning mode.
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RESULTS |
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Molecular cloning of the E. coli X2194 pilus adhesin
To further characterize the E. coli X2194 adhesin by molecular cloning, chromosomal DNA was partially digested with Sau3A and fragments between 6·0 and 12·0 kb were ligated into a BamHI-restricted pBR322. After transformation of CMK603 mannose-resistant haemagglutination of human erythrocytes was used to screen for the expression of the functional adhesin. Adherence-positive bacteria were found to harbour a recombinant plasmid named pAD1 which carried an 8·0 kb chromosomal DNA insert. Directional cloning of the 8·0 kb insert from pAD1 into the pBSIISK(-) vector carrying a multiple cloning site was achieved by excising the insert with the flanking HindIII and SalI sites. Ligation of the HindIIISalI fragment (8·6 kb) encompassing the original 8·0 kb insert with some flanking pBR322 vector sequences into the pBSIISK(-) vector generated plasmid pSK12. The 8·0 kb insert also mediated adhesion of E. coli CMK603(pAD1) and of E. coli JM109(pSK12) to HeLa cells. The 8·0 kb insert harboured by pAD1 (also present in pSK12) contained all the genes necessary for the functional expression of the specific pilus adhesin of E. coli strain X2194 (Pix pili).
Pix pilus characterization by electron microscopy
The wild-type E. coli strain X2194 as well as the recombinant strain harbouring plasmid pAD1 were investigated by electron microscopy using negative staining. As demonstrated in Fig. 1, the original isolate (Fig. 1a, b
) as well as the recombinant type 1 pili-negative HB101 strain harbouring the pAD1 plasmid (Fig. 1f
) expressed numerous filamentous appendages. Inspection of the images revealed the pili to have a diameter of about 10 nm and a maximal length of approximately 3·5 µm. Interestingly, these pili appeared to be not as rigid as pili expressed by other UTI strains, like the Pap pili, for example.
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Nucleotide sequence of the region upstream of pixA
The cloned fragment of the pix operon contained seven complete ORFs which exhibited significant homology to the genes of the well-characterized pilin subunits of the pap operon. Additionally, an incomplete coding sequence for a papB homologue, denoted pixB, was identified upstream of pixA. This suggested that an incomplete operon had probably been cloned, which might have been truncated in the 5'-regulatory sequences. To complete the pixB gene and to further characterize the sequences upstream of pixA, a modified PCR technique was employed for chromosome walking steps for completing the pix operon sequence. After two walking steps we could additionally clone 1518 bp upstream of pixB. Sequence alignment revealed, besides a now completed ORF for pixB, a potential promoter site for a pixBpixA transcript followed by a 370 bp long AT-rich region. This AT-rich sequence contained several direct as well as inverted repeats suggesting a potential effector site for transcriptional regulators (data not shown). In contrast to the corresponding region in the pap operon, the sequence contained no apparent sites for DNA methylation (GATC). Interestingly, a putative coding sequence for an ORF homologous to papI could also not be detected.
Furthermore, the fragment upstream of the AT-rich region showed significant sequence identities to the first part of the R6 gene which had been identified in the PAI of the highly virulent uropathogenic E. coli strain CFT073. The R6 gene of E. coli CFT073 has recently been reported to express significant homology to a transposase gene of Chelatobacter heintzii (Xu et al., 1997). It was interesting to see whether the R6 gene fragment represented just the remnant of a transposable element or if this sequence might be involved in the regulation of Pix pilus expression. Expression of a truncated R6 protein is not possible because the insertion of an additional nucleotide leads to a frameshift mutation. Homologies to ORFs BD detected in the upstream sequence could not be identified.
The R6 fragment participates in pixA transcription
To evaluate the potential influence of the upstream sequence of pixB including the R6 fragment on expression, two different pixAlacZ fusions were constructed (Fig. 2b) and transformed into E. coli DH5
to assess the specific influence of the R6 homology region. In addition to the potential regulator pixB and the AT-rich region, the construct pAL3 contained the sequence downstream of nucleotide 706 and consequently only a truncated R6 fragment. The construct pAL6 harboured the complete sequence for the R6 fragment of E. coli strain X2194. As an indicator for the specific relative influence of the 5'-regions on expression, the
-galactosidase activities were compared in strains DH5
(pAL3) and DH5
(pAL6) grown in M9CA medium containing glucose as sole carbon source. The
-galactosidase activities show that in the absence of the R6 fragment in pAL3, pixA transcription is reduced about 40-fold in comparison to pAL6 containing the R6 fragment (data not shown), suggesting an essential role of the R6 sequence for pix transcription.
Monitoring growth-phase-specific and temperature-dependent PixA expression
To assess whether the effect of the 5'-sequence on the expression of Pix pili is influenced by environmental parameters, the pAL3 and pAL6 plasmids encoding a PixALacZ fusion protein with either a truncated (pAL3) or a complete (pAL6) R6-homology region (see Fig. 2B) were transformed into DH5
. Stationary LB cultures of DH5
(pAL3) or DH5
(pAL6) were diluted 1 : 100 in M9CA medium containing 0·2 % glucose as carbon source. With DH5
(pCB192) serving as vector control,
-galactosidase activity was determined as an indicator of PixA pilin synthesis and bacterial growth was monitored by OD600 (Fig. 3
). During the exponential growth phase of DH5
(pCB192), the rate of
-Gal production appeared to be limited as the enzyme activity increased only twofold. By the late exponential growth phase, transcription of the gene encoding the major pilus subunit PixA was markedly induced in DH5
(pAL6) as indicated by a sevenfold increase in activity of LacZ compared to the basal level. In contrast, no induction of pixA transcription could be detected in DH5
(pAL3).
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DISCUSSION |
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The expression of fimbrial adhesins is usually regulated at the transcriptional level, whereas DNA rearrangement occurs in type 1 pili (Abraham et al., 1985). Methylation-dependent transcriptional regulation has been described for the pap operon, where the gene encoding the transcriptional activator PapB is co-transcribed with the gene of the major pilin subunit PapA and genes encoding additional pilus subunits from a promoter upstream of papB. The amount of the major pilin subunit PapA is a critical parameter for pilus synthesis (Nilsson & Uhlin, 1991
). The second specific regulator for the synthesis of the Pap pilus is PapI. The gene encoding this is located upstream of papB and is transcribed in the opposite direction. Regulation of papBA transcription is dependent on the co-operative binding of Lrp to defined DNA sequences containing GATC motifs, which are differentially methylated by deoxyadenosine methylase (Braaten et al., 1994
). The papB gene product increases pap operon expression and acts as a transcriptional regulator of papI as well. Therefore, PapB has an autoregulatory function (Forsman et al., 1989
). The gene products of fanA and fanB serving as regulatory proteins in K99-pili production share significant homology to PapB but have been shown to work as transcriptional anti-terminators (Roosendaal et al., 1989
). PapI, the second pilus-specific regulator, increases the affinity of Lrp for definite pap DNA sites and thereby induces a translocation of Lrp leading to an activation of pilus transcription. Similar mechanisms were described for the plasmid-encoded fimbriae (Pef) of Salmonella typhimurium (Nicholson & Low, 2000
), but also some modifications were observed, e.g. the function of Pef. Other global regulators of Pap pilus transcription are the catabolite activator protein and H-NS (White-Ziegler et al., 1998
).
Interestingly, although the pix operon exhibits significant sequence homology to the pap operon, Pix pilus synthesis appears to be regulated differently. Sequence analysis of the cloned fragment encoding the whole pix operon identified an AT-rich region containing several direct and inverted repeats upstream of pixB. As AT-rich domains have been suggested to serve as a target for transcriptional regulators, this sequence might harbour a putative regulatory site. AT-rich regions seem to have special properties, e.g. the A tracts of the oligonucleotide 5'-GGAAATTTCC-3' show a gradual compression of the minor groove as determined by NMR (Katahira et al., 1990). Additionally, A or T tracts longer than three nucleotides result in curved DNA when positioned on the same side of the helix (Hagermann, 1990
). Recent studies by Xia et al. (1998)
could demonstrate that the PapB protein recognizes a motif including a 9 bp repeat sequence containing T/A triplets at conserved positions. Inhibitory experiments with distamycin, a minor groove DNA binding drug (Coll et al., 1987
), could demonstrate a competition effect between PapB and distamycin, which further suggested that the DNA architecture might be a factor in the transcriptional control of adhesin expression.
In contrast to the homologous pap or pef operon, no Lrp-binding sites and GATC motifs could be observed within the regulatory region of the pix operon. Lrp is a 19 kDa DNA-binding protein that activates some genes and represses others in the E. coli chromosome (Calvo & Matthews, 1994). The pap-regulatory region contains six binding sites each sharing the consensus sequence GN23TTT. Lrp acts as a repressor when bound to pap DNA sites [1,2,3] and as an activator when bound to DNA sites [5,6]; each of these two binding regions contains a GATC motif which is methylated by Dam. Differential methylation and binding of Lrp enables a transition from phase on to phase off pilus transcription (van der Woude et al., 1996
; Weyand et al., 2001
). In the corresponding regulatory region of the pix operon we did not detect any similar features by sequence alignment suggesting that other regulatory mechanisms might be involved. PapI is the second pilus-specific regulator in the pap operon shown to be essential for the translocation of PapB from Lrp-binding site [1,2,3] to Lrp-binding site [5,6]. We were not able to detect Lrp-binding sites in the pix operon and we could also not identify any ORF corresponding to a PapI homologue. Employing database searches and sequence alignment we identified a 952 bp fragment of the R6 gene which had been found in the PAI of the highly uropathogenic E. coli CFT073 recently reported by Guyer et al. (1998)
. This PAI spans a 58 kb region, and among the 44 ORFs identified it contains several virulence genes, including a complete pap gene cluster. Additionally, the authors described the R6 gene as showing a highly significant homology to a gene inside a gene cluster encoding nitrilotriacetate monooxygenase and NADH : flavin mononucleotide oxidoreductase in C. heintzii ATCC 29600 (Xu et al., 1997
). By sequence alignment an ORF of 1467 bp encoding a protein of 488 aa was suggested. The deduced amino acid sequence had a weak but still significant identity (
25 %) to several transposases, including those from E. coli IS21 and Bacillus thuringiensis IS232. It was therefore suggested that this gene might be part of an insertion element, but no further evaluation of its function has been reported. Interestingly, an IS element has also been identified at the 3'-end of the related sfp operon (Brunder et al., 2001
), which might indicate the presence of a PAI. This might even suggest a distribution of these related genes by horizontal gene transfer as had been reported for pap and prs operons (Marklund et al., 1992
).
To further investigate the relevance of the R6 fragment for Pix pilus transcription in the absence of a second pilin-specific regulator homologue, we carried out initial studies using the vector pCB192 which contains a promoterless lacZ gene. Two pixAlacZ fusions were constructed: the first contained the complete R6 region as well as the ORF encoding PixB; in the second construct the R6 region was deleted. These studies clearly demonstrated the effect of the R6 fragment on Pix pilus expression. pixA transcription was markedly reduced after deletion of this regulatory region and no regulation of expression could be observed. As was found also with the Pap pilus system, PixB proved not to be sufficient for transcriptional activation (Göransson et al., 1988). We conclude that expression of the pix operon possibly involves the interaction of the PapB homologue PixB with the R6 fragment. Dissection of the mechanism of regulation of Pix pili expression will require additional studies that have to elucidate the function of this region in more detail and to address possible interactions with global regulators (Morschhäuser et al., 1994
). Additionally, the pix operon may be part of a PAI on the chromosome of the clinical isolate E. coli X2194.
The expression of fimbrial operons is regulated by environmental signals such as temperature, growth rate, aliphatic amino acids, oxygen level, iron, osmolarity and carbon source (Uhlin, 1994). In this study, we demonstrated that temperature-dependent expression of Pix pili occurs predominantly during the stationary growth phase as has been described for other virulence factors. The effect of growth temperature is mediated in many fimbrial systems by histone-like protein H-NS. Whether temperature regulation of the pix operon is achieved in a similar manner has to be established. However, the observation that in contrast to other pilus systems Pix pili are expressed during the stationary growth phase reflects the growth-phase-dependent regulation of virulence gene expression as an adaptive response to different microenvironments (Puente et al., 1996
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Båga, M., Normark, S., Hardy, J., O'Hanley, P., Lark, D., Olsson, O., Schoolnik, G. K. & Falkow, S. (1984). Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli. J Bacteriol 157, 330333.[Medline]
Braaten, B. A., Nou, X., Kaltenbach, L. S. & Low, D. A. (1994). Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli. Cell 76, 577588.[Medline]
Brunder, W., Salam Khan, A., Hacker, J. & Karch, H. (2001). Novel type of fimbriae encoded by the large plasmid of the sorbitol-fermenting enterohemorrhagic Escherichia coli O157 : H-. Infect Immun 69, 44474457.
Calvo, J. M. & Matthews, R. G. (1994). The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli. Microbiol Rev 58, 466490.[Medline]
Coll, M., Frederick, C., Wang, A. H. & Rich, A. (1987). A bifurcated hydrogen-bonded conformation in the d(AT) base pairs of the DNA dodecamer d(CGCAAATTTGCG) and its complex with distamycin. Proc Natl Acad Sci U S A 84, 83858389.[Abstract]
Cravioto, A., Gross, R. J., Scotland, S. M. & Rowe, B. (1979). An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr Microbiol 3, 9599.
Denich, K., Blyn, L. B., Craiu, A., Braaten, B. A., Hardy, J., Low, D. A. & O'Hanley, P. D. (1991). DNA sequences of three papA genes from uropathogenic Escherichia coli strains: evidence of structural and serological conservation. Infect Immun 59, 38493858.[Medline]
Dominguez, O. & Lopez-Larrea, C. (1994). Gene walking by unpredictable primed PCR. Nucleic Acids Res 22, 32473248.[Medline]
Forsman, K., Göransson, M. & Uhlin, B. E. (1989). Autoregulation and multiple DNA interactions by a transcriptional regulatory protein in E. coli pili biogenesis. EMBO J 8, 12711277.[Abstract]
Foxman, B., Zhang, L., Palin, K., Tallman, P. & Marrs, C. F. (1995). Bacterial virulence characteristics of Escherichia coli isolates from first-time urinary tract infection. J Infect Dis 171, 15141521.[Medline]
Girardeau, J. P., Bertin, Y. & Callebaut, I. (2000). Conserved structural features in class I major fimbrial subunits (pilin) in gram-negative bacteria. Molecular basis of classification in seven subfamilies and identification of intrasubfamily sequence signature motifs which might be implicated in quaternary structure. J Mol Evol 50, 424442.[Medline]
Göransson, M., Forsman, K. & Uhlin, B. E. (1988). Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli. Mol Gen Genet 212, 412417.[Medline]
Guyer, M. D., Kao, J. S. & Mobley, H. L. T. (1998). Genomic analysis of a pathogenicity island in uropathogenic Escherichia coli CFT073: distribution of homologous sequences among isolates from patients with pyelonephritis, cystitis and catheter-associated bacteriuria and from fecal samples. Infect Immun 66, 44114417.
Hagermann, P. J. (1990). Sequence-directed curvature of DNA. Annu Rev Biochem 59, 755781.[CrossRef][Medline]
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Biol Chem 166, 557580.
Hultgren, S. J., Abraham, S., Caparon, M., Falk, P., St Geme, J. W. & Normark, S. (1993). Pilus and nonpilus bacterial adhesins: assembly and function in cell recognition. Cell 73, 887901.[Medline]
Ikaheimo, R., Siitonen, A., Karkkainen, U., Kuosmanen, P. & Mäkelä, P. H. (1993). Characteristics of Escherichia coli in acute community-acquired cystitis of adult women. Scand J Infect Dis 25, 705712.[Medline]
Jacob-Dubuisson, F., Heuser, J., Dodson, K., Normark, S. & Hultgren, S. (1993). Initiation of assembly and association of the structural elements of a bacterial pilus depend on two specialized tip proteins. EMBO J 12, 837847.[Abstract]
Jonsson, A. B., Pfeifer, J. & Normark, S. (1992). Neisseria gonorrhoeae PilC expression provides a selective mechanism for structural diversity of pili. Proc Natl Acad Sci U S A 89, 32043208.[Abstract]
Källenius, G., Möllby, R., Svenson, S. B., Winberg, J., Lundblad, A. & Svensson, S. (1980). The pK antigen as receptor for pyelonephritic E. coli. FEMS Microbiol Lett 7, 297300.[CrossRef]
Källenius, G., Möllby, R., Svenson, S. B., Helin, I., Hultberg, H., Cedergren, B. & Winberg, J. (1981). Occurrence of P-fimbriated Escherichia coli in urinary tract infections. Lancet 26, 13691372.
Kao, J. S., Stucker, D. M., Warren, J. W. & Mobley, H. L. T. (1997). Pathogenicity island sequences of pyelonephritogenic Escherichia coli are associated with virulent uropathogenic strains. Infect Immun 65, 28122820.[Abstract]
Katahira, M., Sugeta, H. & Kyogoku, Y. (1990). A new model for bending of DNAs containing the oligo(dA) tracts based on NMR observations. Nucleic Acids Res 18, 613618.[Abstract]
Korhonen, T. K., Valtonen, M. V., Parkinnen, J., Vaisanen-Rhen, V., Finne, J., Ørskov, I., Ørskov, F., Svenson, S. B. & Mäkelä, P. H. (1985). Serotype, hemolysin production and receptor recognition of Escherichia coli strains associated to neonatal sepsis and meningitis. Infect Immun 48, 486491.[Medline]
Marklund, B. I., Tennent, J. M., Garcia, E., Hamers, A., Baga, M., Lindberg, F., Gaastra, W. & Normark, S. (1992). Horizontal gene transfer of the Escherichia coli pap and prs-operons as a mechanism for the development of tissue specific adhesive properties. Mol Microbiol 6, 22252242.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Morschhäuser, J., Vetter, V., Emödy, L. & Hacker, J. (1994). Adhesin regulatory genes within large, unstable DNA regions of pathogenic Escherichia coli: cross-talk between different gene clusters. Mol Microbiol 11, 555566.[Medline]
Nicholson, B. & Low, D. (2000). DNA methylation-dependent regulation of pef expression in Salmonella typhimurium. Mol Microbiol 35, 728742.[CrossRef][Medline]
Nilsson, P. & Uhlin, B. E. (1991). Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE-dependent endonucleolytic processing. Mol Microbiol 5, 17911799.[Medline]
Nou, X., Braaten, B., Kaltenbach, L. & Low, D. A. (1995). Differential binding of Lrp to two sets of pap DNA binding sites mediated by PapI regulates Pap phase variation in Escherichia coli. EMBO J 14, 57855797.[Abstract]
Parkkinen, J., Korhonen, T. K., Pere, A., Hacker, J. & Soinila, S. (1988). Binding sites in the rat brain for Escherichia coli S pili associated with neonatal meningitis. J Clin Investig 81, 860865.[Medline]
Ponniah, S., Endres, R. O., Hasty, D. L. & Abraham, S. N. (1991). Fragmentation of Escherichia coli type 1 pili exposes cryptic D-mannose-binding sites. J Bacteriol 173, 41954202.[Medline]
Puente, J. L., Bieber, D., Ramer, S. W., Murray, W. & Schoolnik, G. K. (1996). The bundle-forming pili of enteropathogenic Escherichia coli: transcriptional regulation by environmental signals. Mol Microbiol 20, 87100.[Medline]
Roberts, J. A., Marklund, B., Ilver, D. & 7 other authors (1994). The Gal(14)Gal-specific Tip adhesin of Escherichia coli P-fimbriae is needed for pyelonephritis to occur in the normal urinary tract. Proc Natl Acad Sci U S A 91, 1188911893.
Roosendaal, B., Damoiseaux, J., Jordi, W. & de Graaf, F. K. (1989). Transcriptional organisation of the DNA region controlling expression of the K99 gene cluster. Mol Gen Genet 215, 250256.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schneider, K. & Beck, C. F. (1986). Promoter-probe vectors for the analysis of divergently arranged promoters. Gene 42, 3748.[CrossRef][Medline]
Smyth, C. J., Marron, M. B., Twohig, J. M. & Smith, S. G. (1996). Fimbrial adhesins: similarities and variations in structure and biogenesis. FEMS Immunol Med Microbiol 16, 127139.[CrossRef][Medline]
Southern, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503517.[Medline]
Swansson, J. & Koomey, J. M. (1989). Mechanisms for variation of pili and outer membrane protein II in Neisseria gonorrhoea. In Mobile DNA, pp. 743761. Edited by D. E. Berg & M. M. Howe. Washington, DC: American Society for Microbiology.
Uhlin, B. E. (1994). Regulation of E. coli fimbrial expression. In Pili: Adhesin, Genetics, Biogenesis and Vaccines, pp. 171178. Edited by P. Klemm. Boca Raton, FL: CRC Press.
Van der Woude, M. W., Braaten, B. & Low, D. A. (1996). Epigenic phase variation of the pap-operon in Escherichia coli. Trends Microbiol 4, 59.[CrossRef][Medline]
van Die, I., Dijksterhuis, M., deCock, H., Hoekstra, W. & Bergmans, H. (1986). Structural variation of P-fimbriae from uropathogenic Escherichia coli. In Protein-Carbohydrate Interactions in Biological Systems, pp. 3946. Edited by D. L. Lark. London: Academic Press.
Weyand, N. J., Braaten, B. A., van der Woude, M., Tucker, J. & Low, D. A. (2001). The essential role of the promotor-proximal subunit of CAP in pap phase variation: Lrp- and helical phase-dependent activation of papBA transcription by CAP from -215. Mol Microbiol 39, 15041522.[CrossRef][Medline]
White-Ziegler, C. A., Angus Hill, M. L., Braaten, B. A., van der Woude, M. W. & Low, D. A. (1998). Thermoregulation of Escherichia coli pap transcription: H-NS is a temperature-dependent DNA methylation blocking factor. Mol Microbiol 28, 11211137.[CrossRef][Medline]
Willems, R., Paul, A., van der Heide, H. G., ter Avest, A. R. & Mooi, F. R. (1990). Fimbrial phase variation in Bordetella pertussis: a novel mechanism for transcriptional regulation. EMBO J 9, 28032809.[Abstract]
Wullt, B., Bergsten, G., Conell, H., Röllano, P., Gebretsadik, N., Hull, R. & Svanborg, C. (2000). P fimbriae enhance the early establishment of Escherichia coli in the human urinary tract. Mol Microbiol 38, 456464.[CrossRef][Medline]
Xia, Y., Forsman, K., Jass, J. & Uhlin, B. E. (1998). Oligomeric interaction of the PapB transcriptional regulator with upstream activating region of pili adhesin gene promotors in Escherichia coli. Mol Microbiol 30, 513523.[CrossRef][Medline]
Xu, Y., Mortimer, M. W., Fisher, T. S., Kahn, M. L., Brockman, F. J. & Xun, L. (1997). Cloning, sequencing and analysis of a gene cluster from Chelatobacter heintzii ATCC29600 encoding nitrilotriacetate monooxygenase and NADH : flavin mononucleotide oxidoreductase. J Bacteriol 179, 11121116.[Abstract]
Zhang, J. P. & Normark, S. (1996). Induction of gene expression in Escherichia coli after pilus-mediated adherence. Science 273, 12341236.[Abstract]
Received 28 January 2003;
accepted 3 March 2003.
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