INTRODUCTION

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Numerous proteinaceous adhesins have been detected in Escherichia coli (for recent reviews, see references 20 and 27). These adhesins occur in the form of fimbrial filaments or are nonfimbrial proteins of the outer membrane. The adhesins recognize different receptor molecules on the mammalian epithelia or extracellular matrices and function to enable colonization of E. coli at specific ecological niches. Many of the adhesins are associated with E. coli isolates from specific disease manifestations and contribute to the establishment of the infections. Examples of such disease-associated adhesins include the P fimbria of uropathogenic E. coli (UPEC) (55) and the various adhesin types detected in E. coli pathogroups causing diarrheal diseases (reviewed in references 13 and 36). Some adhesin genes, such as those encoding the mannoside-binding type 1 fimbriae (26) and the fibronectin-binding curli (39), are present on and expressed by a large proportion of natural E. coli isolates. The type 1 fimbriae are important for the spread of E. coli from one host individual to another (6) and also enhance bacterial colonization in the human and animal gut and the urinary tract (20, 27). The curli are expressed at low temperature and low osmolarity and may convey a selective advantage for E. coli in the environment and in the early phases of intestinal tract colonization (40).

The natural populations of E. coli exhibit extensive genetic diversity that is organized into a limited number of genetically distinct clonal groups (reviewed in reference 59). Such widespread, homogenous clonal groups have been well characterized in E. coli strains from separate outbreaks of disease. The isolates in the clonal groups are characterized by several identical phenotypic traits, such as serotype, biotype, phage type, outer membrane protein profiles, and production of hemolysins, other toxins, specific iron-scavenging systems, and specific adhesins. Multilocus enzyme electrophoresis has shown that isolates in a given clonal group are genetically conserved (50, 59), and the clonal groups appear sufficiently stable that they have spread into human populations on several continents. Clonal groups in pathogenic E. coli include several UPEC clones (10, 43, 55), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) clones (reviewed in references 13 and 35). The association of specific adhesins with these pathogroups has resulted from horizontal gene transfer of plasmid genes or chromosomal pathogenicity islands from other pathogens. On the other hand, diversification of adhesin alleles may result from within-host evolution of E. coli, in which random point mutations affect the tissue tropism of the bacterium (52).

The O18acK1H7 isolates represent the major clonal group of E. coli associated with newborn meningitis and septicemia (MENEC) (1, 31, 51). Electrophoretic analysis of chromosomally encoded enzymes revealed that the O18acK1H7 MENEC isolates are genetically highly conserved and form a distinct clone, which is phenotypically characterized by expression of the S and the type 1 fimbriae and a conserved outer membrane protein profile, as well as, on the other hand, by lack of the P and the type 1C fimbriae and hemolysin (1, 31, 51). In this report, we describe a novel fimbrial gene that is common in E. coli isolates, including laboratory K-12 strains, but is expressed only in O18acK1H7 MENEC.

	MATERIALS AND METHODS

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Bacteria, growth conditions, and plasmids. The bacterial strains and plasmids used in this work are listed in Table 1. For expression studies, the bacteria were cultivated in static Luria broth at 20 or 37°C and under shaking at 37°C for other purposes. Antibiotics, when necessary, were added at concentrations of 100 (ampicillin), 25 (chloramphenicol), 30 (gentamicin [Gm]), 75 (rifampin [Rif]), 100 (streptomycin [Sm] and streptothricin [St]), or 12.5 (tetracycline) µg ml¹. The antibiotics were obtained from Sigma Chemical Co, St Louis, Mo., except for streptomycin and streptothricin, which were a gift from Helmuth Tschäpe, Robert Koch Institute, Wernigerode, Germany. Induction of mat genes in recombinant E. coli strains was done with 5 µM isopropyl--D-thiogalactopyranoside (IPTG) (Promega Corporation, Madison, Wis.).

DNA techniques. Isolation of plasmid and chromosomal DNA from E. coli cells and DNA manipulations were performed by routine procedures (47). Restriction enzymes (Promega Corp., Madison, Wis., and New England Biolabs, Beverly, Mass.) were used according to the manufacturers' instructions. Cloning of IHE 3034-79 chromosomal DNA into cosmid pHC79 was done according to the manufacturer's instructions (DNA-Packaging kit; Boehringer Mannheim, Mannheim, Germany), resulting in the plasmid pMAT1. A 7-kb EcoRI fragment of pMAT1 that reacted in Southern hybridization (47) with the degenerate oligonucleotides designed on the basis of the peptide sequences obtained from MatB was subcloned into pACYC184 to give pMAT2. The DNA sequences of the degenerate oligonucleotides used in the hybridization were 5' GCIGAIGTIACIGCICAIGCIGTIGCIACITGG 3' and 5' TTIGAIGTIGCIATIGAIGGIGAI 3'. The nucleotide sequence of the DNA region encoding matA and matB in pMAT2 was determined by using a commercially available sequencing service (MedProbe AS, Oslo, Norway). The matB gene in the chromosome of clinical E. coli isolates was detected by PCR with primers designed on the basis of the sequence of the matB homolog of MG1655 (5) and DyNAzyme II DNA-polymerase (Finnzymes, Espoo, Finland) according to manufacturer's instructions. The obtained PCR products were analyzed by Southern hybridization with matB gene of E. coli IHE 3034 as a probe by using the ECL enhanced chemiluminescence direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech, Amersham Place, Little Chalfont, Buckinghamshire, England) according to the manufacturer's instructions.

Sequence comparisons. Homology searches in the databases were performed with the BLAST (available at http://www.expasy.ch/sprot/sprot-top.html) and FASTA (http://www.ebi.ac.uk/fasta3/) package programs. Comparison of MatB with other fimbrillins was done by using Align for global alignment (http: //molbiol.soton.ac.uk/compute/align.html) and SIM for local alignment (http://www.expasy.ch/tools/sim-prot.html). Prediction of the presence of stem-loop structures and signal sequences in the mat genes of MG1655 and IHE 3034 was done by using programs available at http://www.fruitfly.org/seq_tools/promoter.html, http://www.genebee.msu.su/services/rna2_reduced.html, and http://www.cbs.dtu.dk/services/SignalP/. DNA-binding motifs were identified by using the ProfileScan program at http://www.isrec.isb-sib.ch/.

Protein chemical methods. Mat fimbriae were purified from E. coli IHE 3034-79 and E. coli LE392(pMAT1) by using deoxycholate and concentrated urea (29). Prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (47), the type 1 and Mat fimbriae were denaturated at low pH (33).

Immunological methods. The anti-type 1 fimbria and anti-Mat fimbria sera were obtained by immunizing rabbits according to standard procedures with the type 1 fimbriae purified from E. coli IHE 3034-8 and the Mat fimbriae purified from E. coli IHE 3034-79 and E. coli LE392(pMAT1) used as antigens. The rabbit antiserum raised against the S fimbriae of IHE 3034 was available from previous work (56). Polyclonal anti-Mat, anti-type 1, and anti-S fimbria sera were titrated against the purified type 1, S, and Mat fimbriae by enzyme-linked immunosorbent assay (ELISA) as described previously (30) with alkaline phosphatase-conjugated swine anti-rabbit immunoglobulins (Dako A/S, Denmark; diluted to 1:500) as secondary antibodies and phosphatase substrate (Sigma Diagnostics, Inc., St. Louis, Mo.) at a concentration of 1 mg/ml in diethanolamine-magnesium chloride buffer (OY Reagena AB, Kuopio, Finland). Results were recorded with a Multiscan MS ELISA reader (Labsystems OY, Helsinki, Finland). Western blotting of SDS-PAGE-separated fimbrial proteins with anti-Mat fimbria antibodies was done by routine procedures as described in reference 11.

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7

Electron microscopy. For electron microscopy, the bacteria were suspended in Luria broth and immobilized on copper grids coated with Pioloform and carbon. For negative staining, the bacteria were stained with 1% (wt/vol) potassium tungstate (pH 6.5) for 1 min. For IEM, the bacteria were left to react with anti-Mat antibodies diluted to 1:500 in PBS and 10-nm-particle-diameter gold-labeled protein A (Amersham Life Science) diluted to 1:40 and negatively stained as described above. The grids were examined in a JEOL JEM-1200EX transmission electron microscope at an operation voltage of 60 kV. The micrographs were taken at the Section of Electron Microscopy, Institute of Biotechnology, University of Helsinki.

Hemagglutination assays. Treatment of human O₁ erythrocytes with endo--galactosidase, neuraminidase, trypsin, and pronase and agglutination of the erythrocytes by E. coli 3034-79 were performed by routine procedures (28).

Nucleotide sequence accession number. The nucleotide sequence of the 6- to 8-min region of the E. coli MG1655 chromosome (5) is available at GenBank under accession no. U73857, and the sequence of the matAB region of the E. coli IHE 3034 chromosome is available under accession no. AF325731. Identity comparisons were performed with predicted protein sequences of matB and the following adhesin genes of E. coli: MG1655 fimA (9), GenBank accession no. AAC77270; PC31 fimA (25), GenBank accession no.X00981; IHE 3034 sfaA (15), GenBank accession no. S53211; J96 papA (3), GenBank accession no. X03391, K01176, X03392, X61239, and S45610; F17 a-A (32), GenBank accession no. AF022140, M36649, and M62503; MG1655 ppdD, GenBank accession no. AE000119 and U00096; K-12 csgA (40), GenBank accession no. L04979; C921b-1 cooA (44), GenBank accession no. M58550 and M37148; cooB, GenBank accession no. X62495; and LMC10 cooC and cooD (14), GenBank accession no.X76908.

	RESULTS

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Purification and characterization of fimbriae from the fimA sfaA fliC mutant derivative of E. coli IHE 3034. The O18acK1H7 meningitis isolate E. coli IHE 3034 and the S- and type 1-fimbria-negative double mutant IHE 3034-59 have been described earlier (15, 30, 31, 45, 51). In order to obtain a derivative that is devoid of all surface filaments, the H7 flagellin gene fliC of IHE 3034-59 was inactivated by allelic exchange with fliC::cat::sat. The sat cassette was positioned within the cat DNA because it contained a suitable NcoI restriction site. Transconjugants with the correct antibiotic markers were assessed for flagellation by agglutination in anti-H7 antiserum, and a nonflagellate mutant was isolated and named IHE 3034-79. The correct genotype of IHE 3034-79 was confirmed by Southern hybridization of PvuI-digested chromosomal DNA by using fliC and the sat cassette as probes (data not shown). Transmission electron microscopy of IHE 3034-79 cells cultivated at 20°C revealed a lack of flagella, but, unexpectedly, showed the presence of fimbrial filaments on the bacteria (Fig. 1A). The strain IHE 3034-79 was not agglutinated in antisera raised against the type 1 or S fimbriae from the same strain, indicating that the strain expressed a third fimbrial type. This fimbria had a diameter of 5 to 7 nm and was 0.4 to 5 µm in length. Because the expression of this fimbrial type was found to be associated with the major clonal group of MENEC as well as with low temperature (see below), we named it the Mat (meningitis associated and temperature regulated) fimbria.

View larger version (162K): [in this window] [in a new window]   FIG. 1.   Electron microscopy of Mat fimbriae. (A) Negatively stained cells of the fimA sfaA fliC mutant strain E. coli IHE 3034-79 cultivated at 20°C. (B to D) IEM of fimbriae from XL1 Blue MRF' harboring pMAT3 (B), pMAT9 (C), or pMAT5 (D). Size bars, 100 nm.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE (A) and Western blotting (B) of the S fimbria (lane 1), the type 1 fimbria (lane 2), and the Mat fimbria (lane 3) of E. coli IHE 3034. The blotting was done with anti-MAT fimbria antiserum as primary antibodies. The migration distances of molecular mass marker proteins (size in kilodaltons) are indicated on the left.

Cloning and sequencing of mat genes. We next ligated random 40-kb fragments from the genome of 3034-79 into the cosmid cloning vector pHC79, and transfectants of E. coli LE392 expressing the Mat fimbria were identified by bacterial agglutination in anti-Mat fimbria serum and by electron microscopy. A positive recombinant was isolated, and the cosmid was named pMAT1. Cosmid pMAT1 was digested with the EcoRI restriction enzyme, and a 7-kb DNA fragment reacting in Southern hybridization with the oligonucleotide probes was identified and cloned into pACYC184, to obtain plasmid pMAT2. Electroporation of pMAT2 into E. coli LE392 gave a recombinant strain reacting with the anti-Mat-fimbria antibodies (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Gene organization of the 6.8-kb fragment containing mat at the 6- to 8-min region in the chromosome of E. coli MG1665 (5). The nucleotide numbers refer to the gene location in the sequence available in GenBank (accession no. U73857). Arrows below the sequence indicate DNA fragments cloned into pSE380 and tested for in trans complementation of the matB::cat mutation in the chromosome of E. coli IHE 3034-90 as well as for expression in E. coli XL1 Blue MRF' (on the right).

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Predicted amino acid sequence of MatB of E. coli IHE 3034. The sequence has a 22-mer signal sequence. No. 1 indicates the N-terminal amino acid residue of the mature protein. The predicted amino acid sequence from E. coli MG1665 (5) contains methionine at the position 6.

Mutagenesis, complementation, and expression of matB. We next inactivated the matB gene of E. coli IHE 3034 by allelic exchange with a matB::cat fusion. A transconjugant with the correct antibiotic markers as well as genotype was isolated and named IHE 3034-90. The correct genotype of IHE 3034-90 was confirmed by Southern hybridization of BamHI-SacII-digested DNA by using matB and the cat cassette as probes (data not shown). The strain IHE 3034-90 did not express Mat fimbriae, as analyzed by agglutination in anti-Mat fimbriae antisera, as well as by IEM (data not shown) and whole-cell ELISA with anti-Mat fimbria antibodies (Fig. 5A).

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Expression of Mat fimbriae in E. coli, as analyzed by whole-cell ELISA with anti-Mat fimbriae as primary antibodies. (A and C) Expression in cells grown at 20°C. (B and D) Expression in cells grown at 37°C. (A and B) Expression is shown for the wild-type IHE 3034, the spontaneous streptomycin-resistant strain IHE 3034-Sm, the fimA::cat derivative IHE 3034-2, the sfaA::Gm derivative IHE 3034-8, the fimA::cat sfaA::Gm double mutant IHE 3034-59, the fimA::cat sfaA::Gm fliC::St triple mutant IHE 3034-79, and the matB::cat derivative IHE 3034-90. (C and D) Complementation of the matB::cat mutation in IHE 3034-90 by the pMAT-plasmids encoding regions of the putative mat gene cluster. (E) SDS-PAGE (top) and Western blotting (bottom) of cellular proteins in E. coli XL1 Blue MRF'(pSE380) (lanes 1 and 3), E. coli XL1 Blue MRF'(pMAT3) (lanes 2 and 4), E. coli IHE 3034-90(pSE380) (lanes 5 and 7), and E. coli IHE 3034-90(pMAT3) (lanes 6 and 8). The bacteria were cultivated at 20°C (lanes 1, 2, 5, and 6) or at 37°C (lanes 3, 4, 7, and 8). (F) RT-PCR of RNA from E. coli IHE 3034 (lanes 1 and 2), MG1665 (lanes 3 and 4), and IHE 3034-90 (lanes 5 and 6). The bacteria were cultivated at 37°C (lanes 1, 3, and 5) or 20°C (lanes 2, 4,and 6). Amplification was done with a primer pair specific for matBC.

Presence of matB and expression of the Mat fimbria in isolates of E. coli IHE 3034 belongs to the serogroup O18acK1H7, which forms the major clonal group of MENEC. These isolates form a homogenous and distinct group of E. coli, as analyzed by several phenotypic assays and isoenzyme polymorphism (30, 31, 51). We analyzed 27 isolates representing various pathogroups of E. coli (Table 1) for the presence of matB as well as expression at 20 and 37°C of the Mat fimbria. The analyzed strains belonged to the O18K1H7 MENEC clonal group or to the clonal groups of UPEC (O1K1H7, O4K12H7, O6K2H1, O18K5H7, and O6K13H1), or they represented the diarrhea-causing pathogroups EAEC, EHEC, EPEC, and ETEC. Additionally, K-12 strains and the widely used UPEC strain 536 were analyzed. PCR and Southern hybridization showed the presence of a matB homolog in all strains except the two ETEC isolates (Table 1). The apparent size of the amplified matB fragment in the isolates was invariant and equalled the one seen in IHE 3034 (data not shown). However, expression of the Mat fimbria was detected only in the MENEC isolates grown at 20°C (Table 1).

Hemagglutination by E. coli IHE 3034-79. Many fimbrial types of E. coli recognize specific carbohydrate sequences. In order to test whether this might be the case for the Mat fimbria as well, we assessed hemagglutination of human erythrocytes by the strain IHE 3034-79 cultivated at 20°C. No agglutination was detected; neither did enzymatic treatments known to reveal cryptic carbohydrates on the erythrocyte surface induce detectable hemagglutination.

	DISCUSSION

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Mat fimbria represents a novel type of E. coli fimbria that is morphologically similar to the type 1 or P fimbriae, but serologically and in a subunit sequence unrelated to the previously characterized fimbrial proteins of E. coli or other bacteria. Although the matB gene encoding the major subunit was found to be common in E. coli isolates, it was expressed on the bacterial surface only in the genetically conserved clonal group of O18acK1H7 MENEC cultivated at low temperature. The matB gene is the fourth fimbrillin gene detected in laboratory K-12 strains, which previously have been shown to harbor fim genes encoding the type 1 fimbria, the csg genes encoding curli, as well as ppdD encoding the type IV fimbrillin (5, 9,25, 40, 48). Of these, only the type 1 fimbriae are expressed by E. coli K-12 under routine laboratory conditions at 37°C. Curli expression takes place at low temperature and low osmolarity (40). It was recently found that a fraction of E. coli isolates from cases of human adult urosepsis also express curli at 37°C (4). The type IV fimbriae require an exogeneous secretion apparatus for surface expression in E. coli K-12 (49), but are expressed in EPEC that harbor the EPEC adherence factor plasmid (35, 36). The matB fimbrillin gene appears to be common in E. coli and is expressed at low temperature by isolates of a genetically distinct pathogroup.

matB encodes the major subunit of the Mat fimbria. The complete nucleotide sequence of matB was determined and the deduced protein sequence matched the sequence of 57 amino acids obtained from the Mat fimbria. The knockout matB::cat mutation abolished Mat fimbria production, which was complemented in trans by matB-containing DNA together with at least either matA or matC. The predicted sequence of MatB was not related to the fimbrillin sequences deposited in data banks and lacks the N-terminal motifs of type IV fimbrillins (53) and CsgA-related fimbrillins (12, 40). MatB also lacks cysteine residues and the C-terminal -zipper motif of chaperone-assisted fimbrillins (21). These characteristics are also lacking in the CooA fimbrillin of the CS1 filaments and in the other fimbrillins detected on human ETEC strains (46). The fimbrillins on human ETEC share 40 to 50% sequence identity with each other, and their plasmid-borne gene clusters are closely related in gene organization (46). However, we found only 23% sequence identity between MatB and CooA, and the matA and matC gene products showed a similar low sequence identity with the four Coo proteins of the CS1 fimbria (46). The ELISAs, Western blotting, and IEM with anti-Mat fimbria antibodies showed that the Mat fimbria was immunologically distinct from the type 1 and S fimbriae expressed by MENEC. We thus conclude that the Mat fimbria represents a novel fimbrial type of E. coli. We could not detect any carbohydrate binding by the Mat fimbria and are currently analyzing the binding of Mat fimbria to epithelial cells and tissue proteins.

A matB homolog was detected in 25 of the 27 E. coli strains analyzed in this study. The analyzed isolates represented major pathogroups of E. coli as well as K-12 strains; the two matB-negative strains were ETEC isolates. The nucleotide sequence of matB of IHE 3034 was 97.8% identical to the matB from MG1665, and the matB homologs amplified from the genomic DNA of the E. coli isolates were of invariable size, also in isolates found not to express MatB. These findings indicate conservation of the matB gene in E. coli. We detected Mat fimbria expression only in the O18acK1H7 clonal group of MENEC, and it is noteworthy that the O1K1H7 isolates, which on the basis of isoenzyme polymorphism form a clonal group of E. coli strains genetically closest to the O18acK1H7 MENEC group (43, 51), did not express their matB gene under the conditions we tested.

We subcloned matB from IHE 3034-79 on a 7-kb fragment that supported Mat fimbria expression when transformed into E. coli K-12. This DNA fragment has a homolog in the MG1665 chromosome at the 6- to 8-min region and contains six putative genes (5), of which we found matA, matB, and matC affected Mat fimbria formation. The two chromosomal regions are highly similar; nucleotide sequencing showed that the matAB region was 96.6% identical for the sequenced 1,471-bp fragment, and a high level of identity of the whole 7-kb region was also evident in the restriction mapping. At present, the number of genes that are involved in Mat fimbria sythesis and regulation remains open. We also do not know whether genes located at a distance from the matB region are involved in fimbria synthesis. The fimbrial gene clusters of E. coli usually contain four or more genes needed for the structure, assembly, or regulation of the fimbrial filament (reviewed in references 20 and 27). Our complementation assays indicated that matB alone is not sufficient to confer Mat fimbria expression on E. coli XL1 or the strain IHE 3034-90 with the polar matB::cat mutation. Complementation was slightly more efficient in the matAB transformant and further increased in the matBC transformant, but was not significantly enhanced when fragments encompassing more than the matBC genes were introduced into IHE 3034 matB::cat or XL1 Blue MRF'. The RT-PCR analysis indicated that matB and matC are part of the same transcript, whereas we did not detect a matABC transcript, which suggests that matA is transcribed independently from matBC. It is noteworthy that the E. coli strain MG1665 contained a matA transcript, but we failed to amplify a matBC RNA from this strain. IEM did not reveal any major structural differences between Mat fimbrial filaments on E. coli K-12 expressing the pMAT plasmids. Western blotting of the Mat fimbriae showed the presence of a minor protein apparently smaller than MatB; its size, however, did not match those of any of the gene products from the mat region, and it may be a degradation product from MatB. On the other hand, in trans expression of mat genes in IHE 3034-90(pMAT3) showed a minor protein with an apparent size of ca. 20 kDa, which is close to the calculated size of mature MatC (22.4 kDa). The function of MatC in fimbria formation remains open. Lack of the chaperone target motif in the C terminus of MatB suggests that Mat fimbriae are not assembled by the classical chaperone-usher pathway of the P and the type 1 fimbriae (20), and the assembly mechanism of the Mat fimbria remains to be elucidated. The predicted sequence of MatA does not contain a signal sequence, whereas a putative signal sequence is present in the matB and matC gene products. The predicted MatA sequence contained a C-terminal helix-turn-helix DNA-binding motif of the LuxR family of regulatory proteins (17). This indicates that MatA may be a cytoplasmic protein functioning in regulation.

We found that transcription of matBC in IHE 3034 cells was reduced but not abolished at 37°C and that expression of mat genes from the plasmid pMAT3 with the inducible trc promoter also was temperature sensitive. These findings indicate that the temperature regulation of the mat genes involves transcriptional as well as posttranscriptional events. Expression of curli in E. coli K-12 is favored at low temperatures. The transcription of csgA is positively regulated by the RpoS sigma factor and repressed by the histone-like protein H-NS, but the temperature and osmolarity regulation of curli expression involves other factors (40). The mechanisms of the temperature regulation of Mat expression remain to be determined. Temperature is known to regulate translation by affecting mRNA conformation (2, 18) or the translational machinery itself (reviewed in reference 22). The matA sequence contains four copies of putative RNase E target sequences (37), which might become accessible upon temperature-induced conformational change. Temperature may also affect the stability of the MatB protein. The mechanisms behind the repression of mat expression in most E. coli isolates also remain open. We found by DNA sequencing and restriction mapping that the 7-kb mat regions in IHE 3034 and MG1655 are highly similar, which indicates that the lack of Mat fimbria expression in E. coli MG1655 does not result from truncation or lack of genes at this chromosomal region. E. coli IHE 3034 and another O18acK1H7 MENEC isolate were recently found to be rpoS mutants (57); whether this holds true for the entire O18acK1H7 MENEC clone and how RpoS influences mat expression are to be elucidated. RpoS is a positive regulator of curli expression, and we indeed have not detected curli expression in IHE 3034 cells cultivated at 20°C (B. Westerlund-Wikström and R. Pouttu, unpublished data). Our ongoing work has shown that silencing of rpoS does not alone lead to expression of the Mat fimbria. The regulation of the Mat fimbria expression appears complex and is particularly interesting because it seems to be a clonal property in E. coli.