matB, a common fimbrillin gene of Escherichia coli, expressed in a genetically conserved, virulent clonal group

Abstract

A novel fimbrial type in Escherichia coli was identified and characterized. The expression of the fimbria was associated with the O18acK1H7 clonal group of E. coli, which cause newborn meningitis and septicemia when grown at low temperature; hence, it was named the Mat (meningitis associated and temperature regulated) fimbria. The fimbriae were purified from a fimA::cat sfaA::Gm fliC::St derivative of the O18K1H7 isolate E. coli IHE 3034. The purified Mat fimbrillin had an apparent molecular mass of 18 kDa and did not serologically cross-react with the type 1 or S fimbria of the same strain. The matB gene encoding the major fimbrillin was cloned from the genomic DNA of the fimA::cat sfaA::Gm fliC::St derivative of IHE 3034. The predicted MatB sequence was of 195 amino acids, contained a signal sequence of 22 residues, and did not show significant homology to any of the previously characterized fimbrial proteins. The DNA sequence of matB was 97.8% identical to a region from nucleotides 17882 to 18469 in the 6- to 8-min region of the E. coli K-12 chromosome, reported to encode a hypothetical protein. The 7-kb DNA fragment containing matB of IHE 3034 was found by restriction mapping and partial DNA sequencing to be highly similar to the corresponding region in the K-12 chromosome. Trans complementation of the matB::cat mutation in the IHE 3034 chromosome showed that matB in combination with matA or matC restored surface expression of the Mat fimbria. A total of 27 isolates representing K-12 strains and the major pathogroups of E. coli were analyzed for the presence of a matB homolog as well as for expression of the Mat fimbria. A conserved matB homolog was found in 25 isolates; however, expression of the Mat fimbriae was detected only in the O18acK1H7 isolates. Expression of the Mat fimbria was temperature regulated, with no or a very small amount of fimbriae or intracellular MatB fimbrillin being detected in cells cultivated at 37(o)C. Reverse transcriptase PCR and complementation assays with mat genes controlled by the inducible trc promoter indicated that regulation of Mat fimbria expression involved both transcriptional and posttranscriptional events.

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.

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.

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).

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.

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.