Identification and nucleotide sequence of the gene determining the adhesion capacity of Serratia marcescens

Three open reading frames, designated smfE, smfF, and smfG, within the mannose-resistant fimbria gene cluster of Serratia marcescens were identified. smfG, which is responsible for determining the receptor binding of S. marcescens, encodes a 280-amino-acid polypeptide with a typical prokaryotic signal sequence.

and smfG (Fig. 2). The smfG gene appears to code for an adhesin, since the transposon insertion into this gene led to formation of a hemagglutination-negative fimbriated mutant (Fig. 1). The smfG gene consists of an 840-nucleotide coding region with a presumed initiation codon (ATG), preceded by a consensus ribosome-binding sequence (15), at nucleotide position 1348. The sequence of the smfG gene, initiated at this position and terminated at the TAG triplet at position 2187, would code for a polypeptide of 280 amino acid residues. In the amino-terminal region, a typical prokaryotic signal peptide sequence (5) was detected.
To obtain further support for the notion that the smfG gene encodes an adhesin, a small deletion was introduced at * Corresponding author. the SacI site located within this prospective coding sequence. Plasmid pYM821, carrying the 12-kb DNA fragment at the DraI site of pACYC184 (3), was partially digested by SacI and treated with T4 DNA polymerase in the absence of nucleotide substrate to remove the 3' protruding ends. A plasmid clone with a deletion at the Sacd site within the smfG sequence was selected and designated pYM822 (Fig. 1). E. coli P678-54 cells harboring pYM822 did not exhibit MR hemagglutination; however, they did possess fimbriae, as revealed by electron microscopic observations as well as by aggregation with anti-MR fimbria monoclonal antibody (Table 1). Therefore, it is apparent that the smfG gene codes for a protein responsible for the adhesion capacity of MR fimbriae of S. marcescens.
The primary structure of the SmfG protein is homologous to the structure of the PapG protein of E. coli (10), especially in the carboxyl-terminal regions (Fig. 3) Fig. 2) is enlarged, and detailed restriction sites are given in the middle of the figure. Plasmids pYM141 and pYM821 carry the same wild-type DNA fragment at different sites on the pACYC184 plasmid. Plasmid pYM1085 carries the Tn3 transposon (V), whereas pYM822 possesses a deletion at the Sacd site of the smfG gene (V). MRHA, MR hemagglutination; Fim., fimbria formation. distance between two carboxyl-terminal cysteine residues of The adhesin gene (papG) for E. coli Pap pili is the furthest the SmfG and the PapG proteins is 31 residues, which is the downstream of the pap gene cluster (10). The smfG gene, same for the two cysteine residues in the major and the coding for the adhesin of MR fimbriae of S. marcescens, is minor fimbrial proteins (2,9,13,14). SmfG and PapG have a also located the most promoter distal of the smffgene cluster. glycine residue 14 amino acid positions from the carboxyl Upstream of the smfG gene there are two open reading terminus, as was found in the major and the minor E. coli frames, designated smfE and smpl (Fig. 2). Both of the (Pap and type 1, respectively) (2,9,14) and S. marcescens coding sequences are preceded by the consensus sequence fimbrial proteins (Fig. 3 and 4).
for the ribosomal binding site (15) and are probably actively NOTES 3259 L broth agar at 37'C and suspended in phosphate-buffered saline (PBS). b MRHA, MR hemagglutination. To examine MRHA, chicken erythrocytes were suspended at a concentration of 2% in PBS containing 1% D-mannose and mixed with the bacterial suspension on a glass slide. c To determine agglutination by antibody, a bacterial suspension was mixed with anti-MR fimbria monoclonal antibody on a glass slide. All agglutination tests were read within a few minutes.
d Fimbriae present on the bacterial surface were identified by electron microscopy.
expressed. The prospective sizes of the SmfE and SmfF proteins, calculated from the nucleotide sequence, were 163 and 180 amino acids, respectively, and the proteins also carried the typical prokaryotic signal peptide sequence (5) at the amino-terminal regions.
The primary structures of the SmfE and SmfF proteins are homologous to the structure of the SmfA protein (13), which is the major fimbrial subunit of S. marcescens (Fig. 4). It is most likely that the SmfE and SmfF proteins are minor fimbrial subunits, as is the case with the PapE and PapF proteins of Pap fimbriae (8,9).
The sizes and arrangements of the six genes within the smf gene cluster of S. marcescens are similar to those of the E. coli pap fimbria gene cluster. The primary structures of major and minor fimbrial subunits of S. marcescens show a high homology to those of the Pap fimbrial subunits. As for adhesin, there is a homology between MR fimbriae of S.   (13). Amino acid positions are numbered relative to the SmfA sequence. Amino acids are given in the standard one-letter code. Sequences with identical amino acids or conservative amino acid replacements are boxed. Conservative replacements are defined as being within the following groups: D and E; K and R; S and T; F, Y, and W; and I, L, V, and M. Gaps, indicated by dashes, are introduced in order to obtain a maximum fit.
PapG was found to be required for incorporation into the pilus (4).
These results imply that genes encoding the S. marcescens MR fimbriae and the E. coli Pap fimbriae may have evolved from a common ancestor. While genes for the fimbrial structure and the region of the adhesin gene necessary for pilus assembly may have been conserved, the unidentified region related to the binding specificity in the adhesin gene may have changed. Differences in binding specificities of the adhesins may explain the difference in pathogenesis between S. marcescens and E. coli.