THE FIMA LOCUS OF STREPTOCOCCUS-PARASANGUIS ENCODES AN ATP-BINDING MEMBRANE-TRANSPORT SYSTEM

The gene encoding fimA, a 36 kDa fimbrial adhesin of Streptococcus par asanguis FW213, is highly conserved in all four genetic groups of sang uis streptococci, FimA-like peptides were produced by all strains test ed. The nucleotide sequence directly upstream of fimA contains two ope n reading frames, ORF5 and ORF1, whose deduced protein products are ho mologous to members of a superfamily of ATP-binding cassette membrane transport proteins, including both prokaryotic and eukaryotic uptake a nd export systems. The amino acid sequence of FimA contains the consen sus prolipoprotein cleavage site (LxxC) common to the 'periplasmic' bi nding proteins of Gram-positive transport systems, The deduced product of ORF5 is a 28,6 kDa membrane-associated protein that has the consen sus binding site for ATP (GxxGxGKS). It shares significant homology wi th AmiE of Streptococcus pneumoniae as well as with Escherichia coli p roteins involved in iron(lll) uptake. Allelic-replacement mutagenesis of ORF5 resulted in greatly increased resistance to aminopterin. These data demonstrate functionality with the amiE locus as well, The deduc ed product of ORF1 is an extremely hydrophobic integral membrane prote in of 30.8 kDa with a pattern of six potential membrane-spanning regio ns, typical of a component of these types of transport system. The nuc leotide sequence downstream of fimA, ORF3, encodes a 20 kDa protein ha ving 78% identity with the 20 kDa protein encoded downstream of ssaB, a fimA homologue in S. sanguis 12. It also exhibits significant homolo gy with bacterioferritin co-migratory protein (Bcp) of E. coil K-12. A llelic-replacement mutagenesis in the fimA locus of FW213 showed that (i) expression of fimA was initiated at a site far upstream of the fim A start codon, and (ii) expression of fimA was not linked to expressio n of ORF3. Northern blots probed with internal fragments of ORF5, ORF1 , fimA or ORF3 hybridized to the same transcript of 3.3 kb, which sugg ested that these loci were transcribed as a polycistronic message, The ORF3 probe also hybridized to a 540 bp transcript consistent with the size of ORF3 alone and supportive of the mutagenesis data of non-link age. Strains mutated in fimA continued to produce fimbriae, indicating that FimA was not the fimbrial structural subunit. Immunoelectron mic roscopy revealed FimA was localized at the tips of the fimbriae of FW2 13. This is the first study that demonstrates that an adhesin which bi nds a bacterial cell to a substrate is associated with an ATP-binding cassette.