CLONING AND SEQUENCE-ANALYSIS OF THE PFL GENE ENCODING PYRUVATE FORMATE-LYASE FROM STREPTOCOCCUS-MUTANS

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS -5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobicall y. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental str ain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3 -kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same so rbitol-negative phenotype, Nucleotide sequence analysis of the pfl gen e revealed a 2,3-kb open reading frame (ORF) preceded by potential rib osome-binding and promoter-like sequences. The ORF specified a putativ e protein of 775 amino acid residues with a calculated molecular weigh t of 87,533. The amino acid sequence deduced from the ORF exhibited si gnificant similarity to that of the E. coli pfl gene.