Transposon-encoded sucrose metabolism in Lactococcus lactis. Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1.
Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.
Thompson
J
J
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Nguyen
N Y
NY
Sackett
D L
DL
Donkersloot
J A
JA
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D004247:DNA; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008040:Genetic Linkage; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification*; D006868:Hydrolysis; D007525:Isoelectric Focusing; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D015183:Restriction Mapping; D013395:Sucrose / Q000235:genetics / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D004251:DNA Transposable Elements; D015345:Oligonucleotide Probes; D013395:Sucrose; D004247:DNA; D000594:Amino Acid Oxidoreductases; C064774:N(5)-(carboxyethyl)ornithine synthase; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase