Cloning and expression of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans.
The fructose-1,6-diphosphate-dependent lactate dehydrogenase from Streptococcus mutans JH1000 was purified by a modification of published methods. The sequence of 27 amino-terminal amino acids was determined, which allowed us to construct a 17-base DNA probe that had 32-fold degeneracy. The probe was used to screen a genomic library in pBR322. Of 18 reactive clones, 1 was found that expressed lactate dehydrogenase (LDH) activity identical to that of S. mutans with regard to dependence on fructose-1,6-diphosphate, thermal inactivation profile, and inhibition by sodium oxamate. Extracts of this clone possessed a protein band that comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with purified LDH from JH1000. Compared with controls, the clone was shown to produce elevated amounts of L-(+)-lactic acid during growth in the presence of glucose, thereby indicating that the activity was expressed in vivo. This result was substantiated by demonstrating that the activity could complement a mutation in the fermentative D-(-)-LDH of Escherichia coli. Subcloning showed that the S. mutans LDH subunit is encoded by a 1.2-kilobase gene. Our ability to clone this gene is expected to have great practical significance in the construction of an effector strain for use in the replacement therapy of dental caries.
Hillman
J D
JD
Forsyth Dental Center, Boston, Massachusetts 02115.
Duncan
M J
MJ
Stashenko
K P
KP
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005635:Fructosediphosphates; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015183:Restriction Mapping; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D005635:Fructosediphosphates; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate