EVIDENCE THAT L-(-LACTATE DEHYDROGENASE-DEFICIENCY IS LETHAL IN STREPTOCOCCUS-MUTANS())

In order to construct an effector strain for the replacement therapy o f dental caries, we wished to combine the properties of low-level acid production and high-level colonization potential in a strain of Strep tococcus mutans. To this end, we made a deletion in the lactate dehydr ogenase (LDH) gene cloned from the bacteriocin-producing S, mutans str ain JH1000. However, we were unable to substitute the mutant for the w ild-type allele by transformation with linear DNA fragments. The mutat ed gene, carried on a suicide vector, was shown by Southern analysis t o integrate into the JH1000 chromosome to yield transformants carrying both the wild-type gene and mutated LDH gene. Three spontaneous self- recombinants of one heterodiploid strain were isolated by screening 1, 500 colonies for a loss of the tetracycline resistance encoded by the gene used to mark the LDH deletion. In all three cases, Southern analy sis showed that a loss of tetracycline resistance was accompanied by a loss of the mutated LDH gene, resulting in restoration of the wild-ty pe genotype. However, screening the same number of colonies for self-r ecombinants that did not make lactic acid during anaerobic grow-th in Todd-Hewitt broth faded to identify clones in which the wild-type alle le was lost. A second, simpler screening of more than 80,000 colonies grown aerobically on glucose tetrazolium medium to identify low-level- acid-producing colonies was also unsuccessful. These results are inter preted as indicating that LDH deficiency is lethal in S. mutans under the cultivation conditions used in these experiments. The physiologica l bases for this hypothesis are described.