Infect Immun. 1996 Oct; 64(10): 4319-23
Hillman JD, Chen A, Snoep JL

CH4ts is a previously isolated recombinant mutant of Streptococcus mutans NG8 which produces a thermolabile L-(+)-lactate dehydrogenase (LDH) activity. It does not grow at 42 degrees C under a variety of cultivation conditions. In this study, we show that a batch culture of CH4ts shifted from 30 to 42 degrees C underwent rapid cessation of growth and accelerated cell death. The mutant grew at 42 degrees C in continuous culture under glucose-limiting conditions. Under these conditions, lactate production was replaced by production of ethanol and, to a smaller extent, acetoin. The cloned Zymomonas mobilis gene for alcohol dehydrogenase II, placed under the control of the S. mutans spaP regulatory signals, complemented LDH deficiency. The alcohol dehydrogenase-complemented mutant grew as well or better than NG8 on a variety of carbon sources at 42 degrees C and produced significant amounts of ethanol in place of lactic acid. These results are in accord with other approaches indicating that S. mutans has other enzymatic activities, including pyruvate formate-lyase and pyruvate dehydrogenase, for pyruvate metabolism. However, at high glucose concentrations, the levels of activity of these enzymes are apparently insufficient to compensate for the absence of LDH.

Ann Neurol. 1994 Oct; 36(4): 661-5
Tsujino S, Shanske S, Brownell AK, Haller RG, DiMauro S

We identified two new mutations in 2 white patients with muscle lactate dehydrogenase deficiency. Both patients had exercise intolerance, cramps, and recurrent myoglobinuria. One patient was homozygous for a 2-bp deletion in exon 5, resulting in a frameshift with premature termination of translation. The second patient was homozygous for a G-->A substitution at the 3' end of exon 2, leading to exon skipping and splicing of exon 1 to exon 3; the aberrantly spliced messenger RNA contains a frameshift, resulting in premature termination of translation. The present report provides evidence of molecular genetic heterogeneity in white patients with muscle lactate dehydrogenase deficiency.

J Bacteriol. 1994 Mar; 176(5): 1542-5
Chen A, Hillman JD, Duncan M

The previously cloned gene for L-(+)-lactate dehydrogenase (LDH) from Streptococcus mutans was mutagenized in vitro. An Escherichia coli transformant which expressed a thermolabile LDH activity was identified. The ldh(Ts) gene was introduced into S. mutans on a suicide vector to create a heterodiploid expressing both wild-type and thermolabile LDH activities. Self-recombinants which had only one ldh gene were isolated. One of these clones expressed only the thermolabile LDH activity. This isolate grew well at 30 degrees C but did not grow at 42 degrees C under a variety of cultivation conditions, thereby proving that LDH deficiency is lethal in S. mutans in the absence of compensatory mutations.

Infect Immun. 1994 Jan; 62(1): 60-4
Hillman JD, Chen A, Duncan M, Lee SW

In order to construct an effector strain for the replacement therapy of dental caries, we wished to combine the properties of low-level acid production and high-level colonization potential in a strain of Streptococcus mutans. To this end, we made a deletion in the lactate dehydrogenase (LDH) gene cloned from the bacteriocin-producing S. mutans strain JH1000. However, we were unable to substitute the mutant for the wild-type allele by transformation with linear DNA fragments. The mutated gene, carried on a suicide vector, was shown by Southern analysis to 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 analysis showed that a loss of tetracycline resistance was accompanied by a loss of the mutated LDH gene, resulting in restoration of the wild-type genotype. However, screening the same number of colonies for self-recombinants that did not make lactic acid during anaerobic growth in Todd-Hewitt broth failed to identify clones in which the wild-type allele 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 interpreted as indicating that LDH deficiency is lethal in S. mutans under the cultivation conditions used in these experiments. The physiological bases for this hypothesis are described.

J Dent Res. 1989 Jul; 68(7): 1155-61
Kulkarni GV, Chan KH, Sandham HJ

Restriction endonuclease analysis (REA) was performed on the total cellular DNA from each of 396 strains of mutans streptococci (1) to determine its potential usefulness for the study of transmission of the organism and (2) to document the proportions and variety of strains harbored by members of a small group of families. The DNA was digested with restriction enzyme EcoRI and/or HindIII, electrophoresed on agarose gels, and the resulting patterns compared. The strains examined included fresh isolates from 58 subjects, including 19 strains from each member of five families. The sensitivity and reproducibility of REA patterns from the mutans streptococci seemed ideal for studies of their epidemiology and transmission. The pattern of each isolate from humans was unique, except for isolates from the same individual or from the same family. REA types from subjects from different families were always heterogeneous. A high frequency of multiple REA types (up to 5) was observed in many subjects. While evidence for intra-familial transmission was obtained, including transmission between spouses, there was also strong evidence of frequent sources of infection outside of the family. Mutations of strains to streptomycin resistance or to lactate dehydrogenase deficiency caused no detectable change in the REA patterns. The lack of plasmids in any of the 57 fresh isolates that were examined for them suggested that they may have contributed little to the heterogeneity of the patterns seen.

J Dent Res. 1985 Nov; 64(11): 1267-71
Abhyankar S, Sandham HJ, Chan KH

Three lactate-dehydrogenase-deficient mutants of serotype c S. mutans were made by using, as parents, two serotype c strains that produced unusually large amounts of ethanol, acetic acid, and acetoin, and very little lactic acid, when grown in broth containing a limiting amount of glucose. The mutants, obtained with N-methyl-N'-nitro-N-nitrosoguanidine, were stable during 12 weeks of daily subculture in broth. Crude cell-free extracts of the mutants had less than 1% of the LDH-specific activity of their parent strains. The serotype c mutants resembled serotype g mutants in having molar growth yields at least as high as those of their parents. However, in contrast to the g mutants, the c mutants produced cell crops (cell mass per ml medium) that were as high as those of their parent strains.

Ann Intern Med. 1985 Aug; 103(2): 245-57
Valentine WN, Tanaka KR, Paglia DE

The human erythrocyte generates high-energy adenosine triphosphate by anaerobic glycolysis and cycles oxidized and reduced nicotinamide adenine dinucleotide phosphate by the aerobic pentose phosphate shunt pathway. Certain enzymopathies of the pentose phosphate shunt are associated with hemolysis resulting from oxidative denaturation of hemoglobin. Glucose-6-phosphate dehydrogenase deficiency, an X-chromosome-linked disorder, is the prototype of these diseases and is genetically and clinically polymorphic. Six enzymopathies of anaerobic glycolysis cause hemolytic anemia; lactate dehydrogenase deficiency does not. In 2,3-diphosphoglycerate mutase deficiency, 2,3-diphosphoglycerate is greatly reduced and asymptomatic polycythemia is noted. Pyrimidine-5'-nucleotidase deficiency, an enzymopathy of nucleotide metabolism, is characterized by intracellular accumulations of pyrimidine-containing nucleotides, marked basophilic stippling on the stained blood film, splenomegaly, and hemolysis. Lead inhibits the nucleotidase and an identical syndrome occurs during severe lead poisoning. Hemolysis also accompanies an unusual enzymopathy characterized by a 40- to 70-fold increase (not decrease) in adenosine deaminase activity.