Genetic and physiological analysis of the lethal effect of L-(+)-lactate dehydrogenase deficiency in Streptococcus mutans: complementation by alcohol dehydrogenase from Zymomonas mobilis.

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.     

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.     

DNA sequence and in vitro mutagenesis of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans.

Previously, the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase gene of Streptococcus mutans JH1000 was cloned into Escherichia coli (J. D. Hillman, M. J. Duncan, and K. P. Stashenko, Infect. Immun. 58:1290-1295, 1990). In the present study, the nucleotide sequence of 1.29 kb of S. mutans DNA which contained the promoter and protein-coding region of the gene was determined. In vitro disruption of the gene was achieved by deletion of the promoter and a major portion of the protein-coding sequence. Subsequently, a tetracycline resistance gene from S. mutans was inserted at the deletion site as a marker for selection. In addition, evidence from Southern hybridization showed that S. mutans JH1000 contained a single copy of the lactate dehydrogenase gene.     

Oxidant stress in Neisseria gonorrhoeae: adaptation and effects on L-(+)-lactate dehydrogenase activity.

Neisseria gonorrhoeae, an obligate human pathogen, is subjected to oxidant stress when attacked by O2 reduction products formed by neutrophils. In this study, exposure of gonococci to sublethal concentrations of superoxide and hydrogen peroxide (and related O-centered radicals) resulted in phenotypic resistance to oxidant stress. Adaptation required new protein formation but was not related to increases in superoxide dismutase or catalase. We have previously demonstrated that gonococci use phagocyte-derived L-(+)-lactate. Oxidant stress of greater magnitude than that required for adaptation led to a generalized increase in bacterial metabolism, particularly in L-(+)- and D-(-)-lactate utilization and lactate dehydrogenase activity. Increased lactate utilization required new protein synthesis. These results suggest the possibility that lactate metabolism is of importance to N. gonorrhoeae subjected to oxidant stress. Use of lct mutant organisms unable to use L-(+)-lactate should allow examination of this hypothesis.     

Cloning studies on the gene coding for L-(+)-lactate dehydrogenase of Plasmodium falciparum

We show that the L-(+)-lactate dehydrogenase (EC 1.1.1.27;L-lactate: NAD+-oxidoreductase) of Plasmodium falciparum (LDH-P) is encoded in the parasite genome. A monoclonal antibody (McAb 7.2) has been shown to bind the LDH-P subunit which has an apparent molecular mass of 35 kDa. A polyclonal antiserum raised against affinity purified LDH-P has been used to isolate cDNA clones containing LDH-P epitopes from a lambda gt11Tn5 expression library. DNA sequence analysis of one clone, lambda LDH-P.1, reveals a single open reading frame which shows a degree of homology to the N-terminal domain of known LDH amino acid sequences.     

Pyruvate dehydrogenase activity in Streptococcus mutans.

Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.     

Evidence that a low-affinity sucrose phosphotransferase activity in Streptococcus mutans GS-5 is a high-affinity trehalose uptake system.

High-affinity sucrose uptake in the oral pathogen Streptococcus mutans is mediated by the phosphoenolpyruvate-dependent phosphotransferase system. In this report, we provide evidence that a lower-affinity sucrose phosphotransferase system in S. mutans GS-5, previously described by others, is in fact a high-affinity trehalose uptake system that also recognizes sucrose as a substrate.     

Evidence for a disseminated plasmid in Streptococcus mutans

Based on a survey of 86 isolates, approximately 5% of all naturally occurring strains of Streptococcus mutans contains a 3.6 X 10(6)-dalton (3.6-megadalton) multicopy plasmid of unknown function. The amount of plasmid deoxyribonucleic acid per chromosome varies from 2 to 6% depending on the host strain. About 13% of the total covalently closed circular deoxyribonucleic acid in each of the four plasmid-containing strains consists of dimeric molecules, with interlocked circular forms predominating. Site-specific restriction endonucleases have been identified that cleave this 3.6-megadalton plasmid at single and at multiple sites. Each of the four plasmids is cleaved once by the HindIII and BamHI restriction enzymes. The HpaI, TaqI, and HhaI enzymes generate two, five, and six components, respectively, and the digestion products of each of the four plasmids are identical. Because the four plasmid-containing S. mutans strains are physiologically unique with respect to one another, we conclude this plasmid to be a disseminated extrachromosomal element in S. mutans.     

A Streptococcus mutans mutant that synthesizes elevated levels of intracellular polysaccharide is hypercariogenic in vivo.

We used the streptococcal transposon, Tn916 to identify and isolate mutants of Streptococcus mutans with altered intracellular polysaccharide (IPS) accumulation. We report on the isolation and characterization of S. mutans SMS202, a transposon mutant which accumulated the glycogen-like IPS in excess of wild-type levels. Southern blot analysis confirmed a single Tn916 insertion into the SMS202 chromosome. Moreover, quantitative ultrastructural analysis revealed significantly increased concentrations of IPS in SMS202 relative to those of the wild-type progenitor strain, UA130. The activities of ADPglucose pyrophosphorylase (GlgC) and glycogen synthase (GlgA), enzymes required for the biosynthesis of bacterial IPS, were also elevated in the IPS excess mutant. Furthermore, SMS202 was significantly more cariogenic on the molar surfaces of germ-free rats than the wild type (P < 0.01), thus confirming a central role for IPS in S. mutants-induced caries formation. We propose that the increased cariogenic potential of SMS202 is due to constitutive expression of genes which encode glycogen biosynthesis in this oral pathogen. The coordinate expression of GlgC and GlgA along with the results of ongoing nucleotide sequence analysis and Northern hybridization experiments support an operon-like arrangement for the glg genes of this oral pathogen.     

Lactate dehydrogenase mutants of Streptococcus mutans: isolation and preliminary characterization.

Mutants of Streptococcus mutans were isolated which lack the enzyme activity L (+)-lactate dehydrogenase. Reversion studies indicate that the genetic defects are in the structural gene for the enzyme. The mutants produce less titratable acid from glucose, adhere better to hydroxyapatite, and accumulate more plaque when grown in the presence of sucrose than does the parent strain. These findings suggest a possible use for the mutants as effector strains in the replacement therapy of dental caries.     

Expression of Streptococcus mutans gtf genes in Streptococcus milleri.

The Streptococcus mutans glucosyltransferase (GTF) genes gtfB and gtfC were ligated into Escherichia coli-streptococcus shuttle plasmids and introduced into Streptococcus milleri. gtfB transformant KSB8 formed an S. mutans-like rough colony on mitis salivarius agar and expressed an extracellular GTF-I, of 158 kDa, and two cell-bound GTF-Is, of 158 and 135 kDa. gtfC transformant KSC43 formed a semirough colony on mitis salivarius agar and expressed primarily an extracellular GTF-SI, of 146 kDa, and two cell-bound GTF-SIs, of 146 and 152 kDa. The extracellular GTFs from KSB8 and KSC43 were purified and characterized. The two types of GTF also reacted specifically with monoclonal antibodies directed against each enzyme. Both enzymes synthesized significant amounts of oligosaccharides, consisting primarily of alpha-1,6-glucosidic linkages, as well as water-insoluble glucans, containing alpha-1,3-glucosidic linkages. Insoluble-glucan-synthesizing activities of both enzymes were stimulated (three- to sixfold) by the addition of dextran T10 and were inhibited in the presence of 1.5 M ammonium sulfate. The Km(s) for sucrose and the optimal pHs were also similar for both enzymes. However, when the transformants were grown in Todd-Hewitt broth supplemented with sucrose, KSC43 cells, expressing GTF-SI activity, adhered to glass surfaces in vitro, while KSB8 cells, expressing GTF-I activity, did not. These results are discussed relative to the potential role of the gtfB and gftC genes in S. mutans cariogenicity.     

Evidence for an immunological relationship between Streptococcus mutans and human cardiac tissue.

Two-dimensional immunoelectrophoresis, indirect immunofluorescence, and radioimmunoassay were used to demonstrate that antisera from rabbits immunized with some strains of Streptococcus mutans contain antibodies that cross-react with human cardiac tissue. These rabbits were sensitized to a shocking dose of human heart antigen, and anaphylactic deaths were sometimes produced. Myocarditis was also a result of the immunization procedure. Data obtained with all five techniques were comparable. Cross-reactivity could be associated with three antigens designated ID, IF, and HL. Antigens ID and IF were major immunogens of S. mutans Ingbritt, but HL antibodies were produced only after hyperimmunization. Corss-reactivity was of an immunological nature and not the result of nonspecific factors such as bacterial Fc reactive components or antibody elicited to growth medium constituents. These findings support the hypothesis that immunization with S. mutans can induce autoimmune reactions and indicate that antigens must be selected with caution before formulating any dental caries vaccine.     

Plasmid-mediated transformation of Streptococcus mutans.

Streptococcus mutans GS-5 was transformed to erythromycin resistance with streptococcal plasmid pVA736. Transformation frequencies were higher with plasmids reisolated from transformed GS-5 cells relative to plasmid originally derived from S. sanguis Challis.     

Antigens of Streptococcus mutans I. Characterization of a Serotype-Specific Determinant from Streptococcus mutans

A membrane-associated glycerol teichoic acid antigen has been isolated from Streptococcus mutans AHT and a similar antigen has been demonstrated to be present in each of the other Bratthall serotype a organisms studied. Trichloroacetic acid-extracted material was resolved into two phosphorus-containing antigenic fractions (B and C) by agarose chromatography. Fraction B was preliminarily identified as a phospholipid moiety with a glycerol-to-phosphorus ratio of 2:1, and fraction C showed a ratio of 1:1 indicative of a glycerol teichoic acid. This latter fraction also was associated with glucose, galactose, alanine, and fatty acids. Diglycerol triphosphate, the compound characteristically released from 1-3 phosphodiester-linked glycerol teichoic acids by alkaline hydrolysis, was isolated and characterized. Alanine was identified as its alkaline-labile, ester-linked D-isomer. A glyceride was isolated containing a disaccharide of glucose and galactose attached to the 2-hydroxyl group of glycerol. Hapten inhibition analysis demonstrated that beta-galactosides were the greatest inhibitors of the precipitin reaction (>75%), whereas glucose and its derivatives inhibited to a much lesser extent (<30%). Comparative immunodiffusion and immuno-electrophoresis analyses demonstrated that all six Bratthall serotype a organisms tested contained this antigenic determinant and that it was absent in serotypes b, c, and d. It is suggested that the common antigenic determinant of this serotype within S. mutans may be a beta-galactoside associated with a glycerol teichoic acid and possibly other polymers.     

变异链球菌临床株乳酸脱氢酶mRNA水平的表达研究

目的 分析来自不同龋敏感人群变异链球菌不同基因型乳酸脱氢酶（LDH）的基因表达差异,探讨mRNA水平上基因表达与蛋白生物学功能及细菌致龋性的关系.方法 应用半定量RT-PCR两步法和凝胶成像系统定量软件分析,分别对20株来自不同基因型的LDH进行基因表达水平差异的评价和比较.结果 两种不同基因型LDH的mRNA表达水平不同（P＜0.05）,表现为低酶活性的B基因型1dh表达高;高龋和无龋菌株1dh表达的差异无统计学意义（P＞0.05）.结论 变异链球菌乳酸脱氢酶基因表达水平具有遗传异质性,产酸因子1dh基因表达高低与龋病活跃性不呈正相关关系.     

Purification of a Streptococcus mutans protein that binds to heart tissue and glycosaminoglycans.

Proteins of Streptococcus mutans MT703 were isolated by differential filtration from chemically defined culture medium following growth of the bacteria. Incubation of this preparation with cryostat-cut sections of fresh rabbit cardiac muscle resulted in deposition of streptococcal components on basement membranes of sarcolemmal sheaths and capillary walls, as indicated by indirect immunofluorescence assay. Binding of radioiodinated streptococcal proteins to heart in vitro was time dependent and saturable. Unlabeled S. mutans proteins competitively inhibited 72% of heart binding by the radiolabeled proteins, indicating a high level of binding specificity. A selection of components common to tissue basement membranes was tested for their abilities to inhibit the binding of streptococcal proteins to heart tissue. Of the glycosaminoglycans, heparin was the most effective inhibitor, followed by heparan sulfate and chondroitin sulfate. Hyaluronic acid was not inhibitory. Of the glycoproteins tested, laminin and collagen type IV were weakly inhibitory, whereas fibronectin was ineffective. A single polypeptide was purified to homogeneity by affinity chromatography on a column of heparin-agarose. Gel filtration chromatography of the purified protein under nondissociating conditions showed a single component at 31 kilodaltons (kDa), whereas in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one band appeared at 8 kDa. This indicates that the tissue-binding protein may either be a linear polypeptide or be released into the environment by the bacterium as a tetramer of the 8-kDa polypeptide. The purified protein had an isoelectric point of 9.5 and showed binding activity for basement membranes in thin sections of heart. Chemical analyses of the purified binding protein showed it to have high contents of lysine and alanine and to be devoid of half-cystine, methionine, tyrosine, histidine, and both neutral and amino sugars.     

Linkage of sucrose-metabolizing genes in Streptococcus mutans.

Antibiotic resistance markers inserted adjacent to different cloned genes from Streptococcus mutans were used to determine the relative positions of these genes on the chromosome. The results showed that these genes, fru-1 and gbp, are closely linked to the gtfA-ftf-scrB cluster. However, gtfD was linked neither to this cluster nor to gtfB-gtfC.