Mannitol transport in Streptococcus mutans.

A hexitol-inducible, phosphoenolpyruvate-dependent phosphotransferase system was demonstrated in Streptococcus mutans. Cell-free extracts obtained from mannitol-grown cells from a representative strain of each of the five S. mutans serotypes (AHT, BHT, C-67-1, 6715, and LM7) were capable of converting mannitol to mannitol-1-phosphate by a reaction which required phosphoenolpyruvate and Mg2+. Mannitol and sorbitol phosphotransferase activities were found in cell-free extracts prepared from cells grown on the respective substrate, but neither hexitol phosphotransferase activity was present in extracts obtained from cells grown on other substrates examined. A heat-stable, low-molecular-weight component was partially purified from glucose-grown cells and found to stimulate the mannitol phosphotransferase system. Divalent cations Mn2+ and Ca2+ partially replaced Mg2+, while Zn2+ was found to be highly inhibitory.     

MsmE, a lipoprotein involved in sugar transport in Streptococcus mutans.

Metabolic labelling by [14C]palmitic acid showed that growth of Streptococcus mutans LT11 in raffinose, an inducer of the msm operon, resulted in increased production of a 45-kDa lipoprotein corresponding to MsmE, which is believed to be a sugar-binding protein. MsmE was also labelled when an msmE clone was expressed in Escherichia coli. The presence of a lipid anchor on MsmE provides a likely explanation of how the sugar-binding protein component of the msm binding protein-dependent multiple sugar transport system is retained at the cell surface.     

Ferrous iron transport mutants in Escherichia coli K12

A ferrous iron transport system in Escherichia coli is described. Mutants in this transport system were isolated using the antibiotic streptonigrin. The gene locus feo (for ferrous iron transport) was mapped near pncA at 38.5 min on the genetic map of E. coli K12. The transport of ferrous iron was regulated by fur as the siderophore transport systems.     

Oxygen toxicity in Streptococcus mutans: manganese, iron, and superoxide dismutase.

When cultured anaerobically in a chemically defined medium that was treated with Chelex-100 to lower its trace metal content, Streptococcus mutans OMZ176 had no apparent requirement for manganese or iron. Manganese or iron was necessary for aerobic cultivation in deep static cultures. During continuous aerobic cultivation in a stirred chemostat, iron did not support the growth rate achieved with manganese. Since the dissolved oxygen level in the chemostat cultures was higher than the final level in the static cultures, manganese may be required for growth at elevated oxygen levels. In medium supplemented with manganese, cells grown anaerobically contained a low level of superoxide dismutase (SOD) activity; aerobic cultivation increased SOD activity at least threefold. In iron-supplemented medium, cells grown anaerobically also had low SOD activity; aerobic incubation resulted in little increase in SOD activity. Polyacrylamide gel electrophoresis of the cell extracts revealed a major band and a minor band of SOD activity in the cells grown with manganese; however, cells grown with iron contained a single band of SOD activity with an Rf value similar to that of the major band found in cells grown with manganese. None of the SOD activity bands were abolished by the inclusion of 2 mM hydrogen peroxide in the SOD activity strain. S. mutans may not produce a separate iron-containing SOD but may insert either iron or manganese into an apo-SOD protein. Alternatively, iron may function in another activity (not SOD) that augments the defense against oxygen toxicity at low SOD levels.     

A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism.

An 11-kilobase gene region of Streptococcus mutans has been identified which contains eight contiguous genes involved with the uptake and metabolism of multiple sugars (the msm system). Sequence analysis of this region indicates that several of these genes specify proteins with strong homology to components of periplasmic binding protein-dependent transport systems of Gram-negative bacteria. Additionally, this operon is controlled by a regulatory gene (msmR) that acts as a positive effector. The proteins specified by the structural genes of the msm operon include alpha-galactosidase (aga), a "periplasmic-like" sugar-binding protein (msmE), two membrane proteins (msmF, msmG), sucrose phosphorylase (gtfA), an ATP-binding protein (msmK), and dextran glucosidase (dexB). Insertional inactivation of each of these genes along with uptake data indicate that this system is responsible for the uptake of melibiose, raffinose, and isomaltotriose and the metabolism of melibiose, sucrose, and isomaltosaccharides.     

Dual mechanism for stimulation of glutamate transport by potassium ions in Streptococcus mutans.

An ATP-driven primary transport system operative for L-glutamate or L-aspartate in Streptococcus mutans is, through the entire pH range from 5.5 to 8.5, specifically stimulated by extracellular potassium ions. The stimulation by potassium ions observed in the low pH range between 5.5 and 7 has been interpreted to be due to potassium ion-dependent regulation of the intracellular pH (the first mechanism). In the high pH range from 7 to 8.5, on the other hand, the present study demonstrates that potassium stimulation is essentially not associated with such intracellular pH regulation. This conclusion is based on our observation that potassium stimulation in the high pH range is insensitive to a proton conductor, carbonyl cyanide-p-trifluoromethoxy-phenyl-hydrazone. Since none of the other monovalent cations, including sodium, rubidium, ammonium, and Tris ions, could replace potassium ions in significantly stimulating glutamate transport, it is most likely that the influx of potassium ions specifically cancels the membrane potential derived by movement of glutamate with the net negative charges across a membrane and thus facilitates transport (the second mechanism). The second mechanism appears to be operative even in a low pH range, in addition to the first mechanism.     

Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans

Streptococcus mutans, a gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries. Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear. The data reported in this study indicates that iron-deprived saliva (Fe3+ < 0.1 microM) increases S. mutans aggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+ from 0.1 to 1 microM), while iron-loaded saliva (Fe3+ > 1 microM) inhibits both phenomena. Our findings are consistent with the hypothesis that S. mutans aggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 microg/ml) in the apo- or iron-saturated form. Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases. On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S. mutans. Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S. mutans counteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity. In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S. mutans free, aggregated or biofilm on abiotic surfaces. Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S. mutans pathogenicity.     

Regulation of hexitol catabolism in Streptococcus mutans.

Regulation of hexitol catabolism was investigated in Streptococcus mutans, a cariogenic human dental plaque bacterium. Induction of hexitol catabolic enzymes and phosphoenolpyruvate:hexitol phosphotransferase and hexitol phosphate dehydrogenase activities was regulated by an inducer exclusion mechanism initiated by D-glucose and 2-deoxy-D-glucose. Kinetic analysis of the inhibitory effect of 2-deoxy-D-glucose on initial hexitol uptake illustrated that this was a noncompetitive type of inhibition. In mutant strains of S. mutans lacking phosphoenolpyruvate:glucose phosphotransferase activity, 2-deoxy-D-glucose was unable to inhibit hexitol uptake. These observations provide evidence for possible molecular mechanisms for the exclusion process.     

Wall-associated protein antigens of Streptococcus mutans.

When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.     

Glycosyltransferases of Streptococcus mutans strain Ingbritt.

The glycosyltransferases of S. mutans strain Ingbritt have been resolved by SDS-polyacrylamide gel electrophoresis, followed by incubation in the presence of non-ionic detergent to restore enzyme activity. A group of high molecular weight proteins synthesizing glucans has been identified, as well as three distinct fructan-synthesizing activities. The glucan-forming enzymes have been purified by affinity chromatography on insoluble glucan, followed by gel chromatography in SDS, and antiserum to the purified enzymes has shown that they are antigenically identical within serotypes c, e and f, and cross-react strongly with serotype b.     

Genetic heterogeneity in Streptococcus mutans

The genetic homogeneity among eight cariogenic strains of Streptococcus mutans was assessed by deoxyribonucleic acid (DNA)-DNA reassociation experiments. DNA species were extracted from strains GS5, Ingbritt, 10449, FAl, BHT, E49, SLl, and KlR. Labeled DNA ((14)C-DNA) was extracted from strains 10449, FAl, and SLl. Denatured (14)C-DNA fragments were allowed to reassociate, i.e., form hybrid duplexes, with denatured DNA immobilized on membrane filters incubated in 0.45 m NaCl-0.045 m sodium citrate at 67 or 75 C. At 67 C, 10449 (14)C-DNA reassociated extensively only with GS5 and Ingbritt DNA. FAl (14)C-DNA hybridized extensively only with BHT DNA, and SLl (14)C-DNA reassociated with KlR and E49 DNA. DNA which hybridized extensively at 67 C also reassociated to a high degree at 75 C. Thermal elution of (14)C-FAl-BHT duplexes showed that the hybrid duplexes were thermostable. The results indicate that S. mutans is a genetically heterogeneous species. The strains studied can be divided into three (possibly four) genetic groups, and these groups closely parallel antigenic groups.     

Streptococcus mutans murein hydrolase

Allelic replacement of the C terminus of a Streptococcus mutans surface protein affects murein hydrolase activity. The targeted open reading frame encodes a 67-kDa protein (SmaA) with an N-terminal signal sequence and cleavage site, three 46-amino-acid (aa) direct repeats, and two 88-aa direct repeats. The identical autolytic profile was obtained using a sortase mutant (SrtA(-)).     

Glucose-6-phosphate-dependent pyruvate kinase in Streptococcus mutans.

Pyruvate kinase of Streptococcus mutans JC 2 had an absolute and specific requirement for glucose-6-phosphate. Inorganic phosphate was a strong inhibitor. The enzyme required K+ or NH4+ and Mg2+ or Mn2+. S. mutans FIL and E 49, Streptococcus bovis ATCC 9809, and Streptococcus salivarius ATCC 13419 had also glucose-6-phosphate-dependent pyruvate kinases, whereas Streptococcus sanguis NCTC 10904 had an enzyme activated by fructose-1,6-diphosphate.     

A solute binding protein of Streptococcus pneumoniae iron transport

Streptococcus pneumoniae causes considerable morbidity and mortality throughout the world. Iron acquisition is an important virulence factor for bacterial pathogens. Two loci, piu and pia, were identified as responsible for the hemoglobin utilization of S. pneumoniae. The binding activity and surface accessibility of the solute binding protein of PiuA were studied. PiuA is a lipoprotein, binds hemin and hemoglobin, resides on the cytoplasmic membrane, and is not exposed on the surface of S. pneumoniae. The localization of PiuA has implications in its role in hemoglobin utilization and possible use as a pneumococcal vaccine.     

Glucan-binding proteins of Streptococcus mutans serotype c.

Three glucan-binding proteins have been isolated from the extracellular fluid cultures of Streptococcus mutans serotype c. These proteins were adsorbed to glucans containing 1,3-alpha or 1,6-alpha bonds and linked to various chromatographic supports: they were eluted from columns by a dextran solution. Glucosyltransferase activity was associated with two of the glucan-binding proteins.     

Ferrous iron content of intravenous iron formulations

The observed biological differences in safety and efficacy of intravenous (IV) iron formulations are attributable to physicochemical differences. In addition to differences in carbohydrate shell, polarographic signatures due to ferric iron [Fe(III)] and ferrous iron [Fe(II)] differ among IV iron formulations. Intravenous iron contains Fe(II) and releases labile iron in the circulation. Fe(II) generates toxic free radicals and reactive oxygen species and binds to bacterial siderophores and other in vivo sequestering agents. To evaluate whether differences in Fe(II) content may account for some observed biological differences between IV iron formulations, samples from multiple lots of various IV iron formulations were dissolved in 12 M concentrated HCl to dissociate and release all iron and then diluted with water to achieve 0.1 M HCl concentration. Fe(II) was then directly measured using ferrozine reagent and ultraviolet spectroscopy at 562 nm. Total iron content was measured by adding an excess of ascorbic acid to reduce Fe(III) to Fe(II), and Fe(II) was then measured by ferrozine assay. The Fe(II) concentration as a proportion of total iron content [Fe(III) + Fe(II)] in different lots of IV iron formulations was as follows: iron gluconate, 1.4 and 1.8 %; ferumoxytol, 0.26 %; ferric carboxymaltose, 1.4 %; iron dextran, 0.8 %; and iron sucrose, 10.2, 15.5, and 11.0 % (average, 12.2 %). The average Fe(II) content in iron sucrose was, therefore, ≥7.5-fold higher than in the other IV iron formulations. Further studies are needed to investigate the relationship between Fe(II) content and increased risk of oxidative stress and infections with iron sucrose.     

Galactose Metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.