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.     

Gene Regulation by the LiaSR Two-Component System in Streptococcus mutans

The LiaSR two-component signal transduction system regulates cellular responses to several environmental stresses, including those that induce cell envelope damages. Downstream regulons of the LiaSR system have been implicated in tolerance to acid, antibiotics and detergents. In the dental pathogen Streptococcus mutans, the LiaSR system is necessary for tolerance against acid, antibiotics, and cell wall damaging stresses during growth in the oral cavity. To understand the molecular mechanisms by which LiaSR regulates gene expression, we created a mutant LiaR in which the conserved aspartic acid residue (the phosphorylation site), was changed to alanine residue (D58A). As expected, the LiaR-D58A variant was unable to acquire the phosphate group and bind to target promoters. We also noted that the predicted LiaR-binding motif upstream of the lia operon does not appear to be well conserved. Consistent with this observation, we found that LiaR was unable to bind to the promoter region of lia; however, we showed that LiaR was able to bind to the promoters of SMU.753, SMU.2084 and SMU.1727. Based on sequence analysis and DNA binding studies we proposed a new 25-bp conserved motif essential for LiaR binding. Introducing alterations at fully conserved positions in the 25-bp motif affected LiaR binding, and the binding was dependent on the combination of positions that were altered. By scanning the S. mutans genome for the occurrence of the newly defined LiaR binding motif, we identified the promoter of hrcA (encoding a key regulator of the heat shock response) that contains a LiaR binding motif, and we showed that hrcA is negatively regulated by the LiaSR system. Taken together our results suggest a putative role of the LiaSR system in heat shock responses of S. mutans.     

Regulation of ATP-dependent P-(Ser)-HPr formation in Streptococcus mutans and Streptococcus salivarius.

Sugar transport via the phosphoenolpyruvate (PEP) phosphotransferase system involves PEP-dependent phosphorylation of the general phosphotransferase system protein, HPr, at histidine 15. However, gram-positive bacteria can also carry out ATP-dependent phosphorylation of HPr at serine 46 by means of (Ser)HPr kinase. In this study, we demonstrate that (Ser)HPr kinase in crude preparations of Streptococcus mutans Ingbritt and Streptococcus salivarius ATCC 25975 is membrane associated, with pH optima of 7.0 and 7.5, respectively. The latter organism possessed 7- to 27-fold-higher activity than S. mutans NCTC 10449, GS-5, and Ingbritt strains. The enzyme in S. salivarius was activated by fructose-1,6-bisphosphate (FBP) twofold with 0.05 mM ATP, but this intermediate was slightly inhibitory with 1.0 mM ATP at FBP concentrations up to 10 mM. Similar inhibition was observed with the enzyme from S. mutans Ingbritt. A variety of other glycolytic intermediates had no effect on kinase activity under these conditions. The activity and regulation of (Ser)HPr kinase were assessed in vivo by monitoring P-(Ser)-HPr formation in steady-state cells of S. mutans Ingbritt grown in continuous culture with limiting glucose (10 and 50 mM) and with excess glucose (100 and 200 mM). All four forms of HPr [free HPr, P approximately (His)-HPr, P-(Ser)-HPr, and P approximately (His)-P-(Ser)-HPr] could be detected in the cells; however, significant differences in the intracellular levels of the forms were apparent during growth at different glucose concentrations. The total HPr pool increased with increasing concentrations of glucose in the medium, with significant increases in the P-(Ser)-HPr and P approximately HHis)-P-(Ser)-HPr concentrations. For example, while total PEP-dependent phosphorylation [P approximately(His)-HPr plus P approximately (His)-P-(Ser)-HPr] varied only from 21.5 to 52.5 microgram mg of cell protein (-1) in cells grown at the four glucose concentrations, the total ATP-dependent phosphorylation [P-(Ser)-HPr plus P approximately (His)-P-(Ser)-HPr] increased 12-fold from the 10 mM glucose-grown cells (9.1 microgram mg of cell protein (-1) to 106 and 105 microgram mg(-1) in the 100 and 200 mM glucose-grown cultures, respectively. (Ser)HPr kinase activity in membrane preparations of the cells varied little between the 10, 50, and 100 mM glucose-grown cells but increased threefold in the 200 mM glucose-grown cells. The intracellular levels of ATP, glucose-6-phosphate, and FBP increased with external glucose concentration, with the level of FBP being 3.8-fold higher for cells grown with 200 mM glucose than for those grown with 10 mM glucose. However, the variation in the intracellular levels of FBP, particularly between cells grown with 100 and 200 mM glucose, did not correlate with the extent of P-(Ser)-HPr formation, suggesting that the activity of (Ser)HPr kinase is not critically dependent on the availability of intracellular FBP.     

Regulation of glucosyl- and fructosyltransferase synthesis by continuous cultures of Streptococcus mutans

Streptococcus mutans strains Ingbritt, and its derivative B7 which had been passaged through monkeys, have been used to investigate how the synthesis of extracellular glucosyl- and fructosyltransferases is regulated. The most active enzyme from carbon-limited continuous cultures was a fructosyltransferase; enzymes catalysing the formation of water-insoluble glucans from sucrose were relatively inactive. Dextransucrase (EC 2.4.1.5), which catalyses soluble glucan synthesis, was most active in the supernatant fluid from cultures grown with excess glucose, fructose or sucrose, but full activity was detected only when the enzyme was incubated with both sucrose and dextran. Little dextransucrase activity was detected in carbon-limited cultures. It is concluded that glucosyl- and fructosyltransferases are constitutive enzymes in that they are synthesized at similar rates during growth with an excess of the substrate or of the products of the reactions which they catalyse. Although the Ingbritt strain was originally isolated from a carious lesion, it is now a poor source of glucosyltransferase activity. Glucosyltransferases were extremely active in cultures of a recent clinical isolate, strain 3209, and were apparently induced during growth with excess glucose.     

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.     

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Mycobacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.     

Calcification of Selected Strains of Streptococcus mutans and Streptococcus sanguis

Nine strains of cariogenic Streptococcus mutans and two strains of Streptococcus sanguis were tested for their ability to form hydroxyapatite. The cells were examined by X-ray diffraction and electron microscopy for apatite crystals after growth in a synthetic calcification medium. Each of the test isolates, except for one strain of S. sanguis, produced intracellular mineral. Two strains of S. mutans formed both intra- and extracellular crystals. There was no apparent relationship between calcifiability and serotype.     

Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase

Mycobacterial Clp‐family proteases function via collaboration of the heteromeric ClpP1P2 peptidase with a AAA+ partner, ClpX or ClpC1. These enzymes are essential for M. tuberculosis viability and are validated antibacterial drug targets, but the requirements for assembly and regulation of functional proteolytic complexes are poorly understood. Here, we report the reconstitution of protein degradation by mycobacterial Clp proteases in vitro and describe novel features of these enzymes that distinguish them from orthologues in other bacteria. Both ClpX and ClpC1 catalyse ATP‐dependent unfolding and degradation of native protein substrates in conjunction with ClpP1P2, but neither mediates protein degradation with just ClpP1 or ClpP2. ClpP1P2 alone has negligible peptidase activity, but is strongly stimulated by translocation of protein substrates into ClpP1P2 by either AAA+ partner. Interestingly, our results support a model in which both binding of a AAA+ partner and protein‐substrate delivery are required to stabilize active ClpP1P2. Our model has implications for therapeutically targeting ClpP1P2 in dormant M. tuberculosis, and our reconstituted systems should facilitate identification of novel Clp protease inhibitors and activators.     

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.