Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28.

The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 gtfI gene, which encodes a glucosyltransferase that produces an insoluble glucan product. A single open reading frame encodes a mature glucosyltransferase protein of 1,559 amino acids (Mr, 172,983) and a signal peptide of 38 amino acids. In the C-terminal one-third of the protein there are six repeating units containing 35 amino acids of partial homology and two repeating units containing 48 amino acids of complete homology. The functional role of these repeating units remains to be determined, although truncated forms of glucosyltransferase containing only the first two repeating units of partial homology maintained glucosyltransferase activity and the ability to bind glucan. Regions of homology with alpha-amylase and glycogen phosphorylase were identified in the glucosyltransferase protein and may represent regions involved in functionally similar domains.     

Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli.

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.     

Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.

The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.     

Purification of a fourth glucosyltransferase from Streptococcus sobrinus.

Recently, we found a novel primer-independent, water-soluble glucan synthase as a fourth glucosyltransferase (GTF) in a culture supernatant of strain AHT-k of Streptococcus sobrinus (Y. Yamashita, N. Hanada, and T. Takehara, Biochem. Biophys. Res. Commun. 150:687-693, 1988). In the present study, four kinds of purified GTFs, including the novel GTF, were prepared. They were composed of two primer-dependent GTFs and two primer-independent GTFs. Of the primer-dependent GTFs, one was a water-insoluble glucan synthase and the other was a water-soluble glucan synthase; both of the primer-independent GTFs were water-soluble glucan synthases (GTF-Sis). Using antisera against four purified GTFs, we concluded that the immunological properties of each were completely different from those of the others. Additionally, it was shown that the novel GTF-Si, which was previously shown to have a molecular weight of 137,000, was proteolytically degraded and could be isolated at a molecular weight of 152,000 and that Streptococcus cricetus secreted an enzyme that immunologically cross-reacted with GTF-Si. While the product of the novel GTF-Si was not an effective primer for both of the primer-dependent enzymes (water-soluble and -insoluble glucan synthases), the product of the enzyme affected the molecular size of the products of the other GTF-Sis.     

Crystallization of ricin A chain obtained from a cloned gene expressed in Escherichia coli

Ricin is a heterodimeric toxin of the form AB, where B is a lectin which binds cell surfaces, triggering endocytosis. The B chain then aids the A chain in escaping from the endosome. The A chain enzymatically attacks and inactivates ribosomes, thereby killing the intoxicated cell. We have recently solved the three-dimensional structure of whole ricin. Here we report that the A chain, expressed from a gene cloned into Escherichia coli has been crystallized in a suitable form for high resolution x-ray analysis. The crystals are monoclinic space group P2(1) with a = 42.6, b = 68.1, c = 50.2 A and beta = 112.9 degrees. There is evidence that the A chain undergoes a conformational change, resulting in activation, when it is released from the B chain. Comparison of the two structures should facilitate an analysis of this process.     

Construction of Streptococcus mutans glucosyltransferase mutants utilizing a cloned gene fragment

Abstract An internal 1.6-kb BAM HI DNA fragment of the previously cloned Streptococcus mutans GS-5 gtfB gene was utilized to construct plasmids capable of insertion in toto into the GS-5 chromosome. The resultant insertions primarily yielded mutants defective in glucosyltransferase-I activity. These mutants were also defective in sucrose-dependent colonization of smooth surfaces.     

Nigerooligosaccharide acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I

Nigerose and nigerooligosaccharides served as acceptors for a glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus to give a series of homologous acceptor products. The soluble oligosaccharides (dp 5-9) strongly activated the acceptor reaction, resulting in the accumulation of water-insoluble (1-->3)-alpha-D-glucan. The enzyme transferred the labeled glucosyl residue from D-[U-13C]sucrose to the 3-hydroxyl group at the non-reducing end of the (1-->3)-alpha-D-oligosaccharides, as unequivocally shown by NMR 13C-13C coupling patterns. The values of the 13C-13C one-bond coupling constant (1J) are also presented for the C-1-C-6 of the 13C-labeled alpha-(1-->3)-linked glucosyl residue and of the non-reducing-end residue.     

Single-molecule imaging of interaction between dextran and glucosyltransferase from Streptococcus sobrinus

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.     

Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferase from Streptococcus sobrinus

Abstract Oolong tea extract (OTE) and the purified polymeric polyphenols from OTE have been found to inhibit glucosyltransferase (GTase) of mutans streptococci. In view of the partial fermentation characteristic of oolong tea, we describe here an in vitro model reaction system to produce partially fermented products of d-(+)-catechin or green tea extract (GTE) using horseradish peroxidase. A dimeric catechin molecule was identified as dehydro-dicatechin A by instrumental analyses. The molecular size of some oligomeric catechins was estimated by the elution profile with HPLC. These catechin oligomers markedly inhibited GTase from Streptococcus sobrinus 6715. As the degree of polymerization of catechin or GTE increased, GTase was inhibited more effectively. These results suggest that polymeric polyphenols found in OTE are synthesized by partial fermentation due to oxidases/peroxidases present in tea leaves.     

Characterization of the phospholipase C gene of Pseudomonas aeruginosa cloned in Escherichia coli

We have cloned a 4.9-kb fragment of Pseudomonas aeruginosa DNA containing the structural gene of phospholipase C (PLC), by inserting it into the BamHI site of plasmid pBR322. Strains of Escherichia coli carrying this recombinant plasmid produce PLC, but expression of the gene differs from that in P. aeruginosa in two respects: (i) synthesis of the enzyme appears to be constitutive, i.e., not repressible by the presence of inorganic phosphate in the growth medium, and (ii) most of the enzyme remains associated with the outer membrane instead of being secreted. Insertion mutagenesis at a unique restriction site within the PLC gene destroyed the ability of the plasmid to code, in maxicells, for phospholipase C activity and for an Mr 80000 polypeptide.     

Characterization of the cloned Escherichia coli dihydrofolate reductase

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was purified from Escherichia coli strains that carried derivatives of the multicopy recombinant plasmid, pJFM8. The results of enzyme kinetic and two-dimensional gel electrophoresis experiments showed that the cloned enzyme is indistinguishable from the chromosomal enzyme. Therefore it can be concluded that these strains are ideal for use as a source of enzyme for further studies on the biochemistry and regulation of this important enzyme. The plasmid derivatives were constructed by recloning experiments that utilized several restriction endonucleases. From the analysis both of these plasmids and the purified dihydrofolate reductase enzymes it was possible to deduce the location and orientation of the dihydrofolate reductase structural gene on the parent plasmid, pJFM8.     

Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity

Mutans streptococci are oral bacteria with a key role in the initiation of dental caries, because their glucosyltransferases synthesize polysaccharides from sucrose that allow them to colonize the tooth surface. Among the strategies to prevent dental caries that are being investigated are (1) the inhibition of bacterial growth of mutans streptococci or (2) the inhibition of glucosyltransferases involved in polysaccharide formation. Pure fatty acid esters of sucrose, maltose and maltotriose were synthesized by an enzyme-catalyzed process and tested as inhibitors of two glucosyltransferases of great homology, those from Streptococcus sobrinus and Leuconostoc mesenteroides NRRL B-512F. In spite of having their nonreducing end glucose blocked at 6-OH, they did not inhibit dextran synthesis. However, their effect on the growth of S. sobrinus in the solid and liquid phase was notable. 6-O-Lauroylsucrose, 6'-O-lauroylmaltose and 6"-O-lauroylmaltotriose at 100 microg/mL showed complete inhibition of S. sobrinus in agar plates. Consequently, these nontoxic derivatives are very promising for inclusion in oral-hygiene products aimed at disrupting plaque formation and preventing caries.     

Hydrogen production by Escherichia coli containing a cloned hydrogenase gene from Citrobacter freundii

Two hydrogenase genes of Citrobacter freundii complementing different Escherichia coli hyd mutations were cloned on the multicopy-plasmid pBR322. Recombinant plasmids pCBH2 and pCFH1 were obtained. Since hydrogenase activities of E. coli transformant HK-8 (pCBH2) and HK-7 (pCFH1) were much the same as E. coli C600 (wild type cells), the reduction in DNA size of recombinant plasmid pCBH2 (10.7 kb) was investigated. Reduced recombinant plasmids pCBH4 (6.2 kb) and pCBH6 (5.7 kb) were obtained, and a hydrogenase gene was found to be located on the 2.35 kb fragment between AvaI and EcoRI sites. Hydrogenase activity and hydrogen-evolving activity of E. coli HK-8 (pCBH4 or pCBH6) from sodium formate, sodium pyruvate or glucose were approximately 2-fold higher than those of E. coli C600 (wild type cells).On the other hand, a reduced recombinant plasmid pCBH10 (6.0 kb), which contained the adjacent DNA fragment (2.15 kb) to a hydrogenase gene, was obtained. Hydrogenase activity of E. coli C600 harboring pCBH10 was half that of E. coli C600. From these results we estimate that in plasmid pCBH2, the repressor gene suppressing the synthesis of hydrogenase might have been cloned together with a hydrogenase gene.     

Expression of a gene for glucan-binding protein from Streptococcus mutans in Escherichia coli

The structural gene for a glucan-binding protein (GBP) of Streptococcus mutans has been inserted into a bacteriophage lambda vector and expressed in Escherichia coli K12. Lysates of E. coli infected with the recombinant phage contain an antigenic protein of the same size as S. mutans GBP. The GBP synthesized in E. coli can be affinity-purified on immobilized glucan and antiserum raised against it has been shown to precipitate fructosyltransferase activity from S. mutans.     

Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.     

Multiple translational products from a Mycoplasma hyorhinis gene expressed in Escherichia coli.

We analyzed protein expression from a cloned Mycoplasma hyorhinis genomic fragment that produces in Escherichia coli a set of related polypeptides of 110, 100, 65, and 55 kilodaltons from a coding region of just over 3.0 kilobases. Expression of these multiple products resulted from a mechanism operating at the translational level but not from truncation at UGA termination codons, which are known to encode tryptophan in several mycoplasma species. The structural relatedness of the proteins was demonstrated by two-dimensional tryptic peptic mapping, but their generation by posttranslational processing was ruled out by pulse-chase labeling analysis. Examination of proteins expressed from plasmid constructs and tryptic peptide analysis of these polypeptides and the original set of proteins revealed that they share carboxy-terminal regions, an observation inconsistent with truncation at UGA codons. Expression of proteins from this cloned fragment was not dependent on vector sequences and was observed when the coding region was placed under control of a T7 promoter, suggesting that all products were translated from a single message. Expression of related products in mycoplasmas was examined by immunoblot analysis of M. hyorhinis proteins with antiserum against overexpressed recombinant proteins. A single 115-kilodalton mycoplasma protein was detected, which is larger than any of the related proteins expressed in E. coli. Our analysis indicated that translation initiation sites are used in E. coli that are not active in mycoplasmas, thereby defining differences between the translational regulatory signals of mycoplasmas and eubacteria.     

Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli

V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His(6)-tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.