Streptococcus Sobrinus Mfe28 JOSEPH J

JOURNAL OF BACTERIOLOGY, Sept. 1987, p. 4271-4278 Vol. 169, No. 9 0021-9193/87/094271-08$02.00/0 Copyright © 1987, American Society for Microbiology Nucleotide Sequence of a Glucosyltransferase Gene from Streptococcus sobrinus MFe28 JOSEPH J. FERRETTI,'* MARTYN L. GILPIN,2 AND ROY R. B. RUSSELL2 Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190,1 and Dental Research Unit, Royal College of Surgeons of England, Downe, Kent BR6 7JJ, United Kingdom2 Received 3 April 1987/Accepted 2 June 1987 The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 g#f 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. The glucosyltransferases (EC 2.4.1.5) produced by vari- MATERIALS AND METHODS ous species of oral streptococci are of considerable interest Bacteria and media. E. coli MAF1 (containing plasmid because of their production of extracellular glucans from pMLG1) was the initial source of the S. sobrinus MFe28 gtfp sucrose. These glucans are thought to play a key role in the gene (27a). E. coli MAF5 contains plasmid pMLG5, which development of dental plaque because of their ability to has the same 5.0-kilobase (kb) fragment as pMLG1 and an adhere to smooth surfaces and mediate the aggregation of additional 0.5-kb fragment from the bacteriophage lambda bacterial cells and food debris (12). It is known that a single recombinant in which the gtfp insert was first cloned (9). E. strain can produce several distinct glucosyltransferases dif- coli JM109 was used as the recipient for transfection exper- fering in electrophoretic, antigenic, or enzymatic properties, iments with M13 bacteriophage vectors (35) and was rou- although some of this apparent variety may be due to the use tinely grown in 2x YT broth (19). Soft agar overlays of different oral streptococcal strains and different purifica- consisted of 2x YT broth supplemented with final concen- tion procedures and activity assays by different laboratories. trations of 0.75% agar, 0.33 mM isopropyl-3-D-thiogalacto- The properties and characteristics of the glucosyltrans- pyranoside, and 0.02% 5-bromo-4-chloro-3-indolyl-3-galac- ferases of the mutans group streptococci have been reviewed toside for differentiating recombinant and nonrecombinant by Ciardi (3) and Mukasa (18). phages. For the titration of M13 recombinants carrying all or Recently, several glucosyltransferase genes from various part of gtJl, phages were plated on E. coli JM109 on B-broth strains of streptococci have been cloned by recombinant agar (26) to which 1% sucrose was added for detection of DNA techniques and have been shown to be expressed in enzyme activity. Escherichia coli. Robeson et al. (24) have cloned a glucosyl- Enzymes and chemicals. Restriction enzymes were pur- transferase gene (gtfA) from Streptococcus mutans UAB90 chased from Bethesda Research Laboratories, Inc., Gaith- (serotype c) and shown that it produces a protein with a ersburg, Md., and were used in accordance with the speci- molecular weight of 55,000. A similar gtfA gene has also fications of the manufacturer. T4 DNA ligase was purchased been cloned by Pucci and Macrina (23) from S. mutans LM7 from Amersham Corp., Arlington Heights, Ill., or Bethesda (serotype e) and by Burne et al. (2) from S. mutans GS5 Research Laboratories. The Klenow fragment of DNA poly- (serotype c). Aoki et al. (1) reported the cloning of a gluco- merase and the M13 15-base primer were purchased from syltransferase gene (gtfB) from S. mutans GS-5 that pro- Bethesda Research Laboratories. The deoxy- and dideoxy- duces a protein with a molecular weight of about 150,000. nucleotide triphosphates were purchased from P-L Bio- Another glucosyltransferase gene, gtfC, which specifies a chemicals, Inc., and [a-32P]dATP was purchased from New 150,000-molecular-weight polypeptide has been obtained England Nuclear Corp., Boston, Mass.). Isopropyl-3-D-thio- from S. mutans LM7 by Pucci et al. (22). Finally, Gilpin et galactopyranoside and 5-bromo-4-chloro-3-indolyl-3-galac- al. (9) have cloned two glucosyltransferase genes from toside) were purchased from Sigma Chemical Co., St. Louis, Streptococcus sobrinus MFe28 (serotype h): gtfS, which Mo. encodes a glucosyltransferase that synthesizes a water- Subcloning of the gtfl gene and nucleotide sequencing. The soluble glucan, and gtfl, which encodes a glucosyltrans- gtfl gene was obtained for subcloning experiments by diges- ferase that synthesizes a water-insoluble glucan. tion of pMLG1 with HindIII followed by electrophoresis and The availability of these cloned genes allows further isolation of the 5.0-kb fragment from 0.8% type VII agarose characterization of both the genes and gene products, and in gels as previously described (16). The fragment was unidirec- this communication, we report the complete nucleotide tionally degraded with Bal 31 by a modification of the sequence of the gtfl gene from S. sobrinus MFe28. procedure of Gilmore et al. (8), and all subcloning into M13 phages mpl8 and mpl9 was done as described by Ferretti et * Corresponding author. al. (7). A 0.5-kb HindIIl fragment was subsequently isolated 4271 4272 FERRETTI ET AL. J. BACTERIOL. co sis of purified glucosyltransferase was done with an Applied ('4 in P Biosystems 470A protein sequencer with an on-line 120A so. I( 4( PTH analyzer in accordance with the instructions of the manufacturer. IIh-.a ........ ..1.... !X....... RESULTS Cloning of complete gtl gene. The gtpl gene from S. sobrinus MFe28 was originally cloned into the BamHI sites of bacteriophage lambda L47.1 and was located in a 7.6-kb DNA insert. Subsequently, mapping experiments showed that HindIII cleaved the insert at three points and the lambda DNA at two points outside the insert to give four fragments of 5.0, 2.6, 2.5, and 0.5 kb which carried S. sobrinus DNA. Plasmid pMLG1, from which a functional glucosyltransferase is expressed in E. coli, carries the 5.0-kb fragment FIG. 1. SDS-PAGE of glucosyltransferase activity in S. sobrinus (27a). At an early stage of the nucleotide-sequencing pro- MFe28, E. coli MAF4 (carrying pMLG5), and E. coli MAF1 gram, it became clear that pMLG1 contained a long open (carrying pMLG1). The right lane contains the following protein reading frame with no termination codon. These results standards: RNA polymerase 1' subunit 165,000), RNA poly- (Mr, indicated that the entire gtp gene was not present in merase P subunit (155,000), 3-galactosidase (116,000), phosphorylase b (97,400), albumin (66,000), and ovalbumin (45,000). pMLG1, and so a further collection of derivatives of pBR322 carrying HindIII fragments from the bacteriophage lambda recombinants was examined. One of these, pMLG5, en- from pMLG5, cloned into M13 phages, and sequenced. This coded a glucosyltransferase of 173 ki}odaltons (Fig. 1) and fragment contained the remainder of the glucosyltransferase was found to have both the 5.0- and 0.5-kb fragments. sequence not present in the pMLG1 HindIII fragment. Restriction mapping of pMLG5 and subsequent nucleotide Sequencing reactions were performed by the Sanger dide- sequencing confirmed that the 0.5-kb fragment carried the oxy chain termination method (28) by using the procedures information for the C-terminal region of glucosyltransferase. described by Amersham. All sequences were confirmed A partial restriction site map of the 5.5-kb insert containing from at least two overlapping clones, and the entire gene the gft gene is shown in Fig. 2. sequence was determined on both strands. The sequence Nucleotide sequence. The complete nucleotide sequence of information was analyzed by the James M. Pustell DNA/ the 4,995-base-pair (bp) fragment carrying the gtp gene was protein sequencing program obtained from International determined in both orientations and is shown in Fig. 3. Biotechnologies, Inc., New Haven, Conn. Previous evidence had indicated that 0.5 kb of the insert in Gel electrophoresis. Sodium dodecyl sulfate-polyacryl- pMLG1 and pMLG5 was derived from the bacteriophage amide gel electrophoresis (SDS-PAGE) and detection of lambda vector (27a), and this was confirmed by the finding glucosyltransferase activity by incubation of gels with su- that the first 494 bp of the 5.5-kb fragment showed 100% crose in the presence of Triton X-100 was done as described homology with the corresponding part of the lambda se- previously (9), but the sensitivity of the method was en- quence (lambda sequence not shown). An open reading hanced by the use of a periodic acid-Schiffreagent procedure frame containing 4,791 bp codes for the glucosyltransferase modified from published methods (15, 29). After incubation protein. The deduced amino acid sequence, which is coded in sucrose (generally for 40 h at 37°C), the gels were fixed for starting at the ATG codon beginning at position 160 and 30 min in 75% ethanol and treated on a shaker for 30 min extending to the termination codon TAA at position 4951, with 0.7% periodic acid in 5% acetic acid. They were then contains 1,597 amino acids and has a molecular weight of shaken for 60 min in several changes of 0.2% sodium 177,100.

Load more