In vitro and in vivo complementation of Streptococcus mutans mutants defective in adherence

Larrimore, S; Murchison, H; Shiota, Tetsuo; Michalek, Suzanne M.; Curtiss, rd R

doi:10.1128/iai.42.2.558-566.1983

Cited by 28 publications

(18 citation statements)

References 31 publications

(38 reference statements)

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“…Dex also possesses an N-terminal signal peptide and a C-terminal sorting signal in VRs (15,18,35). In addition, Dex is thought to be responsible both for controling the amount and nature of extracellular glucans and for utilizing extracellular glucans as a carbohydrate source (5,21,25,28,34). Consequently, Dex is thought to be one of the virulent factors in mutans streptococci.In this paper we sequenced and characterized the dex gene of S. rattus ATCC19645 and compared the Dex molecule of S. rattus with that of S. mutans, S. sobrinus, Streptococcus downei, and Streptococcus salivarius.…”

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Molecular Characterization of Dextranase from Streptococcus rattus

Igarashi

Morisaki

Goto

2004

Microbiology and Immunology

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Streptococcus rattus is a cariogenic bacterium classified under mutans streptococci (36). This organism is usually isolated from the oral cavities of rats and occasionally of humans. As most studies concerning cariogenic properties of mutans streptococci have focused on Streptococcus mutans and Streptococcus sobrinus, which are frequently isolated from the human oral cavity, little is known about those of S. rattus. Before now many factors associated with cariogenicity of S. mutans and S. sobrinus have been determined (4,8,20); for example, one of them is a dextranase (Dex) that hydrolyzes α-1, 6-linkage in a glucan molecule produced from sucrose by mutans streptococci (3,7,8,13,34). Although a wide range of bacterial species associated with dental plaque is shown to produce Dex (19), oral streptococci, in particular mutans streptococci, are known to be a major producer of Dex (30,34). So far, the Dexs of S. mutans and S. sobrinus have been well characterized by biological and genetic studies (2, 12-15, 23, 35). Previous genetic studies showed structural features and the physiological role of Dex. It has been shown that the Dex molecule is composed of two variable regions (VRs) and a conserved region (CR) (15,18), and that catalytic and glucan-binding sites exist in the CR of the Dex molecule (12, 23). Dex also possesses an N-terminal signal peptide and a C-terminal sorting signal in VRs (15,18,35). In addition, Dex is thought to be responsible both for controling the amount and nature of extracellular glucans and for utilizing extracellular glucans as a carbohydrate source (5,21,25,28,34). Consequently, Dex is thought to be one of the virulent factors in mutans streptococci.In this paper we sequenced and characterized the dex gene of S. rattus ATCC19645 and compared the Dex molecule of S. rattus with that of S. mutans, S. sobrinus, Streptococcus downei, and Streptococcus salivarius. Materials and MethodsBacterial strains and plasmid. Streptococcus rattus ATCC19645, which was isolated from a caries lesion in rat, was grown in Todd Hewitt broth (Difco Laboratories, Detroit, Mich., U.S.A.). Escherichia coli JM109 was cultured in Luria-Bertani broth. Plasmid vector pT7Blue T (Novagen, Madison, Wisc., U.S.A.) was used for gene cloning and sequencing (14). GST gene fusion vector, pGEX-4T-2 (Pharmacia Biotech, Uppsala, Sweden), Molecular Characterization of Dextranase from

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Molecular Characterization of Dextranase from Streptococcus rattus

Igarashi

Morisaki

Goto

2004

Microbiology and Immunology

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“…So far, the dextranases of Streptococcus mutans and Streptococcus sobrinus were well characterized by enzyme purification and gene cloning (2,(14)(15)(16)38). Previous studies suggested important roles of Dex in plaque formation: one was that Dex supplies carbohydrate nutrients through the degradation of glucan (5,14,24) and the other was that Dex modifies the glucan substrate to a more firm and adhesive form (5,21,28,37). Consequently, Dex is thought to be one of the virulent factors in mutans streptococci.…”

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Nucleotide Sequence and Molecular Characterization of a Dextranase Gene from Streptococcus downei

Igarashi

Yamamoto

Goto

2001

Microbiology and Immunology

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DNA fragments encoding the Streptococcus downei dextranase were amplified by PCR and inverse PCR based on a comparison of the dextranase gene (dex) sequences from S. sobrinus, S. mutans, and S. salivarius, and the complete nucleotide sequence of the S. downei dex was determined. An open reading frame (ORF) of dex was 3,891 bp long. It encoded a dextranase protein (Dex) consisting of 1,297 amino acids with a molecular mass of 139,743 Da and an isoelectric point of 4.49. The deduced amino acid sequence of S. downei Dex had homology to those of S. sobrinus, S. mutans and S. salivarius Dex in the conserved region (made of about 540 amino acid residues). DNA hybridization analysis showed that a dex DNA probe of S. downei hybridized to the chromosomal DNA of S. sobrinus as well as that of S. downei, but did not to other species of mutans streptococci. The C terminus of the S. downei Dex had a membrane-anchor region which has been reported as a common structure of C termini of both the S. mutans and S. sobrinus Dex, The recombinant plasmid which harbored the dex ORF of S. downei produced a recombinant Dex enzyme in Escherichia coli cells. The analysis of the recombinant enzyme on SDS-PAGE containing blue dextran showed multiple active forms as well as dextranases of S. mutans, S. sobrinus and S. salivarius. Key words: Dextranase, dex gene, Streptococcus downeiDextranase (Dex) is an enzyme which hydrolyzes o-1, 6-linkage in glucan molecule produced from sucrose by mutans streptococci (4, 10, 14,37). Although a wide range of bacterial species associated with dental plaque have been shown to produce extracellular dextranases (19), oral streptococci, in particular mutans streptococci, has been known to be a predominant producer of the dextranases (32, 37). So far, the dextranases of Streptococcus mutans and Streptococcus sobrinus were well characterized by enzyme purification and gene cloning (2,(14)(15)(16)38). Previous studies suggested important roles of Dex in plaque formation: one was that Dex supplies carbohydrate nutrients through the degradation of glucan (5, 14,24) and the other was that Dex modifies the glucan substrate to a more firm and adhesive form (5, 21, 28, 37). Consequently, Dex is thought to be one of the virulent factors in mutans streptococci.Although the Dex enzyme has abilities of both glucanhydrolysis and glucan-binding, the relationship of structure and function of the Dex molecule has not yet been clarified. To understand the relationship, comparison of the primary structure of the dextranases from different *Address correspondence to Dr. Takeshi Igarashi, Department of Oral Microbiology, Showa University School of Dentistry, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Fax: 03-3784-8012. E-mail: igatakes@dent.showa-u.ac.jp 341 species of mutans streptococci should be helpful. So far, the primary structure data of Dex of mutans streptococci, which is composed of 5 species: S. mutans, S. sobrinus, S. downei, S. rattus, and S. cricetus (39), has been reported only for those of S....

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“…We divided these mutants into at least 13 phenotypic groups based on phenotypic traits (27). Mixed cultivation in sucrose-containing medium of two nonadhering mutants of differing phenotypic traits (UAB95 and UAB230) allowed complementation for adherence, and these mutants complemented each other for caries production in gnotobiotic rats as well (24).…”

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“…One unexpected observation from in vitro complementation studies was that several mutants, namely UAB108, UAB113, UAB124, and UAB244, inhibited the adherence of both mutant and wild-type control strains (24). These mutants fall into the same phenotypic group (27).…”

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In vitro inhibition of adherence of Streptococcus mutans strains by nonadherent mutants of S. mutans 6715

Murchison

Larrimore

Curtiss³

1985

Infect Immun

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Four nonadherent mutants from Streptococcus mutans 6715 mutant UAB66 (serotype g) with similar phenotypes were shown to inhibit the adherence of adherence-proficient S. mutans serotypes c and g strains. One mutant, UAB108, was shown to inhibit adherence by wild-type strains representing serotypes a, d, and e as well. This inhibition of adherence was seen with pairs of strains grown in partially defined (PD) medium supplemented with 1% sucrose in both microtiter plates and glass tubes. The inhibiting factor was present in culture supernatant fluids of inhibiting strains grown in PD medium plus 1% sucrose and was heat stable.Ethanol precipitation of culture supernatant fluids of these strains yielded a water-soluble polymer which effectively inhibited the adherence of UAB66. This polymer, isolated from UAB108, was also shown to inhibit the adherence of UAB66 at lower concentrations than that needed to inhibit adherence with dextran T10. Partially purified glucosyltransferase, isolated from the culture supernatant fluids of glucose-grown UAB108, produced a water-soluble glucan which was shown to inhibit the adherence of UAB66 as well. The methods developed permit rapid screening for strains or mutants of strains or both that inhibit adherence or plaque formation or both by wild-type strains of S. mutans Dental caries, resulting from infection by Streptococcus mutans in the presence of dietary sucrose, are generally considered to be due to the interaction of several factors. These factors include adherence of the bacteria to the tooth surface mediated by proteins (32) and water-insoluble glucans (WIG;3,9,10,12,19,21,25,26,35), aggregation in a plaque matrix (6,8), and acid production with subsequent enamel demineralization (15,18). In an effort to better understand the interaction of these factors, we previously isolated and characterized a number of mutants of S. mutans which are defective in one or more of these virulence characteristics. We divided these mutants into at least 13 phenotypic groups based on phenotypic traits (27). Mixed cultivation in sucrose-containing medium of two nonadhering mutants of differing phenotypic traits (UAB95 and UAB230) allowed complementation for adherence, and these mutants complemented each other for caries production in gnotobiotic rats as well (24).One unexpected observation from in vitro complementation studies was that several mutants, namely UAB108, UAB113, UAB124, and UAB244, inhibited the adherence of both mutant and wild-type control strains (24). These mutants fall into the same phenotypic group (27). Although there are slight differences in their colony morphologies on mitis salivarius (MS) agar, all of these mutant strains produce colonies that are submerged in a clear sticky fluid. All are devoid of dextranase activity and are adherence defective.In this study, we more closely examine this phenomenon of inhibition of adherence by UAB108. Results indicate that an ethanol-precipitable polymer present in the supernatant fluid of UAB108 cultures grown in sucrose-containing...

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In vitro and in vivo complementation of Streptococcus mutans mutants defective in adherence

Cited by 28 publications

References 31 publications

Molecular Characterization of Dextranase from Streptococcus rattus

Molecular Characterization of Dextranase from Streptococcus rattus

Nucleotide Sequence and Molecular Characterization of a Dextranase Gene from Streptococcus downei

In vitro inhibition of adherence of Streptococcus mutans strains by nonadherent mutants of S. mutans 6715

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