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 various species of oral streptococci are of considerable interest because of their production of extracellular glucans from sucrose. These glucans are thought to play a key role in the development of dental plaque because of their ability to adhere to smooth surfaces and mediate the aggregation of bacterial cells and food debris (12). It is known that a single strain can produce several distinct glucosyltransferases differing in electrophoretic, antigenic, or enzymatic properties, although some of this apparent variety may be due to the use of different oral streptococcal strains and different purification procedures and activity assays by different laboratories. The properties and characteristics of the glucosyltransferases of the mutans group streptococci have been reviewed by Ciardi (3) and Mukasa (18).Recently, several glucosyltransferase genes from various strains of streptococci have been cloned by recombinant DNA techniques and have been shown to be expressed in Escherichia coli. Robeson et al. (24) have cloned a glucosyltransferase gene (gtfA) from Streptococcus mutans UAB90 (serotype c) and shown that it produces a protein with a molecular weight of 55,000. A similar gtfA gene has also been cloned by Pucci and Macrina (23) from S. mutans LM7 (serotype e) and by Burne et al. (2) from S. mutans GS5 (serotype c). Aoki et al. (1) reported the cloning of a glucosyltransferase gene (gtfB) from S. mutans GS-5 that produces a protein with a molecular weight of about 150,000. Another glucosyltransferase gene, gtfC, which specifies a 150,000-molecular-weight polypeptide has been obtained from S. mutans LM7 by Pucci et al. (22). Finally, Gilpin et al. (9) have cloned two glucosyltransferase genes from Streptococcus sobrinus MFe28 (serotype h): gtfS, which encodes a glucosyltransferase that synthesizes a watersoluble glucan, and gtfl, which encodes a glucosyltransferase that synthesizes a water-insoluble glucan.The availability of these cloned genes allows further characterization of both the genes and gene products, and in this ...

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SUMMARYDuring infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish. Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen. It was found that the expression of msa encoding the p57 antigen of R. salmoninarum, was constitutive, while the expression of hly and rsh, encoding haemolysins, and lysB and grp was reduced after infection. Macrophages showed a rapid inflammatory response in which the expression of interleukin-1b (IL-1b), major histocompatibility complex class II (MHC II), inducible cyclo-oxygenase (Cox-2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor-a (TNF-a) expression was greatly reduced initially and then increased. After 5 days, except for TNF-a and MHC II, expression returned to levels approaching those of uninfected macrophages. We propose that R. salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF-a-dependent killing pathways. The effects of specific R. salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa, hly, rsh, lysB, or grp. We found that msa reduced the expression of IL-1b, Cox-2, and MHC II but stimulated TNF-a while hly, rsh and grp stimulated MHC II but down-regulated TNF-a. Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF-a. The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R. salmoninarum infections rely on MHC II-and TNF-a-dependent pathways. Moreover, prolonged stimulation of TNF-a may contribute to the chronic inflammatory pathology of bacterial kidney disease.

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, g t f l , previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage 2 vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtjlr was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d ) and 6715.(serotype g ) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-Cr-Dglycosyl residues (-90%). An internal fragment of the g t f l gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group. I N T R O D U C T I O NThe glucosyltransferases (GTFs) of the mutans group of oral streptococci are extracellular enzymes responsible for the synthesis of complex mixtures of water-soluble and insoluble glucans. These glucans are believed to be important in the formation and metabolism of dental plaque (Hamada & Slade, 1980). It was first shown by Guggenheim & Newbrun (1969) that a single strain could produce a number of electrophoretically distinct GTFs. Ciardi et al. (1976Ciardi et al. ( , 1977 showed that some of these enzymes synthesized soluble, and others insoluble, polymers but progress towards understanding the reason for the multiplicity of enzymes has been considerably hampered by technical problems, including the fact that GFTs exist in highmolecular-mass aggregates and have a strong tendency to bind to separation matrices commonly used in column chromatography (Germaine et al., 1977;Russell, 1979; Figures & Edwards, 1981 ; Ono et al., 1984). The latter fact has, however, proved useful in purification procedures involving affinity chromatography (McCabe & Smith, 1977;Russell, 1979;McCabe, 1985). At least some of the heterogeneity of GTFs is a consequence of proteolytic degradation (Asem et al., 1986;Mukasa, 1986;Russell et al., 1986), but many questions remain concerning the number of distinct GTFs and their individual functions.Most progress has been made with strains that originally constituted Streptococcus mutans serotypes d and g, which are now classified as Streptococcus sobrinus (Coykendall, 1983; Coykendall & Gustafson, 1986). The purification from strains of these serotypes of GTFs capable of synthesizing soluble glucans (GTF-S) or insoluble glucans (GTF-I) has been described by a number of workers (Ciardi et al., 1977;Germaine et al., 1977;Hare et al., 1978; Figures& Edwards, 1981 ; Fukushimaetal., 1981 ; Fukui etal., 1982; Kogaetal., 1983;Robyt & Martin, 1983;Furuta et al., 1985;McCabe, 1985; Namiki et al., 1985;Mukasa, 1986). The glucan synthesized by GTF-S (dextran) consists mainly of 1,6-a-linked glucose units while that

The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.Renibacterium salmoninarum is an important cause of clinical and subclinical infections among farmed and wild salmonid populations in North and South America, Europe, and Japan (5). The organism causes a chronic, systemic, and granulomatous infection, bacterial kidney disease (BKD), that is often fatal under conditions which are stressful to the host (11). There is no effective vaccine or chemotherapy, and the presence of subclinical infections complicates attempts to control the disease through programs of eradication. An improved understanding of the transmission and spread of BKD is of considerable importance in policy management issues relating to aquaculture and wildfisheries. There have been a number of studies investigating the presence, prevalence, and means of transmission of BKD within and between fish populations. This work has shown that R. salmoninarum is endemic within many wild salmonid populations as a low-level, subclinical infection; it has been isolated in up to 100% of samples (9,12,15). However, the epidemiology of BKD remains unclear, mainly because of the difficulty of differentiating isolates of R. salmoninarum by biochemical, serological, and multilocus enzyme electrophoresis techniques (1,6,16).We used two approaches to assess the extent of molecular variation among R. salmoninarum isolates from different geographic locations. First, we investigated possible polymorphisms in specific regions within the genome, genes msa (3), rsh (4), and hly (8), and the rRNA genes, including the intergenic spacer (ITS) regions. PCR and DNA sequencing studies have shown that R. salmoninarum has only limited variation in these regions (7). Identifying specific markers of variation in the R. salmoninarum genome, such as insertion sequences or variable numbers of tandem repeats (TR), has been constrained by a paucity of sequence information. Second, we analyzed differences throughout the genome using randomly amplified polymorphic DNA (RAPD) analysis. RAPD analysis is a PCR-based alternative method to the use of species-specific DNA sequences for isolate or strain differentiation. The method uses short random primers for rapidly detecting genomic polymorphisms under low-stringency conditions (18,19). RAPD analysis is widely used for differentiating bacterial isolates (2, 10, 17) and relies on small q...