A new strategy for ordered DNA sequencing based on a novel method for the rapid purification of near-milligram quantities of a cloned restriction fragment

Gilmore, Michael S.; Gilmore, K. S.; Goebel, Werner

doi:10.1016/0735-0651(85)90006-8

Cited by 13 publications

(11 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nested deletions of the cloned pAD1 cytolysin determinant contained in plasmid pRAS28-4 ( Fig. 1) were constructed as described previously (16,20). Nucleotide sequence determinations were made with commercially available kits for single-stranded template-directed reactions (Sequenase 2.0; U.S. Biochemical, Cleveland, Ohio) or double-stranded template-containing reactions (fmol DNA Sequencing System; Promega, Madison, Wis.), as instructed by the manufacturers.…”

Section: Methodsmentioning

confidence: 99%

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants

et al. 1994

View full text Add to dashboard Cite

Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylL9, cylLS, cyiM, and cyiB). The inferred products of two of these open reading frames, Cyll and CylLs, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity.Enterococcusfaecalis isolates derived from infection sites are more frequently hemolytic than isolates from the oral cavity or stools of healthy volunteers (22,26,48). The variable nature of the hemolytic phenotype results from the hemolysin determinant being located on highly transmissible, pheromone-responsive plasmids (e.g., pAD1; recently reviewed in references 8 and 9), although evidence has been obtained recently for its occasional residence on the E. faecalis genome (23). The observation of acute toxicity of hemolytic E. faecalis, when injected intraperitoneally in mice (25), suggested that the hemolysin may contribute to bacterial virulence in models of human disease. Hemolytic E. faecalis strains have been observed to cause a more rapid and fulminant endophthalmitis in a rabbit infection model than isogenic strains rendered nonhemolytic as the result of insertion of a transposon into various areas of the hemolysin determinant (29). Similar observations of an endocarditis model where hemolytic strains were found to be significantly more toxic than isogenic, nonhemolytic mutants have been made (6; for a recent review of enterococcal virulence, see reference 28).Contributing to virulence is a common motif for bacterial hemolysins (3,35,46,47). The E. faecalis hemolysin, however, is unique in that in addition to mediating lysis of erythrocytes, it also possesses antibacterial activity toward a broad range of gram-positive bacteria (4,5,27 Immediately 5' to cylA is cylB, whose nucleotide sequence has been reported (17). cylB is the first member of the HlyB family of ATP-binding transport proteins to have been identified in an operon from a gram-positive bacterium (15,17). CylB was observed to be essential for externalization of the E. faecalis cytolysin precursor activity, component L.Although a substantial amount of information describing (i) the protein that activates the E. faecalis cytolysin precursor extra...

show abstract

Section: Methodsmentioning

confidence: 99%

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants

et al. 1994

View full text Add to dashboard Cite

show abstract

“…The gtfl gene was obtained for subcloning experiments by digestion of pMLG1 with HindIII followed by electrophoresis and isolation of the 5.0-kb fragment from 0.8% type VII agarose gels as previously described (16). The fragment was unidirectionally degraded with Bal 31 by a modification of the procedure of Gilmore et al (8), and all subcloning into M13 phages mpl8 and mpl9 was done as described by Ferretti et al. (7).…”

Section: Methodsmentioning

confidence: 99%

Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28

Ferretti¹,

Gilpin²,

Russell³

1987

J Bacteriol

215

244

View full text Add to dashboard Cite

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 ...

show abstract

“…A novel strategy was used to obtain nested sets of deletion derivatives from the universal priming sites of M13mp8, -9, -18, and -19 (14,31,45) for nucleotide sequence determination by the chain termination method (36). A detailed description of this strategy applied to the tandem B. cereus cytolysin genes, as well as partial cerA nucleotide sequence, has been reported (14). Both strands of the cloned cytolysin determinant were sequenced, and the accuracy of the nucleotide sequence obtained was additionally confirmed by sequence determinations performed from restriction sites located throughout the open reading frames.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid DNA was prepared from B. subtilis as previously reported (25). Purification of singlestranded M13 phage DNA for sequencing templates was done as described in a previous report (14,31). E. coli and B. subtilis strains were transformed by the CaCl2 procedure (30) and by generation of protoplasts (25) for the three buffer systems described (30).…”

Section: Methodsmentioning

confidence: 99%

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage

et al. 1989

Self Cite

View full text Add to dashboard Cite

A cloned cytolytic determinant from the genome of Bacillus cereus Bacillus cereus, a common soil saprophyte, has been recognized as an opportunistic pathogen of increasing importance (reviewed in reference 41). Although food-borne gastroenteritis is the most common malady attributed to B. cereus (41), the most devastating is B. cereus endophthalmitis (1, 4, 17). B. cereus elaborates a variety of extracellular membrane-active enzymes and cytolytic toxins. These membrane-active proteins include a phospholipase C (34), sphingomyelinase (22), phosphatidylinositol phospholipase C (23), cereolysin (7; a cytolysin of the streptolysin 0, thiol-activated class), and a second, heat stabile cytolysin about which little is known (8, 10, 37). Phospholipase C, sphingomyelinase, and cereolysin have been highly purified and used in studies of membrane structure (6,29,43) and in studies on the evolution of cytolysins produced by diverse genera of gram-positive bacteria (13,38). Phospholipase C is a Zn metalloenzyme of 23,000 daltons (34) which shows a high degree of stability in chaotropic agents and surfactants (27,28). The sphingomyelinase produced by B. cereus is a protein of between 41,000 and 23,300 daltons, depending on the method of analysis used (40), and requires divalent cations for activity (21).As a first step in determining the contribution that extracellular membrane-active proteins make to the ecology and virulence of B. cereus, a gene bank was established. Identification of a cloned cytolytic determinant from this B. cereuls GP-4 library has been reported (25). To gain insight into the relationship of the cloned cytolysin determinant to membrane-active proteins of B. cereus, nucleotide sequence and enzyme activity analyses were undertaken. The results reported here show that the cytolytic determinant cloned from B. cereus is composed of tandemly arranged genes for two distinct protein products, the activities of both being required to effect target cell lysis (hemolysis as tested). Moreover, the individual cytolysin components possess * Corresponding author. phospholipase C and sphingomyelinase activity, respectively. These data suggest that although the sphingomyelinase and phospholipase C of B. cereus have been studied in detail individually, their function in nature appears to be as a cytolytic unit representing the heat-stabile hemolysin previously observed. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in these experiments are listed in Table 1. Escherichia coli strains were routinely cultured in 2XYT medium (31) with aeration. Bacillus subtilis cultures were grown in HGP broth as previously described (25). Tetracycline (Sigma Chemical Co., St. Louis, Mo.) was incorporated into liquid and solid (1.2% agar) media at concentrations of 10 ,ug/ml for selection of resistant B. subtilis and E. coli strains. Ampicillin (Sigma) was used at 100 pRg/ml to select for recombinants cloned into the vectors pUC8,45). In addition, to screen for insertional ina...

show abstract

A new strategy for ordered DNA sequencing based on a novel method for the rapid purification of near-milligram quantities of a cloned restriction fragment

Cited by 13 publications

References 9 publications

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants

Nucleotide sequence of a glucosyltransferase gene from Streptococcus sobrinus MFe28

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage

Contact Info

Product

Resources

About