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Genetic analysis and molecular characterization of plasmid deoxyribonucleic acid (DNA) was performed in a toxigenic isolate ofStaphylococcus aureus strain DU4916. Elimination, transduction, and transformation experiments provided us with a series of derivatives similar except for the presence or absence of genes mediating resistance to penicillin (penr), methicillin (mecr), and tetracycline (tetr) and enterotoxin type B (SEB) production (entB+). The derivatives were examined for the presence of a plasmid species which encodes for SEB production. Two distinct species of covalently closed circular DNA of about 2.8 x 106 and 0.75 x 106 daltons were identified in an ethidium bromide-cured, penicillinase-negative (pens) isolate, SN109 (mecr tetr emtB+). Further segregation of either methicillin resistance or tetracycline resistance or of both together resulted in the loss of SEB production and the disappearance of both plasmids. Transduction from strain SN109 showed that determinants for tetracycline resistance are carried by the 2.8 x 106f-dalton plasmid. Transformation with covalently closed circular DNA from strain SN109 yielded mecs tetr entBtransformants harboring the tetracycline resistance plasmid alone and mecr tetr entB+ transformants harboring both the tetracycline resistance and the 0.75 x 106dalton plasmid. Further segregation of methicillin resistance in transformants was not associated with any change in plasmid DNA. The results indicate that a genetic determinant for SEB production is carried by the 0.75 x 106-dalton plasmid. It is possible, however, that this plasmid cannot be maintained in the host independently from the tetracycline resistance plasmid. Methicillin resistance in the strains examined could not be ascribed to any of the covalently closed circular DNA components resolved in strain DU4916.

Genomic DNA frgments encoding 1-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing e-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbeliiferyl-o-D-glucoside. Two genes encoding 13-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a 13-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60°C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-f-D-glucosides and was thermostable, retaining about 70%o of its activity after 48 h at 60°C. BglB activity is activated two-to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bgiB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with 13-glucosidases from glycosal hydrolase family 1. Cellulose, an abundant but recalcitrant biopolymer, is composed of repeating glucose units linked by P-1,4-glycosidic bonds. Cellulases, produced by a wide variety of microorganisms, degrade such polymers and play a major role in the recycling of biomass. Cellulases are multicomponent complexes which are often composed of endoglucanases (1,4-0-D-glucan glucano-hydrolase, EC 3.2.1.4), cellobiohydrolases (1,4-0-D-glucan cellobiohydrolase, EC 3.2. 1.91), and cellobiases (1,4-0-D-glucoside glucohydrolases, EC 3.2.1.21). Cellobiases, while specific for cellobiose, belong to a very diverse family of enzymes (,-glucosidases) capable of hydrolyzing a broad spectrum of ,-glucosides. Cellulase components are thought to act in a stepwise process and can act synergistically to achieve more efficient degradation (10, 53, 54, 56). The major end product of concerted endoglucanase and cellobiohydrolase activity is cellobiose. Cellobiose is then hydrolyzed to glucose by * Corresponding author. 34), and certain of the genes have been cloned and characterized in heterologous hosts (15, 28, 37). To obtain microorganisms that produce 3-glucosidases that are both thermostable and resistant to end product inhibition, we isolated thermophilic cellulolytic actinomycetes from thermal ecological niches and evaluated their enzymes by assaying for ,B-glucosidase activity (against para-nitrophenyl-1-glucoside) in the presence of up to 30% glucose at 60°C (49). Of several isolates, the most attractive was Microbispora bispora (31, 49). In order to study the enzyme components independently and to characterize the genes encoding them, we have cloned several M. bispora cellulase components (56). These include t...

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The cleavage maps of a Staphylococcus aureus plasmid, pSN1 (2.75 megadaltons), conferring tetracycline resistance, were determined. Cleavage maps are given for HpaI and HindIII restriction endonucleases by using the single HpaII site as a reference point. Nucleases EcoRI, BamHI, SalI, and HaeIII have no sites on this plasmid.