• 약학회지
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• 제40권1호
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• pp.59-64
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• 1996

Gram-positive bacteria, Bacillus subtilis BR151 and Staphylococcus aureus RN4220 were transformed with high efficiency by electroporation. The cells were incubated until late log phase, washed three times with 10% glycerol, 1mM HEPES, 12% sucrose and resuspended to $10^{10}{\sim}10^{11}cfu/ml$, then stored at -$70^{\circ}C$. Transformation efficiency of B. subtilis BR151 was $1.03{\times}10^7cfu/{\mu}g$ with cells washed with 10% glycerol and electroporated by 15KV/cm, 0.7msec pulse with pUB110. Transformation efficiency of S. aureus RN4220 was $4{\times}10^6cfu/{\mu}g$ with cells washed with 1mM HEPES + 10% glycerol and electroporated by 15KV/cm, 2.5msec pulse. The number of total transformants was 1000 when B. subtilis BRI51 was transformed with 100ng pUB110 DNA and the number of total transformants was 9000 when S. aureus RN4220 was transformed with 10ng pUB110 DNA

• Biomolecules & Therapeutics
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• 제1권2호
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• pp.177-182
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• 1993

From 84 clinical isolates of Staphylococcus species, ten strains showing inducible resistance to MLS antibiotics were selected by disk agar diffusion method. Colony hybridization was executed using two MLS inducible resistance genes, ermA and ermC, previously identified from S. aureus as probes. S. hemolyticus 401 and S. epidermidis 542 whose genes were not homologous to those probes were finally selected. It was determined that the resistance genes of S. hemolyticus 401 and S. epidermidis 542 were not homologous to ermA, ermC and ermAM by Southern hybridization. S. epidermidis 542 had a plasmid DNA. To know if the plasmid may have genes related to inducible resistance, it was attempted to transform B. subtilis BR151 and S. aureus RN4220 with the plasmid prepared from S. epidermidis 542. It was shown that the gene related to inducible resistance to MLS antibiotics did not exist in this plasmid. These results indicate that two clinical isolates of S. hemolyticus 401 and S. epidermidis 542 had novel genes which were not homologous to MLS resistance genes identified previously. It was assumed that these genes may exist in chromosomal DNA.

• BMB Reports
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• 제40권6호
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• pp.936-943
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• 2007

Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cellwall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region ($P_g$) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the $P_g$-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that $P_g$ in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced $P_g$ efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.