| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We have purified a novel glutamyl endopeptidase from Staphylococcus epidermidis (GluSE) by cation-exchange chromatography, ultrafiltration, and size-exclusion HPLC. An apparent molecular mass of the protease was 27 kDa and the optimal pH of the proteolytic activity was 8.0. The protease specifically catalyzed the hydrolysis of a carbonyl end of glutamic acid of a synthetic peptide, Z-LLE-MCA. The gene encoding GluSE (gse) was cloned by the inverse polymerase chain reaction from genome DNA of S. epidermidis ATCC 14990 in consideration of the N-terminal amino acid sequence of the purified protein and DNA sequence of a relatively conserved region of glutamyl endopeptidases of Staphylococcus aureus. The predicted gse gene-product consists of 282 amino acids, a molecular mass of 30,809, with 66 amino acid residues of a preprosequence. The production of GluSE was preferentially demonstrated when the cells were cultured on the dialysis membrane sheeted on agar medium, but not in culture medium, and this production apparently correlated with formation of biofilms. Therefore, it was speculated that GluSE played some roles in the attachment mechanism of the bacteria by biofilms to the surface of plastic devices. Southern hybridization analysis indicated that the GluSE gene was present as a single copy on the chromosomal DNA. The distribution of the GluSE gene was also examined by PCR in clinical isolates of coagulase-negative staphylococci. The gene was amplified in all the S. epiderinidis isolates (65/65), but not in S. aureus or in the other CNS isolates including S. capitis,S. haemolyticus, S. hominis, and S. warneri. Further, the production of GluSE was demonstrated in 72.3% (47/65) of S. epidermidis isolates. These results suggest that the GluSE gene is ubiquitous and exclusively preserved in S. epidermidis.
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