The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences.
The DNA-encoding alkaline proteinase (AP) of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, the gene-incorporated bacteria expressed high levels of both AP activity and AP antigens. The amino acid sequence deduced from the nucleotide sequence revealed that the mature AP consists of 467 amino acids with a relative molecular weight of 49, 507. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified AP reported previously. The amino acid sequence analysis revealed that both the N-terminal side sequence of the purified AP and several internal lysyl peptide fragments were identical to the deduced amino acid sequences. The percent homology of amino acid sequences between AP and Serratia protease was about S5 %. The zinc ligands and an active site of the AP were predicted by comparing the structure of the enzyme with of Serratia protease, thermolysin, Bacillus subtilis neutral protease, and Pseudomonas elastase.
Antigen-specific suppressor T-ceil hybridomas release soluble suppressor factors (TsF) in the supernatant that modulate both in vivo delayed-type hypersensitivity and in vitro plaque-forming cell responses in an antigen-specific manner.
The alkaline proteinase gene from Pseudomonas Escherichia coli.aeruginosa IFO 3455 was cloned and expressed inThe roles of several extracellular products have been established or implicated in the pathogenicity of Pseudomonas aeruginosa. These include proteases, phospholipase, hemolysin, exotoxin A, and exoenzyme S (6,8). Protease pathogenicity is explained by an aggressin activity (7), whereby the virulence of non-protease-producing strains is increased by the administration of a minute amount of protease to an experimental animal infected with P. aeruginosa. P. aeruginosa can produce two or three proteases (5), one of which is alkaline proteinase. This enzyme was crystallized from fermentation broth of strain PM and from that of IFO 3080. Alkaline proteinase can be regarded as a metalloproteinase, since it is inactivated by the addition of o-phenanthroline (7,8). In the present study we were able to clone the alkaline proteinase gene; the characterization of the cloned gene, as well as its restriction map, is also given.P. aeruginosa IFO 3455 and IFO 3080 were obtained from the Institute for Fermentation, Osaka, Japan. Crystal alkaline proteinase of P. aeruginosa IFO 3080 was obtained from Nagase Biochemical Co., Kyoto, Japan. Anti-alkaline proteinase antibody was produced in rabbits by injecting 0.4 mg of alkaline proteinase, which had been 80% inactivated by Formalin, per animal. The anti-alkaline proteinase antibody was detectable at 32x by Ouchterlony assay.As a first step, we constructed the SphI-digested genomic libraries from P. aeruginosa IFO 3455. The libraries were screened with rabbit anti-alkaline proteinase antibody and then treated with peroxidase-labeled anti-rabbit immunoglobulin antibody. By using this procedure, several clones were obtained out of about 6,000 possible clones. However, when the antibody assays were redone, only one clone [Escherichia coli HB101(pAP101)] reacted repeatedly. This pAP101 plasmid contained a fragment of P. aeruginosa DNA of approximately 5.3 kilobases (kb). From this clone, we constructed the subclone pAPDS2 by unidirectional digestion with exonuclease III (2, 3, 9); this subclone possessed both the antigenicity and enzyme activity of alkaline proteinase. Figure 1 shows the restriction enzyme map of the cloned gene.The alkaline proteinase activity of each clone was studied. E. coli HB101(pAP101), E. coli HB101(pAPDS2), E. coli HB101(pUC18), and parental E. coli HB101 were cultured for 24 h at 37°C in L broth. The bacteria were washed, and these suspensions were sonicated and then ultracentrifuged at 42,000 x g for 10 min. The supernatant fractions were * Corresponding author. assayed to determine enzyme activity as the soluble fraction ( Table 1). The precipitants were solubilized with 8 M urea. Solubilized samples of 42,000 x g precipitants were again ultracentrifuged and dialyzed against 10 mM Tris buffer (pH 8.0). Only these supernatants were used as the bacterial precipitate fractions. Alkaline proteinase activity was determined by a modification of the method...
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