Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa

Abstract

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL308OR clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa ff0 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttranscriptional behavior of this strain.Pseudomonas aeruginosa is an opportunistic pathogen which can cause fatal infections in vulnerable hosts (19). Several components related to its virulence have been identified and characterized (22). Among these, elastase acts to inactivate a variety of biologically important proteins or phenomena (12,14,(16)(17)(18)23). In addition, there is strong evidence that elastase plays an important role in the pathogenesis of P. aeruginosa infections (11,20). Although the regulatory mechanism of elastase production is still unknown, elastase-deficient Pseudomonas strains have been isolated (18). Furthermore, some elastase-positive Pseudomonas isolates often become elastase negative after several passages in vitro.We (6, 29) and another group (2, 25) have independently cloned and sequenced the elastase structural gene using the elastase-producing P. aeruginosa strains IFO 3455 and PA01, respectively. Several elastase-related products (8, 9, 21), the existence of "pro" protein (2,6,9,29), and elastase activation molecules (9, 26) have been described. In addition, Goldberg and Ohm...

Solvent-Stable <i>Pseudomonas aeruginosa</i> PseA Protease Gene: Identification, Molecular Characterization, Phylogenetic and Bioinformatic Analysis to Study Reasons for Solvent Stability

“…1 ) contained 64.2% G + C base pairs, which was found to be similar (67.2%) to the genomic DNA of P. aeruginosa [Palleroni, 1984]. The nucleotide sequence of P. aeruginosa PseA genomic DNA exhibited more than 95% homology with the las B gene of P. aeruginosa PST-01 [Ogino et al, 2000], PAO1 [Bever and Iglewski, 1988], IFO 3455 [Fukushima et al, 1989], IFO 3080, N-10, PA103 [Tanaka et al, 1991] and PA14 (GenBank accession No. NZ_AABQ07000001).…”

“…A search of databases by the BLASTP revealed that the P. aeruginosa PseA protease was most similar in sequence to the zinc metalloprotease (elastase) secreted from various P. aeruginosa strains as PST-01 [Ogino et al, 2000], PA14 and then with that of PA103, IFO 3455 and N-10 [Tanaka et al, 1991].…”

Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion

“…Elastase production and processing are facilitated by a growth medium containing both zinc and calcium ions (22). On the basis of the inferred amino acid sequence (1,5,24) and crystallographic structure (25), elastase shares a high degree of sequence and functional homology with the zinc metalloprotease thermolysin of Bacillus thermoproteolyticus. These similarities to thermol-* Corresponding author.…”

Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions

“…The sequence (1,6,31) and crystallographic structure (32) of elastase are known. It shares a high degree of sequence and functional homology with thermolysin, a zinc metalloprotease produced by Bacillus thermoproteolyticus.…”