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Abstract

CLONING PROTEASE GENE FROM PSEUDOMONAS AERUGINOSA ISOLATED FROM HUMAN CORNEAL ULCERATION CASE

Rabab Omran*

ABSTRACT

previous paper, clinical strain Pseudomonas aeruginosa was isolated from human corneal ulceration case. The bacterial cells secreted the extracellular alkaline protease that was partially purified and characterized. The aim of this paper is determined some antibiotics resistance and pyocyanin pigment production , as well as introduced protease encoding gene or genes into Escherichia coli HB101 to study and understand the role of protease enzymes in bacterial pathogenicity to develop therapeutics against P. aeruginosa future researches. Results: The isolate appeared pyocyanin pigment production and extracellular protease and has multiple drug resistances to twelve antibiotics including ampicillin, amikacin, amoxicillin, carbencillin, chloramphenicol, cefotaxime, erythromycin, lincomycin, penicillin, streptomycin, tetracycline, and trimethoprim and it is sensitive to ciprofloxacin, gentamycin, neomycin and rifampicin. The plasmid profile of the isolate revealed that the presence of single large plasmid on agarose gel electrophoresis. Plasmid transfer experiment including plasmid transformation and bacterial conjugation into the plasmidless competent cells of E. coli MM 294 (Rifr), as the recipients strain at a frequency 5x 10-3 and1.2x10-4 respectively, appeared that transformed E. coli cells acquired all antibiotic resistances but lincomycin and pyocyanin production. While the transconjugant cells acquired pigment production (pyocyanin) on King's medium and all studied antibiotic resistances including lincomycin that may be indicated multiple resistance genes related to the presence of the conjugative plasmid R68.45 or a derivative of R68 that available in P. aeruginosa. This plasmid is able to mobilize the bacterial chromosome genes from many origins and may be the lin resistance gene and pigment genes included in this transfer into E. coli MM294 strain. Pseudomonad genes were introduced into E. coli HB101 Smr, after purified total DNA and cloning vector pACYC184 were cleft by EcoR I restriction endonuclease at frequency 4.3 x 10-3. Subsequently, these clones screened on skimmed milk agar to select protease – producing clones. Only 32 clones showed variable ability to produce protease enzyme out of 86 clones. These clones phenotypically divided into two groups, the first one appeared complete casein digestion (12 clones) on skimmed milk agar plate (clear zone) and the other group (20 clones) has low caseinolytic activity that may be indicated the DNA fragment encoding protease miss certain sequence caused less activity of protease enzyme or most enzyme molecules accumulate intracellular or in periplasmic space of bacterial cells and few enzyme molecules secreted from cells to the medium or may be these clones encoding different protease enzymes.

Keywords: Pseudomonas aeruginosa, protease gene, cloning, antibiotic resistance.


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