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The Genes Involved In The Initiation Of Biofilm Formation In Paeudomonas Aeruginosa

Posted on:2005-08-15Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z Y DanFull Text:PDF
GTID:1100360125955512Subject:Genetics
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Pseudomonas aeruginosa is an important opportunistic pathogen of humans. The bacteria can colonize a variety of surfaces and form biofilm, which causes persistent infection in the host Swimming motility mediated by flagellum and twitching motility mediated by type IV pili play very important role in the initiation of biofilm formation, mutants deficient in twitching and swimming loss ability of initiating biofilm formation. In this study, Mu transposition complexes technique was used to study the genes involved in twitching and swimming of P. aeruginosa strain PA68, which was isolated from a patient with bronchiectasis.Mu transposition complexes technique had not previously been applied to Pseudomonas spps and electrotransformation of the clinical isolate PA68 had not been done previously, so the electroporation conditions were optimized first. Optimum conditions including growth stage of the strain, electric field strength, concentration and preservation of competence cells were defined using plasmid pSMC28 instead of MuA transposition complexes. It showed that the highesttransformation efficiency of pSMC28 could be obtained up to 1.68 x 10 CFU/ug DNA under the optimum conditions in which the competent cells were collected at logarithm stage (OD54o=0.7-0.8) and concentrated up to about 1011cells/ml, field strength was set to be 13 kV/cm. The optimal condition was confirmed using MuA transposition complexes as well. With the optimal conditions, Mu transposition complexes have been successfully introduced into P. aeruginosa strain PA68 and obtained efficiency as high as 3.66 104 CFU/ug DNA, and a pool of 6000 mini-Mu insertion mutants were established, which is large enough to isolate swimming or twitching deficiency mutants of PA68.4 mutants deficient in swimming motility and 11 mutants deficient in twitching motility were isolated out of about 2000 mini-Mu insertion mutants. Southern blotting confirmed that the insertions had occurred as single events. DNA sequencing of the region flanking the insertion revealed that the mini-Mu transposon had inserted into four different genes (PA0171, zwf, phzF1 and uvrD) in swimming motility deficiency mutants, and 8 different genes (PA0171, PA0413, PA1821, PA1822, PA4959, pilV, pilQ and algR) in twitching motility deficiency mutants. The function of six of these genes has been previously demonstrated; While the function of PA1821andPA0413 is predicted by their putative protein structure feature or conserved motif, but short of experimental evidence; PA4959 is recently proven to be a signal protein required for twitching motility. The remaining two genes, PAOl 71 and PA1822 are entirely novel with unknown function.The complete nucleotide sequences of PAO171 and PA1822 were determined. Alignment analysis showed gene PAO171 and its putative protein have no significant homology with any gene or protein with known function in P. aeruginosa or other bacteria, while the predicted product of PA1822 shows 51% similarity and 38% identity with a signal transduction protein PilL.The twitching motility defect in the PA1822 mutant was partially complemented by providing the PA1822 gene in trans, and the twitching and swimming defect in the PAO171 mutant was fully complemented when PAO171 was provided. A PAO171 mutant and a PA1822 mutant were constructed by gene replacement in the P. aeruginosa PAOl strain, motility assay showed that PAO1PA1822 mutant deficient in twitching, while PAOlPA0177mutant loss the ability of twitching and swimming.Flagellaand pili examination were performed using transmission electron microscopy. Type IV pili of PAO171 mutant C54 could not be detected while PA1822 mutant K2 contains regular type IV pili, both C54 and K2 contain flagellum just like wide type strain PA68, suggesting that PAOl 71 is probably a regulation gene which control both the biogenesis of type IV pili and motility of P.aeruginosa; PA1822 is probably a signal protein which involved in the regulation of twitching motility.In this study, Mu transposition complexes technique was first...
Keywords/Search Tags:Pseudomonas aeruginosa, Mu transposition complex, swimming motility, twitching motility, trans-complementation
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