| Summary: | 碩士 === 國立交通大學 === 生物科技系所 === 102 === As the other pathogenic bacteria, Klebsiella pneumoniae often encounters the challenge of iron depletion surroundings in the host cells. The iron transport system encoding operon feoABC, sitABCD, and efeUOB are present in the genome of K. pneumoniae CG43. Under iron rich cultured condition such as LB medium, the expression of feoABC, efeUOB and sitABCD are repressed by the global regulator Fur (ferric uptake regulator). In the study, ∆feoA∆fur, ∆feoB∆fur, ∆feoC∆fur, ∆sitCD, ∆sitCD∆fur, ∆efeUOB, ∆efeUOB∆fur, ∆feoB∆sitCD, ∆feoB∆efeUOB, ∆sitCD∆efeUOB, ∆efeoUOB∆sitCD∆feoB and ∆efeoUOB∆sitCD∆feoB∆fur derived from K. pneumoniae CG43S3 have been generated. The mutation effects on the bacterial growth and virulence-related properties such as capsular polysaccharide (CPS) production, oxidative stress responses, type 3 fimbriae expression, and biofilm formation were analyzed and compared. The putative promoter of the three iron transport systems were isolated and individually cloned in front of the promoterless lacZ on the reporter plasmid pLacZ15 for the optimal expression and condition analysis using β-galactosidase activity. All three iron transport systems could be induced by the absence of Fur repressor and depleting iron from the culture medium. The feo promoter was negatively influenced under iron-depleted and microaerobic condition by FeoC, a predicted Fe-S sensor regulator. The microaerobically-induced sitABCD showed the highest promoter activity among these three systems. SitABCD plays important role in protecting bacteria from oxidative stress, with combined loss of sit and feo further decreased bacterial oxidative-resistance. EfeUOB is induced by weak acid and negatively regulated by the two-component regulator CpxR. This indicates its specific role in aerobic, iron-depleted and low pH environment. Although iron-availability affects CPS biosynthesis, FeoABC, SitABCD and EfeUOB transport systems do not involved in regulation of CPS biosynthesis. Except ∆sitCD∆efeUOB and ∆feoC, deletion of genes from these acquisition systems decreased biofilm formation of K. pneumoniae CG43S3, wherein iron chelators deferoxamine exerted more decreasing effects of biofilm formation. Interestingly, these deletion mutants did not decrease the expression of MrkA, the major pilin of type3 fimbriae which is iron dependent and the major determinant of biofilm formation. These results imply that these ferrous iron transport systems regulate biofilm formation independently of the expression of type 3 fimbriae.
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