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Identification of genes required for...

Hunt, Tracey A.

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Identification of genes required for survival of Burkholderia cenocepacia strain K56-2 in vivo using a modified method of signature-tagged mutagenesis.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Identification of genes required for survival of Burkholderia cenocepacia strain K56-2 in vivo using a modified method of signature-tagged mutagenesis./
Author:	Hunt, Tracey A.
Description:	198 p.
Notes:	Source: Dissertation Abstracts International, Volume: 65-11, Section: B, page: 5534.
Contained By:	Dissertation Abstracts International65-11B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=NQ96829
ISBN:	0612968294

Identification of genes required for survival of Burkholderia cenocepacia strain K56-2 in vivo using a modified method of signature-tagged mutagenesis.
Hunt, Tracey A.

Identification of genes required for survival of Burkholderia cenocepacia strain K56-2 in vivo using a modified method of signature-tagged mutagenesis. - 198 p.

Source: Dissertation Abstracts International, Volume: 65-11, Section: B, page: 5534.

Thesis (Ph.D.)--The University of Western Ontario (Canada), 2004.

Bacterial isolates of the Burkholderia cepacia complex are opportunistic pathogens commonly associated with chronic lung infections in cystic fibrosis patients. Bacterial factors that allow persistence and survival of these microorganisms are not well understood and only some virulence factors have been characterized to date. We hypothesized that some virulence determinants are preferentially expressed following interactions with the host and, therefore, some virulence factors may be discovered using in vivo approaches that would otherwise not be identified using conventional in vitro techniques. We tested the feasibility of a modified version of signature-tagged mutagenesis (STM) to uncover bacterial factors required for survival of B. cenocepacia in vivo. For this, we constructed specialized plasposons carrying unique oligonucleotide tags that can be used to track transposon insertions by PCR. We analyzed the survival of pools of transposon mutants of B. cenocepacia strain K56-2 in a rat agar bead lung infection model. We also established conditions for the rapid and specific screening of recovered mutants by real-time PCR. 2627 B. cenocepacia transposon mutants in pools of 37 were screened and, following two rounds of infections, we identified 101 mutants that showed attenuated growth in vivo. These results suggest that the modifications we applied to the traditional STM method are effective for the identification of factors required for the in vivo survival of isolates of the B. cepacia complex. We analyzed the chromosomal regions flanking the transposon insertion in each of the 101 mutants to identify genes involved in cellular metabolism, regulatory genes, and genes involved in DNA replication and repair. We found at least 16 genes of unknown function, some of which contain homologues in other microorganisms. We also found that many genes encoding bacterial surface structures, such as transmembrane proteins and enzymes that synthesize lipopolysaccharide, are required for survival in this animal model. This study is the first to identify genes required for survival of B. cepacia complex isolates in vivo and the genes identified will provide valuable insight into the pathogenesis of this opportunistic pathogen, as well as a better understanding of the interactions of the microorganism and the host during an infection.

ISBN: 0612968294Subjects--Topical Terms:

1017734
Biology, Microbiology.

Identification of genes required for survival of Burkholderia cenocepacia strain K56-2 in vivo using a modified method of signature-tagged mutagenesis.
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100 1 $a Hunt, Tracey A. $3 1931135
245 1 0 $a Identification of genes required for survival of Burkholderia cenocepacia strain K56-2 in vivo using a modified method of signature-tagged mutagenesis.
300 $a 198 p.
500 $a Source: Dissertation Abstracts International, Volume: 65-11, Section: B, page: 5534.
500 $a Adviser: Miguel A. Valvano.
502 $a Thesis (Ph.D.)--The University of Western Ontario (Canada), 2004.
520 $a Bacterial isolates of the Burkholderia cepacia complex are opportunistic pathogens commonly associated with chronic lung infections in cystic fibrosis patients. Bacterial factors that allow persistence and survival of these microorganisms are not well understood and only some virulence factors have been characterized to date. We hypothesized that some virulence determinants are preferentially expressed following interactions with the host and, therefore, some virulence factors may be discovered using in vivo approaches that would otherwise not be identified using conventional in vitro techniques. We tested the feasibility of a modified version of signature-tagged mutagenesis (STM) to uncover bacterial factors required for survival of B. cenocepacia in vivo. For this, we constructed specialized plasposons carrying unique oligonucleotide tags that can be used to track transposon insertions by PCR. We analyzed the survival of pools of transposon mutants of B. cenocepacia strain K56-2 in a rat agar bead lung infection model. We also established conditions for the rapid and specific screening of recovered mutants by real-time PCR. 2627 B. cenocepacia transposon mutants in pools of 37 were screened and, following two rounds of infections, we identified 101 mutants that showed attenuated growth in vivo. These results suggest that the modifications we applied to the traditional STM method are effective for the identification of factors required for the in vivo survival of isolates of the B. cepacia complex. We analyzed the chromosomal regions flanking the transposon insertion in each of the 101 mutants to identify genes involved in cellular metabolism, regulatory genes, and genes involved in DNA replication and repair. We found at least 16 genes of unknown function, some of which contain homologues in other microorganisms. We also found that many genes encoding bacterial surface structures, such as transmembrane proteins and enzymes that synthesize lipopolysaccharide, are required for survival in this animal model. This study is the first to identify genes required for survival of B. cepacia complex isolates in vivo and the genes identified will provide valuable insight into the pathogenesis of this opportunistic pathogen, as well as a better understanding of the interactions of the microorganism and the host during an infection.
590 $a School code: 0784.
650 4 $a Biology, Microbiology. $3 1017734
650 4 $a Health Sciences, Public Health. $3 1017659
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710 2 0 $a The University of Western Ontario (Canada). $3 1017622
773 0 $t Dissertation Abstracts International $g 65-11B.
790 1 0 $a Valvano, Miguel A., $e advisor
790 $a 0784
791 $a Ph.D.
792 $a 2004
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=NQ96829