Document Type

Dissertation

Date of Degree

Fall 2009

Degree Name

PhD (Doctor of Philosophy)

Degree In

Microbiology

First Advisor

Steven Clegg

Abstract

DNA adenine methyltransferase (Dam) plays an important role in different bacterial functions. It has been shown that Dam is required for regulation of bacterial replication initiation and is required for proofreading newly synthesized DNA through methylation directed mismatch repair. Dam is also involved in the regulation of different genes and is required for virulence in several different bacterial genera though its degree of importance depends on the specific bacteria being studied. During this work, a Dam-negative strain (JSM1) was constructed in Klebsiella pneumoniae strain 43816 to ascertain its importance for K. pneumoniae viability and virulence.

To test JSM1 for expression of fimbrial virulence factors, agglutinations were used to detect the presence of type three and type one fimbriae, respectively. No differences between 43816 and JSM1 were discernable. Similarly, JSM1 production of capsular material appeared to be unaltered.

K. pneumoniae JSM1 virulence in a murine model was examined following intranasal or intraperitoneal inoculation, and it was determined that JSM1 is partially attenuated. Quantitative analysis of 43816 and JSM1 biofilm growth revealed only slight decreases in JSM1 biofilm mass and thickness, but live/dead staining of developed biofilms showed decreased JSM1 biofilm viability over time compared to 43816 biofilms. JSM1 was also examined for alterations in the frequency of spontaneous antibiotic resistance mutations and tested for increased susceptibility to various DNA damaging agents, and statistically significant differences were found for some of the spontaneous antibiotic resistance mutation frequencies tested.

Fimbriae in K. pneumoniae are important virulence factors which facilitate respiratory and urinary tract infections in vivo. They also contribute to formation of biofilms which are believed to cause chronic infections and increased antibiotic resistance. Searches for homologous regions within the Klebsiella chromosome using the chaperone and usher components of E. coli type 1 fimbriae revealed five putative fimbrial gene clusters on the Klebsiella chromosome which had not been characterized. Mutations created within select gene clusters did not yield detectable deficiencies in biofilm formation or murine respiratory virulence. However, based on the multiplicity of fimbrial expression observed in Salmonella enterica serovar Typhimurium, combinational mutations may be required prior to detection of a discernable phenotype.

Keywords

Dam, DNA adenine methylase, DNA adenine methyltransferase, fimbriae, Klebsiella

Pages

xi, 131 pages

Bibliography

Includes bibliographical references (pages 123-131).

Comments

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Copyright

Copyright 2009 Joanna Sue Kuehn

Included in

Microbiology Commons

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