Dissertation

Characterization of Gene Expression During Biofilm Development in Mycobacterium smegmatis, and Genetic Analysis of a Surface Translocation – Defective Transposon Mutant

Balachandran, Amrita

2012

Overview

Tuberculosis is the leading cause of death due to a single infectious agent, and over one-third of the global population is infected with Mycobacterium tuberculosis, the etiologic agent of human tuberculosis. In recent years, the likelihood of biofilm-based infections contributing toward bacterial persistence and increased drug tolerance, has gained some recognition. M. tuberculosis and its non-pathogenic fast-growing relative, M. smegmatis, have been found to form biofilms that harbor bacteria that are more resistant to anti-tuberculosis agents than free-living cells. Biofilm formation involves the development of several distinct morphological structures, with associated physiological features. Bacteria growing within biofilms exist as heterogeneous populations, dependent on the distinct micro-environments within the complex community structure. Consequently, gene expression profiles differ between sub-populations of cells, and also during distinct stages of biofilm development. Gene expression in biofilms also differs greatly from the gene expression profiles of planktonically growing cells of the same species. M. smegmatis biofilms can serve as a model for other mycobacterial biofilms. Transcriptome analyses of M. smegmatis biofilms have led to the identification of several genes that were induced in a biofilm-specific pattern. Iron plays an essential role in mycobacterial growth, metabolism and infection. Taken together with its significance in biofilm development, the detailed profiling of the expression of iron acquisition genes in mature biofilms would provide insight into the physiological state of the bacterial cells within these structures. Using stable fluorescent reporter constructs, we have provided a detailed profile for the expression of the intramembrane-associated siderophore, mycobactin. Our results suggest that mycobactin biosynthesis is differentially induced in biofilms and in liquid cultures. In mature biofilms, a significant proportion of cells induce mycobactin biosynthesis in spite of the availability of iron-rich conditions. Our analyses also attempt to sort out subsets of cells within the biofilm that differentially induce mycobactin biosynthesis. In a related study undertaken to understand the relationship between biofilm formation and surface translocation in M. smegmatis, we have isolated a transposon mutant that is defective in sliding motility, but proficient in biofilm formation. This mutant suggests that biofilm formation in M. smegmatis does not depend on sliding motility.

• Publisher: ProQuest Dissertations & Theses
• Subject: Microbiology / Molecular biology
• ISBN: 9781267660626, 1267660627