Title of Dissertation: TOWARD A BETTER UNDERSTANDING OF THE CELLULAR, MOLECULAR AND GENETIC BASES OF THE RUGOSE MORPHOLOGY OF SALMONELLA TYPHIMURIUM

Title of Dissertation: TOWARD A BETTER UNDERSTANDING OF THE CELLULAR, MOLECULAR AND GENETIC BASES OF THE RUGOSE MORPHOLOGY OF SALMONELLA TYPHIMURIUM Yuda Adha Anriany, Doctor of Philosophy, 2005 Dissertation directed by: Professor Sam W. Joseph Department of Cell Biology and Molecular Genetics The origin of rugose (wrinkled) colony morphology of Salmonella Typhimurium, which is formed only at stationary phase, low temperature, and under low osmolarity, is attributed to the production of an extracellular matrix and is associated with biofilm and pellicle formation, likely for survival strategies. The regulator CsgD is required for the synthesis of both matrix components: curli, encoded by the csgBAC operon, and cellulose, encoded by the bcs operon. Transcription of csgD, in turn, depends upon a number of transcriptional regulators such as σ, OmpR and HNS. Using random mutagenesis, two groups of mutants with altered rugose phenotype were identified. The two mutations in the first group caused retardation of rugosity and altered waaG and ddhC, which are required for synthesis of the core and O antigen of lipopolysaccharide synthesis, respectively. Both mutants exhibited lack of motility, decreased levels of curli, and, especially in the waaG mutant, increased cellulose production. In media containing high osmolarity, both mutants produced more biofilms. Non-polar gene knockout and complementation performed on the waaG further confirmed these phenotypes in this transposon mutant. Thus, alteration in the LPS seemed to influence both curli and cellulose in opposing manners, and appeared to direct cells toward alternative pathways to produce biofilm matrix. The regulatory mutation in the second group affected hfq, and produced only minimal amounts of cellulose and curli protein. This phenotype was confirmed in an hfq deletion mutant. Transcriptional fusion between the csgB or csgD promoter and lacZ showed a drastic reduction in activity of both promoters in an hfq mutant compared to that in the wt. These were surprising results given the known function of Hfq as a posttranscriptional regulator, including in the regulation of σ-encoding gene rpoS. However, when the promoter activity was measured in an rpoS hns background, where transcription continues under σ, significant reduction was still shown in the hfq mutant. Deletion of the gene that codes for DsrA, a sRNA which, together with Hfq, is required for translation of rpoS at low temperatures, had minimal effect in both promoters. These results indicate that Hfq may regulate both promoters independent of σ. TOWARD A BETTER UNDERSTANDING OF THE CELLULAR, MOLECULAR AND GENETIC BASIS OF THE RUGOSE MORPHOLOGY OF SALMONELLA TYPHIMURIUM