Regulation of gene expression in Bacillus subtilis macrofiber by environmental physical stimuli.
AdvisorMendelson, Neil H.
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PublisherThe University of Arizona.
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AbstractExtensive studies indicate that both genetic and epigenetic (physiological and biomechanical) factors play a role in the development of twist state which must correspond to the establishment of cell surface conformational state at the level of cell wall assembly. Therefore, in order to identify the unknown factors that control the macrofiber production, twist states and hand inversion, genetic studies concerning regulation of macrofiber production and macrofiber structural states seemed to be appropriate. Genetic studies were carried out by using an insertional mutagenesis method. Bank(s) of insertions were obtained that carry the Tn917 transposon at random locations in the genome. Selected isolates were characterized with respect to macrofiber production and twist, and helix hand inversion stimulated by various physiological factors. The bank(s) of insertional mutants were searched for those defective or impaired in response to ion-induced hand inversion. None were found to exhibit the desired phenotype. Clones with altered static state were not rare. Another approach was to take advantage of the transposon "lac system" and to use the bank of insertion mutants to study regulation of gene expression. The chromogenic substrate for β-galactosidase, X-gal, made possible the search for factors governing gene expression during macrofiber morphogenesis in a manner similar to the way in which developmental biologists study regulation of gene expression during embryogenesis. First, insertion strains were screened for lac-Z expression on TBAB (Tryptose Blood Agar Base) X-gal plates. Isolates were then characterized by growth in fluid media. One strain (3:1) was found that expressed the E. coli lac-Z structural gene when grown on solid media (TBAB X-gal), but not when grown in fluid media. These observations led us to an examination of the role the medium may play in the regulation of gene expression. Evidence was obtained indicating that a number of insertion strains respond to growth in viscous media by expression of lac-Z+ indicating that different host gene promoters can be regulated by a physical component of the environment. The degree of expression moreover was positively correlated with the degree of viscosity. Environmental physical forces applied to the "body" of a bacterial cell must therefore play a role in gene expression. In at least one strain, 5:7Oring, gene expression was found only in right-handed structures suggesting that either specific genes are involved in the twist state and hand determination or that helix hand itself may govern gene expression. Finally, the 5:7Oring strain shows also the presence of a probable intercellular signalling through a diffusible chemical that causes gene expression to occur only in certain cells found at specific locations within the population.
Degree ProgramMicrobiology and Immunology