Title

Development of genetic methods in the hyperthermophilic bacterium {\it Thermotoga neapolitana\/}

Date of Completion

January 1995

Keywords

Biology, Genetics|Biology, Cell|Biology, Microbiology

Degree

Ph.D.

Abstract

Thermotoga neapolitana is a hyperthermophilic bacterium that branches very early from the Bacterial lineage, suggesting that some of its features may be ancestral to all bacteria. Physiological studies in this ecologically and evolutionarily intriguing organism would be facilitated by development of a gene transfer system.^ Uniform growth and high plating efficiencies of T. neapolitana were achieved using a mineral medium with the addition of 0.7% Gelrite (a solidifying agent) and cysteine (a reducing agent). A number of analog-resistant and auxotrophic mutants were generated to serve as markers for gene transfer. In addition, a vector was constructed conferring chloramphenicol resistance that could serve as a potential shuttle vector between Thermotoga and Escherichia coli. Methods of gene transfer tested included conjugation, natural transformation and electrotransformation. No transformants were observed using any method; electrotransformation was attempted under a variety of biological, electrical and physical conditions.^ $\beta$-galactosidase ($\beta$-gal) was induced when cells were grown on lactose, galactose and cellobiose. Cells grown on 0.5% glucose, maltose, fructose, sucrose, xylose, ribose or starch had no measurable $\beta$-gal activity. Cells grown on the combination of 0.5% lactose or galactose and 0.05% glucose had no detectable $\beta$-gal activity, whereas up to 0.5% glucose did not prevent expression of $\beta$-gal activity in cells induced with 0.5% cellobiose. Therefore, $\beta$-gal synthesis induced by lactose or galactose is subject to catabolite repression.^ Levels of cyclic AMP (cAMP) measured in T. neapolitana cells grown on various defined media ranged from 6-20 fmol/mg of protein irrespective of the carbon source. By comparison, E. coli grown on lactose contained 3.1 pmol/mg of protein. Like gram-positive bacteria, T. neapolitana displays a cAMP-independent mechanism for catabolite repression and this may represent the more ancient mode of regulation. ^