The long term goal of this research is to understand how stationary phase genes are regulated in the extremely halophilic Archaea. In order to do this the investigator has chosen to utilize the expression of halocin Hal R1 in Halobacterium spp. GN101 as a model system. Halocins are """"""""protein antibiotics"""""""" that are externalized by the cell and kill or inhibit other halobacterial cells. Halocin Hal R1 is first detected in stationary phase and the amount of halocin activity continues to increase throughout this period. Dr. Shand has identified 5 specific aims with respect to halocin Hal R1 expression. First is to isolate the halR1 gene using """"""""reverse genetics"""""""". Degenerate inosine containing oligodeoxynucleotides will be designed from the N- and C- terminal sequences, then used as primers in a PCR reaction to amplify the HalR1 gene from the total DNA To recover the entire gene, probes will be made from the PCR product and used to identify restriction fragments on Southern blots.
The second aim will be to correlate the levels of halR1 mRNA with halocin protein production. In addition, treatments that inhibit growth and 3H-uridine uptake in strain GN101 will be analyzed for their suitability for use in mRNA half-life experiments.
The third aim i s to isolate Hal R1 non-producing mutants. Mutants that fail to produce Hal R1 will be isolated from EMS-, MNNG-, or ultraviolet- mutagenized Halobacterium spp. GN101 that have been screened on top agar lawns of sensitive cells for lack of halocin production. Various tests such as western blots, northern blots, and marker rescue will be used to differentiate the various classes of mutants.
The fourth aim i s to characterize the expression of cloned halR1 gene in a halocin Hal R1 non-producing strain. This will allow for the determination of the minimum regulatory region required for expression using nested deletions and site directed mutagenesis. In the final aim Southern blots will be performed to determine if the halR1 gene resides on the 270 kbp megaplasmid of Halobacterium spp. GN101.
| Price, L B; Shand, R F (2000) Halocin S8: a 36-amino-acid microhalocin from the haloarchaeal strain S8a. J Bacteriol 182:4951-8 |
