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Studies in the regulation of the gene transfer agent (GTA) of Rhodobacter capsulatus Florizone, Sarah Marie

Abstract

The gene transfer agent (GTA) of Rhodobacter capsulatus acts as a system of genetic exchange in this purple, non-sulphur, photosynthetic bacterium. GTA is a small bacteriophage-like particle that transfers random 4.5 kbp segments of the donor cell’s genome to recipient cells where allelic replacement can occur. The structural gene cluster for GTA encodes 15 open reading frames, most of which are homologous to known phage genes. Three proteins have been found that regulate the expression of GTA genes: GtaI, CtrA and CckA. GtaI is involved in quorum sensing while CtrA and CckA are thought to be part of a sensor kinase/response regulator signalling pathway. I set out to elucidate this poorly understood regulatory pathway. My initial hypothesis was that there are other regulatory proteins besides GtaI, CtrA and CckA, as mutations in any of the genes disrupts, but does not completely abolish, GTA structural gene expression. However, during mutant screening, it became apparent that the reporter plasmid used to monitor GTA gene expression (pYP) caused a decrease in GTA transduction, which I termed the pYP effect. Promoter analysis was performed to determine which part of plasmid pYP was causing this effect and to study cis-active regulatory sequences of the GTA structural gene cluster. Sequencing the GTA promoter from 3 strains that exhibit different levels of GTA gene expression and transduction did not find any differences, suggesting a trans-acting factor causes the observed differences. 5’ mRNA mapping was used to find the 5’ end of the GTA transcript, which overlaps with the upstream gene. Capsid levels throughout the growth curve were measured for Y262 (GTA overproducer), B10 (wild type), YCKF2 (ctrA⁻), and YKKR2 (cckA⁻). The immunoblots from these strains showed interesting differences in capsid production that give us a better understanding of GTA production and release from the cell in different stages of growth. Although I was unable to provide a clear pathway for the regulation of GTA, the data in this thesis gives us a better understanding of the complexity of GTA production and regulation.

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