The Hda protein plays an essential role in inactivation of the initiator protein DnaA from its active, ATP bound form to the inactive DnaA-ADP in E. coli. Cells deficient in Hda suffer from overinitiation, asynchronous initiation and cell death as a consequence of an increased DnaAATP/ DnaA-ADP ratio . E. coli can suppress the growth defects caused by Hda deficiency by several different mechanisms. The focus of this Ph.d. thesis is to understand the mechanisms that underlie suppression of Hda deficiency in E. coli. These approaches are described in two manuscripts and one published paper.
Over expression of Ribonucleotide reductase encoded by either nrdAB or nrdEF has been shown to suppress Hda deficiency. The nrdAB promoter contains four consensus binding sequences for DnaA and a 45bp inverted repeat important for cell cycle regulation of nrdAB transcription. In manuscript 1 we show that deletions of the DnaA-boxes or the 45bp inverted are likely to lead to a decreased transcription of nrdAB and that these mutants are synthetic lethal in combination with loss of the hda gene. Furthtermore we show that suppression of Hda deficiency is dependent on the degree of nrdAB overexpression.
Deletion of the hda gene causes cells to accumulate suppressor mutants (hsm). In manuscript 2, we characterize the two strains iscUC63F and freΔ68 that contain mutations in the iscU gene encoding an iron sulfur cluster scaffold enzyme and in the fre gene encoding flavin reductase respectively. We find that suppression of Hda deficiency is a consequence reduced gene function in iscUC63F and loss of gene function in freΔ68. We suggest that the mechanism of Hda suppression is based on a mimicked anaerobic growth in both strains.
Lastly we show in paper I that the otherwise lethal overinitation of replication in Hda deficient cells can be tolerated under anaerobic conditions so that a Δhda strain can maintain growth without accumulating any further mutations in the chromosome. We also show that deletion of mutM that is a part of to GO repair system and is responsible for repair of DNA damages caused by reactive oxygen species, suppresses the growth deficiencys in a Δhda strain.