The Physical Nature of F Plasmid TraI and its Effect During Conjugative Transfer

F plasmid;TraI;Biology

Feehery, Tegan KatharineSchroer, Trina A. ;

Johns Hopkins University

关键词: F plasmid;    TraI;    Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37182/FEEHERY-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

F plasmid TraI (192 kD) is essential for DNA transfer during bacterial conjugation. TraI is composed of a relaxase domain, ssDNA binding domain, helicase domain, and C-terminal domain. Some TraI mutants, created by the Traxler lab, have a 31 AA insertion (i31) in different domains that exhibit higher mating efficiencies than wild-type (WT) TraI.We used a live cell SeqA-YFP fusion protein system to investigate the observed higher mating efficiency. DNA appeared earlier in the recipient in the i31 mutants compared to WT, and the change was not due to a difference in second strand synthesis. To investigate where the transfer process is altered, we employed a TraI fusion protein system to determine if altering the stability of a protein inserted within the i31 of the TraI mutants would alter mating efficiency. Utilizing TraI-YFP cloned into the i31 sites, we created YFP point mutants with altered stability, G67A and I161A. Mating efficiency is reduced when YFP is inserted into TraI. However, when the YFP chromophore is destabilized (G67A), or a hydrophobic cluster is altered (161A) mating efficiency recovers to near i31 levels. These data indicate that altering the stability of the protein can affect transfer efficiency. These data also suggest that altered stability may play a role in producing the i31 phenotype. To investigate the stability and folding of the i31 mutants, a region of TraI (309-858) containing various i31 insertion points were cloned and expressed. These fragments were analyzed by circular dichroism and chemical titration denaturation to determine the ∆G of unfolding for each fragment. There were no significant differences in the ∆G values, however subtle differences in the data are consistent with stability playing a role for the increased mating efficiency for i681. These data also indicate the increased mating efficiency seen for all the i31 mutants analyzed may have multiple different causes, the nature of which is dependent on the location of the insertion.