Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Robert E. Andrews, Jr.
Tn916 is a 18 kb, broad-host range conjugative transposon originally isolated from Enterococcus faecalis. Transposon TnphoA was used to identify genes in Tn916 encoding for membrane-targeted proteins or for secretion. Sixty five independent PhoA+ gene fusions were isolated. These insertions clustered into two regions, open reading frames 13 and 15 (Flanagan et al., Plasmid 32:350-354). These TnphoA insertional mutations in resulted in a Tn+ (ability to transpose intracellularly) and Tra- (inability to transfer conjugatively) phenotype. The hydrophathy analysis of ORF13 and ORF15 revealed that these proteins lack the classical signal peptides necessary for protein export, suggesting that these two proteins might be exported out of the cytoplasm independent of the classical signal peptides. The absence of classical signal peptides from these two proteins suggested that ORF13 and ORF15 were to be located in the inner membrane of Escherichia coli; this observation was confirmed by TnphoA gene fusion studies. From the amino acid analysis of ORF13 and ORF15, the membrane topologies of these two proteins were predicted, with ORF13 having one membrane spanning region and ORF15 eight membrane spanning regions. Furthermore, TnphoA insertional mutagenesis in open reading frame (orf)13 and orf15 blocked the conjugal transfer of Tn916 from B. subtilus into B. thuringiensis. Moreover, Tn916-dependent mobilization of non-conjugative plasmid pC194 was also reduced by about 11-fold and 680-fold, respectively. The insertion of Tn5 in the orf2l of Tn916 was shown to reduce the transfer of Tn916 by about 7000-fold and the Tn916-mediated conjugal transfer of pC194 by about 250 fold. Because the sequence analysis of orf21 revealed that the putative ORF21 has a strong homology with the SpoIIIE of the B. subtilus, the function of ORF21 is proposed to be similar to the SpoIIIE. ORF21 may play a crucial role as a DNA transport protein either within the cell or across the cell membrane during the conjugation.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Loe, Suganda, "Identification of genes in Tn916 encoding membrane-targeted proteins" (1997). Retrospective Theses and Dissertations. 11484.