H-089. Regulation of the Convergent Pint and P2 Promoters of the Tn21 Class I Integron

C. A. Cagle, A. O. Summers;
Univ. of Georgia, Athens, GA.

Background: An integron is a genetic element encoding a site-specific tyrosine recombinase called an integrase that inserts and excises small, mobile gene cassettes at an adjacent attachment site, called attI. There are four defined integron classes and the Class I integron In2 of Tn21 has two cassette promoters that converge with its Pint promoter; a weak promoter Pc, is 175 bp 5’ of the attI, and a strong promoter, P2, is 56bp from the insertion point and its -10 overlaps with the Pint -10. P2 is rare, occurring in only 6 of 50 Class 1 integrons; all others have a 3-base deletion within the P2 spacer which, interestingly, gives them a LexA site absent in In2. However, the control region of In2 has sites for FIS (2 sites overlapping the -10 regions) and IHF (in the -35 of P2). Methods: We deleted the distant, weak Pc cassette promoter to focus on the overlapping Pint and P2 promoters, fusing them to bidirectional transcriptional reporters, LacZ and PhoA, respectively. We examined the wildtype promoters and mutants disabling P2 (removal of -35 or of 3 bp in the spacer) or optimizing Pint (to consensus -10) over the entire culture cycle in wildtype, fis- ,ihf-, and lexA- hosts. Results: In all host backgrounds Pint and P2 expression increased in late log phase and plateaued in stationary phase. Wildtype Pint was very weak, but removing 3-bp from the P2 spacer did not increase Pint although P2 lost 94% of its activity. Optimizing the Pint -10 hexamer increased both Pint and P2 expression (they share 3 bp). Expression of wildtype Pint increased in fis- and decreased in ihf- hosts but neither affected P2 expression. Expression of wildtype Pint and P2 did not change in lex- host, but shortened spacer P2 had increased expression. Conclusions: Oddly, convergent Pint and Pc appear not to impede each other, and although Pint is activated by IHF (abundant in early to mid-log) and repressed by FIS (declines in late log), neither regulator affects overlapping P2 expression. Also, the absence of LexA does not affect either wildtype promoter, but does affect P2 3bp deletion promoter. These first observations on Pint and P2 expression indicate this system is under intricate and subtle control.