IS186 insertion at a hot spot in the lon promoter as a basis for lon protease deficiency of Escherichia coli B: identification of a consensus target sequence for IS186 transposition

Abstract

The radiation sensitivity of Escherichia coli B was first described more than 50 years ago, and the genetic locus responsible for the trait was subsequently identified as lon (encoding Lon protease). We now show that both E. coli B and the first reported E. coli K-12 lon mutant, AB1899, carry IS186 insertions in opposite orientations at a single site in the lon promoter region and that this site represents a natural hot spot for transposition of the insertion sequence (IS) element. Our analysis of deposited sequence data for a number of other IS186 insertion sites permitted the deductions that (i) the consensus target site sequence for IS186 transposition is 5'-(G)(> or =4)(N)(3-6)(C)(> or =4)-3', (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N(3-6) sequence, and (iii) in a majority of instances, at least one end of the duplication is at the G-N (or N-C) junction. IS186-related sequences were absent in closely related bacterium Salmonella enterica serovar Typhimurium, indicating that this IS element is a recent acquisition in the evolutionary history of E. coli.

FIG. 1

PCR analysis of lon locus in various E. coli strains. Primers A and B (corresponding to two sites in lon) and primer C (corresponding to an internal site in IS186) were used for PCR in the pairwise combinations indicated with genomic DNA templates of the following strains: 1, MG1655 (K-12 wild type); 2, GJ1823 (K-12 lon); 3, AB1899 (K-12 lon); 4, B/r (lon). Methods for PCR and electrophoresis on 0.9% agarose gel were as described previously (22). At the left are shown the positions of DNA markers (sizes in kilobases). Estimated sizes of the observed PCR products were as follows: MG1655 (primers A and B), 0.7 kb; all lon mutants (primers A and B), 2.0 kb; AB1899 and GJ1823 (primers B and C), 1.0 kb; E. coli B/r (primers A and C), 1.1 kb.

FIG. 2

Molecular identities of various lon:: IS186 insertions. Shown on top is the (double-stranded) sequence of the relevant region of the wild-type lon⁺ locus (4), and the −10 motif in its promoter region is overlined. Beneath are given (lines 1 through 4) the lon locus sequences determined in this study (using an automated DNA sequencer and associated protocols) for K-12 strains AB1899 and GJ1823 and for E. coli B/r, as well as that reported earlier (13) for plasmid pBLI. In each case, the IS186 sequence (uppercase) is denoted in an abbreviated form by a few nucleotides from the inverted repeat at either end and by the orientation of IS insertion (marked, respectively, as I or II depending on whether the PstI site in IS186 is to the left or to the right of the BamHI site within the element); the orientation in pBLI (∗) was not reported (13). The IS186 sequence itself is represented as having a 25-bp pair of inverted repeats as its ends (15). The flanking host sequence duplications are boxed. Arrows, postulated pairs of staggered endonucleolytic cleavage sites (1 through 4, as above) in the target to generate the four different IS186 insertions.

FIG. 3

Southern blot hybridization of genomic-DNA preparations from various strains using the IS186 probe. Experimental methods were as described previously (22). The DNA preparations were digested with EcoRV, BglI, or PvuII as indicated and subjected to Southern blot hybridization with a ³²P-labeled PstI-BamHI internal fragment of IS186 (obtained from plasmid pHYD138 [21]), followed by autoradiography. Strains employed: 1, MG1655 (K-12 wild type); 2, B/r; 3, AB1899 (K-12 lon); 4, GJ1823 (K-12 lon); 5, BL21 (E. coli B); 6, S. enterica LT2. At the left are shown the positions of migration of DNA markers (sizes in kilobases). Arrows, lon-specific bands in the lanes corresponding to AB1899 and GJ1823; their sizes (5.6 [EcoRV] and 8.4 kb [BglI]) are consistent with those expected from IS186 insertion in the cognate lon-bearing restriction fragments of wild-type K-12 (4, 20).