Two amino acid residues of transposase contributing to differential transposability of IS1 elements in Escherichia coli

Abstract

Escherichia coli W3110 contains four types of IS1 elements in the chromosome. Using an insertion element entrapping system, we collected 116 IS1 plasmid insertion mutants, which resulted from a minimum of 26 independent IS1 insertion events. All of them had insertions of IS1 of the IS1A (IS1E and IS1G) type. Inspection of the transposase sequences of the four IS1 types and the IS1 of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS1 elements in W3110. The two amino acid residues of the transposase in IS1A (IS1E and IS1G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS1A (IS1E and IS1G). Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS1 elements in W3110 was confirmed.

FIG. 1

Distribution of the 116 IS1 insertions isolated from eight independent subcultures in the 2.6-kb sacR-and-sacB-containing BamHI-PstI DNA fragment. The top line represents the 2.6-kb fragment, which is further divided into eight restriction fragments according to nucleotide sequence (22). For the indicated subcultures, numbers in parentheses above each restriction fragment indicate how many IS1 insertions were detected in that fragment. Insertions from subcultures 1, 3, 4, 6, 7, and 10 were isolated and mapped previously (5), but in this study they were also mapped with HincII. An insertion in subculture 6 was found mapped at a specific HincII recognition site and is indicated by an asterisk. Insertions from subcultures 11 and 12 were isolated and mapped in this study.

FIG. 2

(A) Alignment of the transposase amino acid sequences of IS1A (IS1E and IS1G), IS1B (IS1C), IS1D, IS1R, and IS1F (IS1T). An amber at position 164 in IS1F (IS1T), which could be suppressed in a supE44 background and result in a transposase of 232 amino acids, is indicated by an asterisk. (B) Structures of IS1A (IS1E and IS1G), IS1B (IS1C), IS1D, and IS1R and positions of the primers used in constructing the transposase mutants. The top two horizontal lines represent the two open reading frames insA and insB′, encoded by the four IS1s, with the frames indicated in parentheses. The −1 frameshift A₆C motif for generation of the InsAB′ transposase protein is shown as a hatched box. The four IS1 elements (768 bp) are shown as horizontal lines, with filled triangles indicating the two terminal inverted repeats, IRL and IRR. For clarity, restriction enzyme recognition sites and the −1 frameshift A₆C motif, present in all four IS1 elements, are shown only in IS1A (IS1E and IS1G). The number in parentheses above each restriction enzyme recognition site is the IS1 coordinate for that site. For IS1B (IS1C), IS1D, and IS1R, only bases different from IS1A (IS1E and IS1G) are shown. Vertical arrows indicate amino acid residues that are altered by the base substitutions. Primers are represented by horizontal arrows below their regions, according to the sequences. Primers IS1DLO and IS1DRO were designed according to the host sequences flanking IS1D, which are not shown. IS1F (IS1T) has 73 bases different from IS1A (IS1E and IS1G) and is not shown.