Systematic mutagenesis of the Escherichia coli genome

Abstract

A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the lambdaRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.

FIG. 1.

General strategy for systematic creation of insertion alleles and replacements. (A) Insertion mutants are made by in vitro transposition of Tn-Kan-I-SceI (black double-headed arrows) into PCR products corresponding to each ORF (shaded bars). The mixture is then electroporated into wild-type cells expressing the λRed proteins and introduced into the chromosome via homologous recombination. (B) Replacement of insertion alleles with markerless mutations by using I-SceI counterselection. A gene deletion strategy is depicted. Linear fragments (bar) consisting of a 5′ homology (solid portion of the bar) fused to a 3′ homology (open portion of the bar) are coelectroporated with pBC-I-SceI into the appropriate insertion strain. I-SceI-mediated cleavage of the genome (gapped double-headed arrow) stimulates λRed-mediated recombination at the targeted locus.

FIG. 2.

Insertion site characteristics. (A) Histogram displaying the relative position and orientation of isolated insertion alleles. x-axis values refer to the position of transposon insertion as a percentage of gene length. The solid and open portions of each bar correspond to insertions in the sense and antisense orientations, respectively. (B) Sequence logo (28) representation of the Tn5 recognition motif obtained from coding strand only analysis. The sequence is oriented 5′ to 3′, and the target site refers to the region that is duplicated upon transposition. (C) Sequence logo representation of the motif derived from independent consideration of both strands by using a weighted matrix. Note that the y-axis scales differ between panels A and B.

FIG. 3.

Viable insertion mutants in essential genes. (A) Sequence and reading frames at the transposon end downstream of the Kan^r gene. The Shine-Dalgarno sequence is underlined, and bars above the sequence indicate initiation codons. Single-letter designations are used for amino acids, and the stop codon is denoted by an asterisk. (B) Insertions into 5′ ends of essential ORFs. Schematic shows orientation of transposon insertion relative to the Kan^r gene and gene transcription for two isolated insertion alleles. Sequences flanking the putative fusion protein start point show transposon (lowercase) and gene (capitals) segments with the predicted amino acid sequence shown below. Underlined nucleotides denote potential Shine-Dalgarno sites, and the position of first ORF nucleotide downstream of the insertion is marked. (B) Insertions into the 3′ ends of essential genes. Schematics show the insertion points and orientation of transposon insertions into indicated essential genes. Numbering refers to amino acid positions corresponding to the upstream insertion site and the wild-type length of the protein.

FIG. 4.

Distribution of lacZ insertion alleles. The gene is depicted as an elongated box, and numbers refer to the nucleotide position within the ORF. Bars indicate insertion site locations, with those on top or below the gene denoting transposons oriented in the sense or the antisense direction, respectively. The four longer bars mark positions where two insertions were found. Asterisks denote alleles with partial function, and increasing font size corresponds to increasing enzymatic activity.