Application of Cloning-Free Genome Engineering to Escherichia coli

Abstract

The propagation of foreign DNA in Escherichia coli is central to molecular biology. Recent advances have dramatically expanded the ability to engineer (bacterial) cells; however, most of these techniques remain time-consuming. The aim of the present work was to explore the possibility to use the cloning-free genome editing (CFGE) approach, proposed by Döhlemann and coworkers (2016), for E. coli genetics, and to deepen the knowledge about the homologous recombination mechanism. The E. coli auxotrophic mutant strains FB182 (hisF892) and FB181 (hisI903) were transformed with the circularized wild-type E. coli (i) hisF gene and hisF gene fragments of decreasing length, and (ii) hisIE gene, respectively. His⁺ clones were selected based on their ability to grow in the absence of histidine, and their hisF/hisIE gene sequences were characterized. CFGE method allowed the recombination of wild-type his genes (or fragments of them) within the mutated chromosomal copy, with a different recombination frequency based on the fragment length, and the generation of clones with a variable number of in tandem his genes copies. Data obtained pave the way to further evolutionary studies concerning the homologous recombination mechanism and the fate of in tandem duplicated genes.

Keywords: evolutionary mechanisms; genetic engineering; histidine biosynthesis; homologous recombination.

Figure 1

Nucleotide sequences of the hisF (777 bp), hisF2 (609 bp), hisF3 (408 bp), and hisF4 (217 bp) fragments. The nucleotide which is deleted in E. coli FB182 (hisF892) is highlighted in yellow.

Figure 2

PCR amplicons, obtained using coli_hisF_ext FW and coli_hisF_ext REV primers, of some His⁺ revertants obtained from the CFGE experiments. Lanes: (1) GeneRuler 1 kb DNA ladder (ThermoFisher Scientific, Waltham, MA, USA); (2–15) hisF amplicons from fourteen HisF⁺ revertants; (16) PCR negative control.

Figure 3

A possible molecular rearrangement leading to a His⁺ clone harboring two in tandem hisF and retaining the mutated copy. The red star corresponds to the E. coli FB182 hisF892 single nucleotide deletion.

Figure 4

(A) Average numbers of His⁺ colonies obtained from the 5 experiments, divided on the basis of the length of the hisF fragment used for the transformation. (B) Average numbers of His⁺ colonies obtained from the transformation with the hisF gene across the 5 experiments, divided on the basis of the different groups. Bars represent standard errors. Significant differences were evaluated through analysis of variance (ANOVA) performed using Tukey’s pairwise test. Asterisks indicate significant differences (*: p-value < 0.01; **: p-value < 0.001; ***: p-value < 0.0001).

Figure 5

Nucleotide sequence of the hisIE gene from E. coli wild-type (black) and the mutant strain E. coli FB181 (red). The amino acid sequence of the encoded protein is reported in upper case. Asterisk indicates the stop codon in E. coli hisIE sequence.

Figure 6

Prediction of the three-dimensional structure of E. coli FB181 HisIE protein (in cyan) superimposed on the three-dimensional structure of wild-type E. coli K12 HisIE available on the AlphaFold2 Protein Structure Database (accession number P06989) (in pink).