A New Suite of Allelic-Exchange Vectors for the Scarless Modification of Proteobacterial Genomes

Abstract

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB-based vector systems are often used for allelic exchange, but counterselection escape, which prevents isolation of cells with the desired mutation, occasionally limits their utility. To circumvent this, we engineered a series of “pTOX” allelic-exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome-binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the gene encoding blue AmilCP or magenta TsPurple nonfluorescent chromoprotein facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens, Escherichia coli O157:H7, Enterobacter cloacae, and Shigella flexneri. Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 paralogous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC β-lactamase activity and the corresponding β-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high-level ampC derepression and β-lactam resistance. These data suggest why β-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens, like E. cloacae. Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria. IMPORTANCE Targeted modification of bacterial genomes is critical for genetic analysis of microorganisms. Allelic exchange is a technique that relies on homologous recombination to replace native loci with engineered sequences. However, current allelic-exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic-exchange vectors, pTOX, which were validated in several medically important proteobacteria. They encode visible nonfluorescent chromoproteins that enable easy identification of colonies bearing integrated vectors and permit stringent selection for the second step of homologous recombination. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on β-lactam antibiotic resistance.

Creation of Golden Gate constructs for gene doctoring

“…5) and 6) The donor plasmid is used for Gene Doctoring as described by Lee et al [1] Although we have retained the original Flp recombinase-based technique for removal of the selectable marker in our experiments, scarless genome editing is possible using tetA as a counterselection marker [18]. Alternatively, any other selectable marker(s) can be used instead, to suit individual experimental requirements [19][20][21][22][23]. Regions HR1 and HR2 will vary between experiments and can easily be produced either as chemically synthesised DNA fragments, PCR products (with BsaI sites included on the primers) or from genetic parts libraries.…”

Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics

“…Phenotypic screening markers can be used as an alternative to drug R selectable markers. Expression of chromoproteins such as amilCP or magenta tsPurple results in blue and purple colonies respectively, enabling selection by colony coloring (Lazarus et al, 2019). While a palette of engineered chromoproteins was recently assessed in E. coli, it is noteworthy that optimal conditions for attaining intense coloring, low fitness cost, and fast maturation have not yet been described (Liljeruhm et al, 2018).…”

“…Of note, in difficult-to-transform bacterial strains (e.g., Serratia marcescens), conjugation constitutes an alternative approach to introduce the plasmid DNA editing substrates (Snyder et al, 2013). Here, the plasmid DNA is transferred from a donor strain to a recipient strain in a process dependent on a pilus structure (Lazarus et al, 2019).…”

Genetic analyses link the conserved inner membrane protein CvpA to the σEextracytoplasmic stress response

“…A gentamicin-resistant mutant of E. coli O157:H7 strain EDL933 (∆lacI::aacC1) (22) was used in all experiments in this study as the wild-type (WT). All mutants were constructed in this strain background using standard allelic exchange techniques (60) with the pTOX plasmid system (61) or lambda-red recombineering (62).…”