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 counter-selection escape, which prevents isolation of cells with the desired mutation, limits its utility. To circumvent this limitation, 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 blue amilCP or magenta tsPurple non-fluorescent chromoproteins 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 orthologous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC beta-lactamase activity and corresponding beta-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high level ampC derepression and beta-lactam resistance. These data suggest why beta-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.\n\nImportanceTargeted modification of bacterial genomes is critical for genetic analyses of microorganisms. Allelic exchange is a technique that relies on homologous recombination to substitute native loci for 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 non-fluorescent chromoproteins that enable easy identification of colonies bearing integrated vector, and permit stringent selection for the second step of homologous recombination, yielding modified loci. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on beta-lactam antibiotic resistance.