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

Abstract

20Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through 21 homologous recombination remains an important tool for genetic analysis. Currently, sacB-based 22 Importance 40Targeted modification of bacterial genomes is critical for genetic analyses of microorganisms. 41Allelic exchange is a technique that relies on homologous recombination to substitute native loci for 42 engineered sequences. However, current allelic exchange vectors often enable only weak 43 selection for successful homologous recombination. We developed a suite of new allelic exchange 44 vectors, pTOX, which were validated in several medically important Proteobacteria. They encode 45 visible non-fluorescent chromoproteins that enable easy identification of colonies bearing 46 integrated vector, and permit stringent selection for the second step of homologous recombination, 47 yielding modified loci. We demonstrate the utility of these vectors by using them to investigate the 48 effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on beta-lactam 49 antibiotic resistance. 50 51The ever-increasing availability of bacterial genome sequence data has driven the demand for 52 widely applicable and facile techniques enabling site-specific targeted mutagenesis. In general, 53 such techniques can be divided into those that rely on exogenous enzymes versus those that 54 depend exclusively on endogenous enzymes. Examples of methods in the former category include 55 those utilizing the Lambda Red recombinase ("recombineering" (1)), those employing clustered 56 regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems (2), or a combination of the 57 two (3, 4). These systems can be fast and reliable, but often require organism-specific 58 modifications, rely on efficient transformation, and can leave genetic scars or result in off-target 59 mutations. 60 61In contrast, "allelic exchange" utilizes endogenous homologous recombination enzymes to facilitate 62 the replacement of a native genomic region with a foreign sequence of interest. This is a versatile 63 technique that can routinely yield mutations ranging from kilobase-scale deletions or insertions to 64 the generation of precise point mutations. The early allele exchange vectors resulted in antibiotic-65 marked strains (5, 6); subsequent advances using counter-selectable cassettes allowed the 66 generation of truly scarless, unmarked mutant strains. However, many genes used in counter-67 selection strategies (e.g. rpsL, pheS, thyA, ccdB) require a specific host genotype, limiting their 68 widespread utility (7). Background-independent counter-selection strategies utilizing tetAR (8), 69 sacB (9), or a combination of the two (10) are valuable but often require considerable optimization. 70Moreover, counter-selection escape, where the integrated allelic exchange vector remains lodged 71 in the genome, preventing isolation of the desired mutant, remains common with such schemes 72 even after optimization. This has been a key tech...