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.

Keywords: AmilCP gene; Serratia marcescens; allelic exchange; ampC; ampD; antibiotic resistance; beta-lactamase; sacB; toxin-antitoxin; type VI toxin.

FIG 1

Allelic exchange with pTOX. (A) (Top) Map of plasmid pTOX1. R6Kori, R6K origin of replication; mobRP4, mobilization region from the RP4 conjugative plasmid; rhaS, rhamnose transcriptional activator gene; MCS, multiple-cloning site; Cam-R, chloramphenicol resistance cassette; pRha, rhamnose promoter. The vertical black bars of various widths represent terminators. (Bottom) Expanded polylinker with restriction sites unique to pTOX1 (yhaV), shown. Red arrow, forward transcriptional terminator. (B) pTOX workflow. (Step 1) The desired allele is inserted into the MCS using isothermal assembly and transformed into donor E. coli (yellow bacillus). (Step 2) Conjugation is performed between the donor E. coli and the organism of interest (red coccobacillus). (Step 3) pTOX integrates into the appropriate chromosomal locus. (Step 4) Merodiploids are isolated, and toxin is induced. (Step 5) The desired clone is identified by colony PCR.

FIG 2

Induction of specific bacterial toxins inhibit S. marcescens growth. S. marcescens wild type (Wt) or merodiploid (Merodip) bacteria harboring the indicated pTOX-carrying toxin were diluted from exponential-phase growth in LB into either 2% (wt/vol) glucose (gluc) or rhamnose-containing (rham) LB and incubated with agitation at 37°C. Note that the Wt (gluc) curve is obscured by the Wt (rham) curve in panel A and the error bars in panel C are smaller than the line for all but Merodip (gluc). Means and SEM are depicted for at least 3 independently generated merodiploids.

FIG 3

pTOX for genomic modification in multiple pathogens. (A) S. marcescens colony coloration in Wt (left) and ΔhexS (right) bacteria grown at 37°C for 1 day. HexS inhibits expression of the red prodigiosin characteristic of S. marcescens. (B) E. coli O157:H7 colony coloration in Wt (left) and ΔlacZ (right) bacteria grown on X-Gal-containing medium. Blue-green colony color indicates lactose fermentation. (C) S. flexneri colony PCR and results of 1% agarose gel electrophoresis demonstrating deletion of ipgH from an S. flexneri virulence plasmid. M, marker; Wt, wild type; Δ, ΔipgH. (D) E. cloacae β-lactamase (βLA) activity in total clarified sonicate from 3 Wt double-crossover colonies and 3 ΔampC colonies induced with 50 μg/ml clavulanate prior to harvesting. The sonicates were incubated with nitrocefin, a chromogenic cephalosporin substrate that absorbs at 495 nm when hydrolyzed. An OD of 1 corresponds to 31 nmol hydrolyzed nitrocefin.

FIG 4

A chromoprotein module facilitates monitoring of conjugation. (A) Plasmid map of pTOX4. R6Kori, the R6K origin of replication; mobRP4, the mobilization region from the RP4 conjugative plasmid; rhaS encodes the rhamnose transcriptional activator; amilCP, AmilCP gene, encoding the blue AmilCP chromoprotein; MCS, multiple-cloning site; Cam-R, chloramphenicol resistance cassette; pRha, rhamnose promoter. The vertical black bars of various widths represent terminators. (B) The tac promoter and apFAB46-B0030 allow optimal AmilCP gene expression. Shown is the relative color saturation at 24 h and 48 h of pTOX4-containing colonies with various promoters and ribosome-binding sites (RBS) (described in more detail in Materials and Methods). (C) Depiction of donor E. coli containing (clockwise from bottom) pTOX without chromoprotein, with the tac-AmilCP gene, and with the apFAB46-B0030-TsPurple gene after 24 h at 37°C. (D) E. cloacae pTOX merodiploids (clockwise from bottom) without chromoprotein, with the tac-AmilCP gene, and with the apFAB46-B0030-TsPurple gene after 24 h at 37°C and an additional 24 h at 25°C.

FIG 5

S. marcescens peptidoglycan amidohydrolase deletions lead to differential derepression of ampC. (A) Phylogenetic analysis performed using the maximum-likelihood method and a JTT matrix-based model in MEGA X (30). An unrooted tree with the lowest log likelihood (−4,913) is shown. (B) Clarified sonicates from the indicated strains were incubated with equal amounts of nitrocefin, a chromogenic cephalosporin β-lactam, and absorbance was measured in the kinetic mode for 10 min. The slope of the line from the first 5 data points was used to calculate β-lactamase (βLA) activity, which was then normalized to the Wt, which corresponds to 31.2 nmol nitrocefin/min/mg total protein. Measurements are shown without preinduction and with induction with 4 μg/ml cefoxitin for 2 h prior to harvesting. The data represent the means and SEM of the results of 4 independent experiments. Comparisons were made between all uninduced mutants and the Wt and between each induced sample and its uninduced control. *, P < 0.05 after performance of the Bonferroni correction. All the induced samples were also significantly different from the corresponding uninduced samples, except for the ΔampD ΔamiD and ΔampD ΔamiD2 mutants and the triple mutant (these asterisks are not shown for clarity).