Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

Abstract

Correlating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis and in vitro assays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCE A great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

Keywords: Tn7; allelic exchange; bacterial genetics; conjugation; counterselection; domestication; genetic manipulation; microbiome; modular tools; proteobacteria; symbiosis; zebrafish.

FIG 1

Construction and application of domestication-free counterselection systems. (A) Temperature-based counterselection was achieved by replacing the R6K origin of replication (ori_R6K) of pUC18R6KT-mini-Tn7T-GM (pTW56 [28]) with the temperature-sensitive origin of replication ori₁₀₁/repA101^ts. Tn7L and Tn7R inverted repeats flank the Tn7 transposon (gray stroke). gent^R, gentamicin (gent) resistance gene; amp^R, ampicillin resistance gene; oriT, origin of transfer. (B) (Left) Triparental conjugation between SM10 donor strains carrying either a temperature-sensitive Tn7-tagging vector (donor^Tn) or transposase helper vector (donor^helper) and a Vibrio target strain. The gentamicin phenotype of each strain is indicated as resistant (R) or sensitive (S). Mating reactions were incubated at 30°C on a filter disc on a trypic soy agar (TSA) plate. (Right) Postconjugation counterselection of donor cells was done on TSA/gent plates at 37°C. (C) Kill switch-based counterselection was achieved by inserting a LacI-regulated toxin array, comprised of the genes hokB, ghoT, and tisB, into the backbone of pUC18T-mini-Tn7T-GM (pTW54 [28]). ori_ColE1, high-copy-number origin of replication. (D) (Left) Triparental conjugation performed as described for panel B, except that donor^Tn carried a kill switch Tn7-tagging vector. (Right) Postconjugation counterselection of donor cells was done on TSA/gent/IPTG plates at 30°C.

FIG 2

Gene expression scaffold design features. Each interchangeable element is flanked by restriction sites (cyan arrowheads). Promoter, constitutively active P_tac promoter without lac operator sequence (O-) driving transcription; the 5′ untranslated region (UTR), epsilon enhancer sequence, and consensus ribosome binding site (i.e., Shine-Dalgarno sequence) promote strong translation. ORF, open reading frame (encoding a single fluorescent protein); 3′ UTR, trpL attenuator sequence terminating transcription.

FIG 3

Performing allelic exchange with a fluorescent merodiploid tracker. (A) Outline of recombination events during allelic exchange with a fluorescent tracker. An allelic exchange vector is depicted that expresses GFP and carries a cassette comprised of a mutant allele (Δ, magenta) flanked by regions (hashed and solid cyan strokes) homologous to regions flanking a target gene located on the bacterial chromosome (chr). The first recombination event—which randomly occurs between either homology region—integrates the vector into the chromosome, producing a GFP-expressing merodiploid. The second recombination event results in GFP loss. If it occurs in the unused homology region (i.e., the “solid” region in this scenario), then allelic exchange is successful. If it occurs in the same region (i.e., the “hashed” region), the original wild-type locus is restored. Black arrows shown above the final allelic exchange products denote primer annealing sites for PCR-based genotyping depicted in panel B. (B) The top row illustrates the procedural steps of allelic exchange performed using a fluorescent merodiploid tracker. The bottom row shows example images acquired during the engineering of a gene deletion in Vibrio ZOR0035. White arrowheads indicate colonies with partial or complete loss of GFP expression. WT, wild-type Vibrio ZOR0035; MD, merodiploid; Δ, ΔcheA mutant.

FIG 4

Rational design of customizable allelic exchange vectors. “vector design” illustrates the vector architecture. Features include customizable molecular scaffolds for holding antibiotic selection markers (Abx^R), a merodiploid tracker, a single Smal restriction site for insertion of allelic exchange cassettes, and an option for kill switch-based counterselection of donor cells. pAX1 was initially constructed and carries two antibiotic selection markers encoding resistance to gentamicin (gent^R) and chloramphenicol (clm^R) along with a GFP tracker. pAX2 was derived via the insertion of a tet-inducible kill switch. oriT, origin of transfer; ori₁₀₁/repA101^ts, temperature-sensitive origin of replication; amp^R, ampicillin resistance gene.

FIG 5

Gene deletion and complementation with modernized engineering vectors. (A) (Top) Wild-type pomAB locus in Vibrio ZWU0020. (Bottom) Result of markerless pomAB deletion via allelic exchange. Black arrows mark approximate primer annealing sites for genotyping, and the size of each amplification product is indicated. (B) Agarose gel showing PCR-based genotyping of the wild-type strain (WT), a merodiploid strain (MD), and a ΔpomAB (Δ) mutant. Migration distances of WT and mutant alleles are indicated. ns, nonspecific amplification product. (C) Swim motility of the WT strain, the ΔpomAB mutant, and the complemented ΔpomAB^{attTn7::pomAB} variant in 0.2% tryptic soy agar at 30°C. (D) Shown is a schematic of the Tn7 transposon from pTn7xTS-sfGFP used for complementation, which was modified to carry the native pomAB locus of Vibrio ZWU0020. Also depicted are the relative positions where the pomAB genes were deleted and reintroduced at the Tn7 insertion site (attTn7) on chromosome 1 of Vibrio ZWU0020. T, transcriptional terminators; Tn7L and Tn7R, Tn7 inverted repeats; P_pomA, native pomA promoter; gent^R, gentamicin resistance gene; sfGFP, fluorescent tag.

FIG 6

Intestinal colonization patterns and growth modes of zebrafish symbionts. (A) Cartoon diagram of a 5-day-old larval zebrafish. The purple dashed box outlines the region imaged in panel C. (B) Diagram showing the boundaries of the bulb, midgut, and distal gut within the larval intestine. The estimated bulb-to-midgut boundary is located where the bulb begins to become patently narrow. The midgut-to-distal-gut boundary is approximately located where intestinal epithelial cells begin transitioning to a more colonic epithelial cell type (59). (C) Three maximum intensity projections of three-dimensional (3D) image stacks acquired by light sheet fluorescence microscopy for the indicated bacterial strains. An orange dotted outline marks the intestine in each image. Scale bar: 200 μm.

FIG 7

Phylogenetic relatedness and summary of motility phenotypes. Shown is a phylogenetic tree generated using nucleotide sequences of the 16S rRNA gene from all strains manipulated in this study. Strains used for live imaging are indicated in bold black type. Symbols denote whether genes associated with flagellar motility were detected by genomic analysis and if motility behaviors were observed in vitro and/or in vivo.

FIG 8

Summary of the genetic tools described in this work. The functions and features of each engineering vector are briefly summarized. The guide is organized based on technique or intended use.