The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila -colonizing Orbaceae

Abstract

Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli , a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying new host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species.

Importance: To fully understand how microbes have evolved to interact with their environments, one must be able to modify their genomes. However, it can be difficult and laborious to discover which genetic tools and approaches work for a new isolate. Bacteria from the recently described Orbaceae family are common in the microbiomes of insects. We developed the Pathfinder plasmid toolkit for testing the compatibility of different genetic parts with newly cultured bacteria. We demonstrate its utility by engineering Orbaceae strains isolated from flies to express fluorescent proteins and characterizing how they colonize the Drosophila melanogaster gut. Orbaceae are widespread in Drosophila in the wild but have not been included in laboratory studies examining how the gut microbiome affects fly nutrition, health, and longevity. Our work establishes a path for genetic studies aimed at understanding and altering interactions between these and other newly isolated bacteria and their hosts.

FIG 1

The Pathfinder plasmid system. (A) Plasmid maps and workflow for multiplex conjugation into a bacterium of interest. (B) Visualizing Pathfinder reporters. The same agar plate containing streaks of E. coli MFDpir donor strains, each with a plasmid expressing a different fluorescent protein or chromoprotein is shown in all images. The leftmost panel shows the plate under white light, the second shows the plate on a blue light transilluminator, and the last two panels show the plate imaged using a Typhoon 9500 FLA system with two different excitation and emission settings.

FIG 2

Recently isolated Orbaceae strains can be engineered with the Pathfinder plasmid system. (A) 16S rRNA gene sequence phylogeny showing the relationship between the isolated species in this paper (in bold) and other members of the family Orbaceae. Strains are color-coded based on the taxonomic order of their original insect host. For each Orbaceae strain first reported in this study the closest taxonomic identifier that could be established for the host is shown in parentheses. Bootstrap values are depicted next to their respective branches. (B) Table depicting the compatibility of the bacteria in this study with each of the Pathfinder plasmids. Compatibility is defined as isolation and verification of at least a single transconjugant colony for each plasmid. Dots are used to represent compatibility and are color-coded according to the origin of replication and reporter gene on the plasmid. (C) The efficiency of conjugation under each of the antibiotic conditions in the study is plotted as a percentage of transconjugants relative to growth on nonselective media. Data are plotted on a log scale. Except in the Kan condition, where multiple plasmids may be present as shown in (B), all plasmids are RSF1010-RCP. (D) Bar chart showing the level of GFP expression for each strain engineered with pSL1-GFP (RSF1010-GFP) normalized to an OD600 reading for that same strain. Images above the chart show the appearance of each strain on a blue light transilluminator. Letters above each bar designate groups that are significantly different at the p < 0.01 level calculated using Dunn’s test with Bonferroni correction.

FIG 3

Engineered Orbaceae strains can colonize D. melanogaster. Images were taken of bacterial growth on selective media containing kanamycin following the growth of bacteria from crushed flies in the days following inoculation. Ability to colonize the host is determined by the presence of many GFP-expressing colonies. Days on which we were unable to collect data or stopped the experiment are indicated by “ND”.

FIG 4

Dynamics of D. melanogaster colonization by two newly isolated Orbaceae strains. CFU/fly was measured following inoculation with either (A) lpD02 or (B) lpD01. CFUs in each of ten flies per arena were measured at each time point. Data are plotted on a pseudo-log scale so the full range of colonization levels can be shown. Results from three independent arenas are shown in subpanels labeled 1, 2, and 3. Letters above each boxplot represent groupings that are significantly different from one another (p < 0.05, Dunn’s test with Bonferroni correction).

FIG 5

Confocal microscopy of the D. melanogaster gut colonized with fluorescent lpD01. (A) Schematic of the Drosophila gut with colors designating three regions, foregut, midgut, and hindgut. The crop (cr) and proventriculus (pro) are labelled due to their relevance for the localization of the strain. Malpighian tubules (mt) are depicted in green. (B-K) Confocal images of the dissected gut of one uncolonized (B, F, I) and three colonized flies. Images (C, G, J and K) are from the same fly, (D, H) are from a second fly, and (E) is a from a third. Images (B-D, F-K) were captured at 10× magnification, and image (E) was captured at 40×. Outlines for each of the relevant gut regions are represented by white dashed lines and arrows were added to (J) to point out individual bacterial cells. The Malpighian tubules (mt) exhibited autofluorescence in all flies. GFP intensities were linearly adjusted in each image to highlight bacterial localization. The scale bar for each image represents 100 µm except where indicated.