XanthoMoClo─A Robust Modular Cloning Genetic Toolkit for the Genera Xanthobacter and Roseixanthobacter

Abstract

Interest in Xanthobacter species is increasing due to their unique metabolic capabilities. They can grow in both heterotrophic and fully autotrophic environments, including carbon dioxide, dinitrogen gas, and hydrogen as the sole carbon, nitrogen, and energy sources, respectively. Academic and industrial groups looking to leverage these metabolic properties are already using Xanthobacter strains for the sustainable production of food and commodities. However, only a handful of genetic parts and protocols exist in scattered genetic backgrounds, and there is an unmet need for reliable genetic engineering tools to manipulate Xanthobacter species. Here, we developed XanthoMoClo, a robust modular cloning genetic toolkit for Xanthobacter and Roseixanthobacter species and strains, providing extensive tools to transform them, manipulate their metabolism, and express genes of interest. The toolkit contains plasmid parts, such as replication origins, antibiotic selection markers, fluorescent proteins, constitutive and inducible promoters, a standardized framework to incorporate novel components into the toolkit, and a conjugation donor to transform Xanthobacter and Roseixanthobacter strains easily with no or minimal optimization. We validated these plasmid components in depth in three of the most commonly studied Xanthobacter strains: X. versatilis Py2, X. autotrophicus GZ29, and X. flavus GJ10, as well as in R. finlandensis VTT E-85241. Finally, we demonstrate robust toolkit functionality across 21 different species of Xanthobacter and Roseixanthobacter, comprising 23 strains in total. The XanthoMoClo genetic toolkit is available to the research community (through AddGene) and will help accelerate the genetic engineering of Xanthobacter to further their applications in sustainability and bioremediation efforts.

Keywords: MoClo; Xanthobacter; genetic engineering; genetic toolkit; sustainability; synthetic biology.

Figure 1

XanthoMoClo—A Golden Gate modular cloning genetic toolkit for Xanthobacter and Roseixanthobacter. (A) Backbone schematic of an assembled, functional Xanthobacter plasmid made with the toolkit, including regions for origins of replication, selection markers, and gene expression cassettes. (B) New plasmid parts can be easily incorporated into the genetic toolkit either by BsmBI assembly or Gibson assembly to be compatible with other toolkit components. (C) Functional Xanthobacter plasmids are assembled from toolkit part plasmids and/or PCR products via BsaI assembly into ready-to-use constructs after a single round of cloning. Additional cassettes can be incorporated through modulator parts as described in the Supporting Information. (D) Plasmid parts and backbones included in the starter XanthoMoClo genetic toolkit.

Figure 2

Bacterial conjugation using an auxotrophic E. coli donors implifies the engineering of Xanthobacter and Roseixanthobacter species. Cultures of an auxotrophic (e.g., diaminopimelic acid/Dap) donor E. coli harboring conjugative machinery (e.g., pTA-Mob or DH10B-MOB) containing the Xanthobacter plasmid were mixed with recipient Xanthobacter on a nonselective plate, allowing both strains to grow. The nonselective plate was then scraped and replated onto a selective plate without Dap, only permitting transformed Xanthobacter recipients to grow for downstream selection.

Figure 3

Characterization of antibiotic selection markers across Xanthobacter and Roseixanthobacter species. (A) The plasmid backbone used to test antibiotic selection markers included a broadly functional kanamycin resistance cassette in addition to a swappable variable antibiotic selection marker. (B) Serial dilution growth of representatives X. versatilis Py2, X. autotrophicus GZ29, X. flavus GJ10, and R. finlandensis VTT E-85241 with gentamicin, kanamycin, spectinomycin, streptomycin, or tetracycline as a selection markers. (C) Functional test of the five antibiotic selection markers across the 23 species and strains of Xanthobacter and Roseixanthobacter. An asterisk with the kanamycin condition for X. tagetidis indicates that 100 μg/mL kanamycin was used instead of 30 μg/mL due to a higher natural resistance.

Figure 4

Rhizobial repABC origins of replication can be used to simultaneously propagate multiple stable single-copy plasmids in Xanthobacter and Roseixanthobacter species. (A) Schematic of plasmid for testing the origins of replication in Xanthobacter (pMB1 is nonfunctional in Xanthobacter, but necessary to replicate repABC plasmids in E. coli). (B) Successful single and combinatorial propagation of plasmids harboring different origins of replication in representatives X. versatilis Py2, X. autotrophicus GZ29, X. flavus GJ10, and R. finlandensis VTT E-85241. For multiplasmid strains, the plasmids carried the following markers in addition to KanR: RK2/RP4—GenR, repABC-pMla—TetR, and repABC-pMlb—SpcR. (C) Wide-spread functionality of the RK2/RP4 broad host origin, and two repABC origins across the 23 species and strains of Xanthobacter and Roseixanthobacter. An asterisk indicates the plates for X. tagetidis that contain tetracycline 12 μg/mL instead of kanamycin due to a naturally higher resistance to kanamycin.

Figure 5

Multiple fluorescent proteins are detectable over the background in Xanthobacter and Roseixanthobacter. (A) The plasmid backbone was used for testing the fluorescent proteins in Xanthobacter and Roseixanthobacter species. (B) Macroscope images of different fluorescent proteins in representatives X. versatilis Py2, X. autotrophicus GZ29, X. flavus GJ10, and R. finlandensis VTT E-85241 on agar plates. (C) Quantitation of microscopy images of cells expressing multiple fluorescent proteins in the same representative species. The fold change in expression level for each fluorescent protein is in arbitrary fluorescence units (AFU) and normalized to median wild-type background fluorescence for the given channel for the representative strain. For X. versatilis Py2 EBFP2, the microscopy was repeated on another day and scaled using a reimaged wild-type control for comparison (please see the Methods for details). (D) Macroscope images of mRFP and sfGFP (expressed on the same plasmid) across the 23 species and strains of Xanthobacter and Roseixanthobacter. A white asterisk indicates the plates for X. tagetidis that contain tetracycline (12 μg/mL) instead of kanamycin due to a naturally higher resistance.

Figure 6

Constitutive promoters span more than a 50-fold range of protein expression and are consistent across Xanthobacter and Roseixanthobacter species. (A) The plasmid backbone was used for testing the constitutive promoter strength in Xanthobacter and Roseixanthobacter species. (B) Wild-type normalized quantitation of 13 constitutive promoters of different strengths using microscopy in representatives X. versatilis Py2, X. autotrophicus GZ29, X. flavus GJ10, and R. finlandensis VTT E-85241. The expression level of mRFP is in arbitrary fluorescence units (AFU). (C) Wide-spread functionality of a low (nptII), medium (bla-v1), and high (rpsM) expression constitutive promoter using microscopy across the 22 species and strains of Xanthobacter and Roseixanthobacter. Data are normalized to their appropriate wild-type control.

Figure 7

Anhydrotetracycline (aTc)-inducible protein expression in Xanthobacter and Roseixanthobacter. (A) The plasmid backbone was used for testing the aTc-inducible promoter in Xanthobacter and Roseixanthobacter species. (B) An aTc concentration curve indicates concentration-dependent inducible expression in representatives X. versatilis Py2, X. autotrophicus GZ29, X. flavus GJ10, and R. finlandensis VTT E-85241. Wild-type normalized quantitation of the expression level of mRFP is in arbitrary fluorescence units (AFU). (C) A time course with the aTc-inducible promoter plasmid indicates maximal expression around 18 h in the Xanthobacter representatives and 12 h in R. finlandensis. (D) Robust functionality of the aTc-inducible promoter system across the 22 species and strains of Xanthobacter and Roseixanthobacter. Data are normalized to their appropriate wild-type control.

Figure 8

Several terminators can stop transcription in Xanthobacter and Roseixanthobacter. (A) The plasmid backbone for testing terminator strength in Xanthobacter and Roseixanthobacter species tests transcriptional read-through the terminator by comparing mRFP to sfGFP expression levels within a cell. (B) Functionality of different terminators in representatives X. versatilis Py2, X. autotrophicus GZ29, X. flavus GJ10, and R. finlandensis VTT E-85241. Data are based on the quantitation of expression levels from microscopy and are normalized to their appropriate wild-type control.