Conjugal DNA Transfer in Sodalis glossinidius, a Maternally Inherited Symbiont of Tsetse Flies

Abstract

Stable associations between insects and bacterial species are widespread in nature. This is the case for many economically important insects, such as tsetse flies. Tsetse flies are the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis-a zoonotic disease that incurs a high socioeconomic cost in regions of endemicity. Populations of tsetse flies are often infected with the bacterium Sodalis glossinidius Following infection, S. glossinidius establishes a chronic, stable association characterized by vertical (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of this association, S. glossinidius has been long sought as a means for the implementation of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the genetic modification of S. glossinidius has hindered progress in this area. Here, we establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that conjugation can be used as a DNA delivery method to conduct forward and reverse genetic experiments in this bacterium. This study serves as an important step in the development of genetic tools for S. glossinidius The methods highlighted here should guide the implementation of genetics for the study of the tsetse-Sodalis association and the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies.IMPORTANCE Tsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping sickness-a zoonotic disease that inflicts a substantial economic cost on a broad region of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. glossinidius to establish an asymptomatic chronic infection. This infection can be inherited by future generations of tsetse flies, allowing S. glossinidius to spread and persist within populations. To this effect, S. glossinidius has been considered a potential expression platform to create flies which reduce T. brucei stasis and lower overall parasite transmission to humans and animals. However, the efficient genetic manipulation of S. glossinidius has remained a technical challenge due to its complex growth requirements and uncharacterized physiology. Here, we exploit a natural mechanism of DNA transfer among bacteria and develop an efficient technique to genetically manipulate S. glossinidius for future studies in reducing trypanosome transmission.

Keywords: Sodalis glossinidius; Trypanosoma brucei; conjugation; gene disruption; genetic modification; insect endosymbiont; mutation; paratransgenesis; plasmid transfer; symbiont; transformation; transposition.

FIG 1

Representative workflow of the conjugation procedure developed for Sodalis glossinidius. Following the direction of the arrows, an E. coli hemA dapA donor strain (gray) is mixed with S. glossinidius (orange) and grown on medium containing DAP and ALA. Following the transfer of a conjugative plasmid (small circle) from the donor to the recipient cells, the mixture is exposed to E. coli-specific lytic bacteriophage T7. Following T7 absorption, cells are washed and plated on medium containing antibiotic but lacking DAP and ALA. The absence of these metabolic intermediates restricts growth of E. coli donor cells that escape killing by bacteriophage. Antibiotic selects for S. glossinidius transconjugants (blue). Suicide conjugative plasmids promote genetic modification (via transposition or homologous recombination) prior to being lost by segregation (top row). Replication-competent conjugative plasmids are maintained as autonomously replicating genetic elements (bottom row).

FIG 2

Counterselection of E. coli hemA dapA donor on BHIB agar. (A) Schematics depicting the reactions catalyzed by HemA (left side) and DapA (right side), enzymes required for the biosynthesis of heme and peptidoglycan, respectively. Complementation of growth medium with the metabolic intermediates depicted in red is used to support the growth of hemA and dapA mutants. (B) Growth of wild-type S. glossinidius, E. coli hemA (MP1182), and E. coli dapA (BW29427) on BHIB agar lacking or containing DAP. Plates were photographed after 8 days of incubation at 27°C under microaerophilic conditions. Red arrows indicate residual growth. (C) Growth of wild-type E. coli (MG1655) and S. glossinidius following 120 min of incubation, at room temperature, in 10 mM MgCl₂ or 10 mM MgCl₂ containing phage T7. Cell suspensions were diluted, and 5 μl was spotted on plates. Escherichia coli was incubated at 37°C on LB for 16 h. Sodalis glossinidius was incubated under microaerophilic conditions at 27°C on BHIB for 8 days. (D) Growth of E. coli dapA hemA (MP1554) on BHIB agar with various combinations of ALA and DAP. Cells were incubated at room temperature for 120 min in 10 mM MgCl₂ or 10 mM MgCl₂ containing phage T7. Cultures were diluted, and 5 μl was plated on BHIB agar. Plates were incubated at 37°C for 16 h. Red arrows indicate residual growth. (E) Growth of S. glossinidius and E. coli dapA hemA (MP1554) on BHIB agar lacking ALA and DAP. Bacteria were grown separately on plates in a mock conjugation experiment, subsequently exposed to phage T7, washed, diluted, and spotted on BHIB as described for panel C. Plates were incubated for 8 days at 27°C under microaerophilic conditions. Images depict representative plates of at least 3 independent experiments.

FIG 3

Transposition mutagenesis in S. glossinidius. (A) Serial dilutions of conjugation mixtures of S. glossinidius and E. coli dapA hemA (MP1554) harboring the suicide vector encoding a Mariner transposon system (Himar1), pMarC9-R6k. Five microliters of cell suspension was spotted on BHIB agar (left panel) or BHIB agar supplemented with kanamycin (middle panel). Individually grown conjugation partners, S. glossinidius and E. coli dapA hemA (MP1554) pMarC9-R6k, were also spotted on BHIB agar supplemented with kanamycin (right panel). The red box indicates plates containing kanamycin. Note that dots at the donor lane correspond to locations where pipette tips punctured the agar. Similar dots are present in lane R2 and on some lanes of panel B. (B) Serial dilutions of conjugation mixtures of S. glossinidius and E. coli dapA hemA (MP1554) harboring the suicide vector encoding the Tn5-based promoter-probe transposition system, pUTmini-Tn5-luxCDABE-Spc. Five microliters of cell suspension was spotted on BHIB agar (left panel) or BHIB agar supplemented with spectinomycin (middle panel). Individually grown S. glossinidius and E. coli dapA hemA (MP1554) pUTmini-Tn5-luxCDABE-Spc were spotted on BHIB agar supplemented with spectinomycin (right panel). Red box indicates plates containing spectinomycin. (C) Transconjugants obtained in a conjugation experiment described for panel B were purified on BHIB agar supplemented with spectinomycin. Luminescence signals of four distinct clones are depicted on the right side of the figure. Plates were incubated for 8 days at 27°C under microaerophilic conditions. Images depict representative plates of at least 3 independent experiments. (D) Quantification of luminescence signals derived from selected S. glossinidius mini-Tn5-luxCDABE-spc^R transconjugants obtained as described for panel B. Error bars represent standard deviations from three technical replicates. (E) Schematic illustration depicting locations of mini-Tn5-luxCDABE-spc^R transposition insertions in selected S. glossinidius clones—hnh (SGGMMB4_03814), clpX (SGGMMB4_01523), pld (SGGMMB4_05728), and amsH (SGGMMB4_02193).

FIG 4

Gene targeting in S. glossinidius by insertional inactivation. (A) General schematic depicting the integration of a suicide vector harboring an antibiotic-resistant marker (ab^R, orange) into a specific chromosomal region. A homologous recombination event (1) between homologous fragments on the chromosome (dark gray) and plasmid (light gray) is shown. (B) General schematic cartoons depicting the annealing locations of the confirmatory primers (P1 and P2) on the targeted chromosome location and suicide vector. (C and D) Agarose gel images of electrophoresed PCR products obtained with primers P1 and P2. Lanes indicate DNA templates used in each PCR: negative control [(−)], wild-type S. glossinidius genomic DNA (wild-type), suicide plasmid vector (plasmid), and transconjugant S. glossinidius clones (clone 1, clone 2, and clone 3). Bands corresponding to PCR products spanning an insertion point of the suicide plasmid are indicated with red arrows. In panel C, note the presence of unspecific bands on the lanes corresponding to S. glossinidius wild-type and plasmid DNA. The identity of PCR bands has been verified by DNA sequencing.