Conjugal DNA transfer in the maternally inherited symbiont of tsetse flies Sodalis glossinidius

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 endemic regions. 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 to 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 as 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.


Introduction 58
African trypanosomiasis or sleeping sickness is a zoonotic disease caused by the 59 parasitic protozoa Trypanosoma brucei. Trypanosoma brucei is transmitted by tsetse flies 60 (Glossina spp.; Diptera: Glossinidae), viviparous insects that feed exclusively on 61 vertebrate blood (1, 2). In addition to T. brucei, natural populations of tsetse flies are 62 often infected with strains of the Gram negative bacterium Sodalis glossinidius (3-7). The 63 establishment of S. glossinidius infection leads to a stable association, where the 64 bacterium colonizes a number of tsetse fly tissues, including the salivary glands 65 inhabited by T. brucei, without imposing a measurable burden to the flies (4,(8)(9)(10)(11)(12). 66 Importantly, while S. glossinidius undergoes a predominantly maternal mode of 67 transmission, being passed from mother to offspring during gestation (3,(8)(9)(10)(11)(12), this 68 bacterium is also capable of paternal transmission during copulation (13), a 69 phenomenon that may facilitate its colonization and spread within uninfected tsetse 70 populations. Due to these particular characteristics, S. glossinidius has emerged as an 71 attractive candidate for the implementation of tsetse fly paratransgenesis-a 72 bioremediation strategy where bacteria capable of colonizing tsetse populations are 73 used to express traits that inhibit Trypanosoma transmission (14)(15)(16)(17)(18)(19). 74 6 requirement for δ-aminolevulinic acid (ALA) or heme, respectively ( Fig. 2A) (29,30). 125 As DAP and ALA are usually not present in complex microbial medium components, E. 126 coli donor strains containing these mutations are often used to select transconjugants on 127 rich medium such as Luria Bertani (LB) (31,32). Sodalis glossinidius forms colonies 5 to 128 10 days following plating on rich media, such as brain heart infusion-blood (BHIB) agar. 129 Therefore, we sought to determine if E. coli donor strains containing mutations in dapA 130 and/or hemA were able to grow on BHIB agar. Escherichia coli dapA and hemA strains 131 were streaked on BHIB agar alongside with S. glossinidius and incubated for 8 days 132 under microaerophilic conditions. Following incubation, S. glossinidius formed small 133 colonies as expected (Fig. 2B). By contrast, dapA and hemA strains displayed residual 134 growth at the inoculation sites on the plates (Fig. 2B). Control BHIB plates 135 supplemented with DAP supported the growth the E. coli dapA strain, which formed 136 large colonies following 8 days of incubation (Fig. 2B). 137 The aforementioned results suggested that it might be possible to counter select a 138 dapA or hemA E. coli donor strain on BHIB agar following conjugation with S. 139 glossinidius. We therefore attempted to recover S. glossinidius transconjugants under a 140 number of mating conditions. We found that E. coli dapA suppressor mutants that are 141 able to grow in the absence of DAP emerge at high frequency following 5 or 16 h of 142 mating, where strains are mixed at ratios of 50 Sodalis to 1 E.coli or 2,500 Sodalis to 1 143 E.coli, respectively (Fig. S1A) (Fig. 2D). The presence of phage T7 alone also decreased the survival of the E. 168 coli donor under all conditions tested (Fig. 2D). In the absence of DAP and ALA, phage 169 T7 lowered the number of donor cells by over nine orders of magnitude, effectively 170 preventing the emergence of E. coli dapA hemA suppressors that can grow in the absence 171 of DAP and ALA ( Fig. 2D and S1B). Importantly, after population expansion of the E. 172 coli donor for 16 h in a mock conjugation experiment, exposure to phage T7 was results suggested that S. glossinidius cells can be isolated from conjugation mixtures 176 with an E. coli dapA hemA donor following exposure to T7 phage. 177 178

Conjugation of transposition systems for random mutagenesis of Sodalis glossinidius 179
Transposable elements have played a pivotal role in the development of forward 180 genetics studies in bacterial species (34,35), and have been previously used in studies of 181 S. glossinidius (22). We therefore attempted to use conjugation for the delivery of stable 182 transposition systems encoded within mobilizable suicide vectors into this bacterium. 183 Following conjugation, S. glossinidius transconjugants were readily recovered by 184 selecting for the antibiotic markers encoded within each transposon ( Fig. 3A and B). 185 A number of controls indicated that these S. glossinidius cells were true 186 transconjugants resulting from random transposition events, originating from the 187 mobilized suicide vector. First, no antibiotic resistant clones were recovered from S. 188 glossinidius cells which were not conjugated with the E. coli donor. Hence, the 189 emergence of antibiotic resistance was linked to a physical interaction with the donor 190 strain ( Fig. 3A and B). Second, antibiotic resistant clones of S. glossinidius remained 191 sensitive to ampicillin, indicating that they did not retain the suicide vector, either as an 192 autonomous replicating episome or as a vector integrated into the chromosome (Table  193 1). Third, conjugation experiments involving promoter-probe transposition systems, 194 such as the Tn5-luxCDABE-Spc (36), yielded a population of antibiotic resistant S. 195 glossinidius clones displaying heterogeneous reporter-gene expression ( Fig. 3C and D). (1.40 x 10 -3 to 1.84 x 10 -2 ) ( Table 1 and Table S1). Together, these results demonstrate 202 that conjugation can be reliably used to deliver transposition systems into S. glossinidius. 203 204

Conjugation of suicide vectors for targeted gene disruption in Sodalis glossinidius 205
We tested whether we could use conjugation for the delivery of replication-deficient 206 suicide plasmids designed for targeted gene disruption. In contrast to transposition, 207 this reverse genetic strategy relies on homologous recombination functions encoded by 208 the host bacterium (37). In its simplest form, insertional disruptions can be generated 209 through single homologous recombination events between the target gene and a 210 homologous fragment cloned in a suicide vector-i.e. a Campbell-like integration (Fig.  211 4A). We employed this strategy to target the transcriptional regulators encoded by S. 212 glossinidius cpxR and ompR genes. Following conjugation, we were able to recover 213 antibiotic resistant S. glossinidius clones which, upon polymerase chain reaction (PCR) 214 analyses, were shown to harbor plasmid insertions in the expected chromosomal 215 locations ( Fig. 4B and C). Taken together, these results demonstrate that conjugation 216 can be used for the delivery of suicide vectors for targeted gene disruption in S. 217

glossinidius. 218 219
Discussion 220 In the current study, we established conditions permitting the counter-selection of E. 221 coli on BHIB agar. We use these conditions to hinder the growth of E. coli DNA donor 222 strains following mating, and demonstrate that the slow-growing, fastidious bacterium 223 glossinidius, effectively implementing efficient methods to carry out forward and reverse 226 genetics. Similar procedures can be developed for the delivery of any mobilizable 227 genetic elements harboring an origin of transfer (oriT) to this bacterium. These include 228 replication-competent plasmids or other episomes encoding an array of functions, such 229 as targeted transposition (38)

Construction of suicide vectors for targeted gene disruption 258
Oligonucleotide sequences used in this study are presented in Table S3. Phusion® product, generated with primers 212 and 213 and plasmid pKD3 as the template (45). 284 Recombinants were recovered on LB plates supplemented with 20 μg/mL of 285 chloramphenicol, 60 μg/mL of DAP and 100 μg/mL ALA. Chloramphenicol clones 286 were screened for the inability to grow on LB in the absence of ALA. 287 288

Preparation of bacteriophage T7 solutions 293
Escherichia coli MG1655 cultures were grown overnight in LB broth at 37˚C and 250 294 rpms. One mL of overnight cultures were used to inoculate 100 mL of fresh LB broth. 295 Cultures were allowed to grow for 2 h at 37˚C and 250 rpms to an OD 600 ~0.3-0.4, and 296 to conical tubes and 1/1000 volume of chloroform was added to each tube. Tubes were 298 vortexed for 1 minute, cell debris were pelleted by centrifugation (7,000 rpm for 2.5 min 299 at room temperature), and the lysate was filtered through a 0.22 μm polyethersulfone 300 membrane filter. Bacteriophage lysate was concentrated using an Amico® Ultra-15 301 centrifugal filter (Millipore) and LB broth was replaced with a solution of 10 mM MgCl 2 . 302 Lysates were sterilized by filtration through a 0.22 μm polyethersulfone membrane and 303 stored at 4˚C. 304 305 Bacteriophage T7 killing assay 306 Cultures of E. coli MG1655 and S. glossinidius were exposed to T7 phage lysate for 30 307 minutes. Cells were collected by centrifugation, resuspended in fresh medium, diluted 308 and spotted on agar plates. For E. coli, plates were incubated for 16 h at 37˚C; for S. 309  Table S1. 355 Table S2. 356 Table S3. Bacteria were grown separately on plates in a mock conjugation experiment, 569 subsequently exposed to phage T7, washed, diluted and spotted on BHIB as described 570 in (C). Plates were incubated for 8 days at 27°C under microaerophilic conditions. 571 Images depict representative plates of at least 3 independent experiments. Representative BHIB agar plates seeded with 2 x 10 9 CFUs of E. coli dapA hemA. Where 611 indicated, plates were supplemented with DAP or ALA, or cells were pre-treated with 612 bacteriophage T7 lysate. Plates were incubated for 7 days, at 27°C and under 613 microaerophilic conditions. Plates displaying confluent growth were imaged following 614 2 days of incubation, when growth became apparent. 615