Conjugal DNA transfer in the maternally inherited symbiont of tsetse fliesSodalis glossinidius

Abstract

25Stable associations between insects and bacterial species are widespread in nature. This 26 is the case for many economically important insects, such as tsetse flies. Tsetse flies are 27 the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis-a 28 zoonotic disease that incurs a high socioeconomic cost in endemic regions. Populations 29 of tsetse flies are often infected with the bacterium Sodalis glossinidius. Following 30 infection, S. glossinidius establishes a chronic, stable association characterized by vertical 31 (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of 32 this association, S. glossinidius has been long sought as a means for the implementation 33 of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the 34 genetic modification of S. glossinidius has hindered progress in this area. Here we 35 establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that 36 conjugation can be used as a DNA delivery method to conduct forward and reverse 37 genetic experiments in this bacterium. This study serves as an important step in the 38 development of genetic tools for S. glossinidius. The methods highlighted here should 39 guide the implementation of genetics for the study of the tsetse-Sodalis association and 40 the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies. 41 42 Importance 43 Tsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping 44 sickness-a zoonotic disease that inflicts a substantial economic cost to a broad region 45 of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. 46 glossinidius to establish an asymptomatic chronic infection. This infection can be 47 inherited by future generations of tsetse flies allowing S. glossinidius to spread and 48 persist within populations. To this effect, S. glossinidius has been considered as a 49 3 potential expression platform to create flies which reduce T. brucei stasis and lower 50overall parasite transmission to humans and animals. However, the efficient genetic 51 manipulation of S. glossinidius has remained a technical challenge due to its complex 52 growth requirements and uncharacterized physiology. Here we exploit a natural 53 mechanism of DNA transfer among bacteria and develop an efficient technique to 54 genetically manipulate S. glossinidius for future studies in reducing trypanosome 55 transmission. 56 57 Results 100

DNA transduction inSodalisspecies: implications for the genetic modification of uncultured endosymbionts of insects

“…By contrast, the establishment of P1 mediated transduction provides a considerable advancement in our ability to genetically manipulate S. glossinidius. This is because S. glossinidius is recalcitrant to DNA transformation by standard techniques such as heat-shock and electroporation Kendra et al, 2020). Whereas we have recently developed a method for DNA transfer to S. glossinidius via conjugation (Kendra et al, 2020), P1-mediated transduction provides an alternative, simpler method for the introduction of exogenous DNA into this bacterium.…”

“…Given the large DNA packaging capability of P1 (up to 100 Kbp) (Thomason et al 2007), this bacteriophage can be efficiently used for a variety of applications, including the delivery of bacterial artificial chromosomes (BAC vectors) or large plasmids encoding multiple genome editing CRISPR systems (Adiego-Pérez et al, 2019) that are not easily transferred by conjugation (Kendra et al, 2020). Additionally, the P1 packing sequence can be incorporated into DNA fragments used in insertional mutagenesis (Huang and Masters, 2014), enabling rapid and efficient combination of mutations via P1 "guided transduction."…”

“…Notably, classical protocols of bacterial genetics require the manipulation of large numbers of cells and the subsequent isolation of rare genetic events as bacterial colonies on selective agar plates. Consequently, the implementation of genetics for the study of insect endosymbionts has remained scarce and limited to species that can grow in axenic culture, form colonies on agar plates and are receptive to exogenous DNA (Pontes and Dale, 2006;Kendra et al, 2020;Masson et al, 2018).…”

“…While this may empower the use of genetics to study these obscure bacteria, it has also clear translational applications. P1-mediated DNA delivery into insect endosymbionts may allow the engineering of bacterial traits aimed at modifying aspects of insect ecology (Pontes and Dale, 2006;Kendra et al, 2020), mitigating their burden on economic activities and human health.…”

“…Because this process is accompanied by the loss of metabolic capability and stress response pathways (McCutcheon et al, 2019;Moran et al, 2008;Toh et al, 2006;Clayton et al, 2012;Pontes and Dale, 2006), S. glossinidius has proven refractory to harsh artificial DNA transformation procedures that are commonly employed in model organisms such as Escherichia coli (Pontes and Dale, 2006). Consequently, this bacterium has remained genetically intractable (Kendra et al 2020).…”