Lambda Red-Mediated Genetic Modification of the Insect Endosymbiont Sodalis glossinidius

Abstract

In the current study, we adapted and optimized the lambda Red recombineering strategy to genetically manipulate the fastidious insect endosymbiont Sodalis glossinidius. This work greatly facilitates the application of genetics to the study of insect symbionts and should also prove useful in the context of long-awaited paratransgenic insect control strategies.The lifestyle switch from facultative to obligate host association is often accompanied by a process of bacterial genome degeneration and size reduction. Whereas this process has a streamlining effect, eliminating genes that are no longer required for the symbiotic lifestyle, it also drives a reduction in metabolic plasticity, yielding fastidious bacteria that have proven to be difficult to manipulate in the laboratory due to their low growth rate, high level of susceptibility to contamination, and complex nutritional requirements (12). To date, only a few studies have utilized genetic techniques to explore the nature of host-symbiont interactions (4,5,14), and the implementation of these techniques has proved arduous and unreliable over the long term. In the current study, we report on the adaptation and optimization of the lambda Red recombineering strategy (6) for the genetic manipulation of the tsetse fly endosymbiont Sodalis glossinidius (Fig. 1).A plasmid harboring lambda Red functions under the control of the P BAD arabinose-inducible promoter (pKD46) (6) was transformed into S. glossinidius using heat shock (7). Sodalis glossinidius is a fastidious bacterium that grows optimally in rich medium formulations containing glucose or N-acetyl-D-glucosamine (NAG) as a carbon source. Since S. glossinidius cannot use arabinose as a carbon source (3) and glucose and NAG can potentially interfere with the expression of genes under the regulation of the P BAD promoter through catabolite repression (8), we examined the expression of the bet lambda Red gene by quantitative PCR (4) in the presence of different sugars and 3Ј-5Ј-cyclic AMP (cAMP). As expected, we found that the expression of the bet gene is catabolite repressed by glucose and NAG but that catabolite repression can be overcome by the addition of cAMP (Fig. 2). Indeed, we were unable to obtain any recombinants following lambda Red recombineering without the addition of cAMP.In comparison to model organisms such as Escherichia coli, S. glossinidius divides very slowly under standard culture conditions. This is largely due to the fact that S. glossinidius cultures are maintained under anaerobic or microaerophilic conditions because the bacterium fails to grow on agar plates under atmospheric levels of oxygen (3). However, in a recent study, we found that S. glossinidius has a quorum-sensing system that regulates a large number of genes involved in the bacterial response to oxidative stress (11). Subsequently, we found that it was possible to significantly increase the growth rate of S. glossinidius by placing cultures in a shaking incubator once they had reached a cell density sufficient to ensure activa...

Endosymbiont evolution: predictions from theory and surprises from genomes

“…While such genetic manipulation has not been performed for most endosymbionts, including long-term primary mutualists of insects, genetic manipulation of uncultured bacteria is an area of active research, and progress has been made in secondary symbionts of insects 135, 136 . Expanding comparative genomic and experimental approaches across bacteria, including uncultured free-living groups, will shed light on whether endosymbiotic and free-living species indeed occupy distinct evolutionary trajectories, or if shared processes contribute to genome changes across lifestyles.…”

Tsetse-Wolbachia symbiosis: Comes of age and has great potential for pest and disease control

“…Sodalis can be cultured in vitro (Welburn et al, 1987) and genetically transformed (Beard et al, 1993a; Dale et al, 1995). Both intron mutagenesis based and lambda red-mediated genetic modification methods have been applied to Sodalis for functional studies (Pontes and Dale, 2011; Runyen-Janecky et al, 2010). A paratransgenic methodology has been developed in order to express trypanocidal products, which can be expressed in tsetse midguts when reconstituted with recombinant Sodalis (Beard and Aksoy, 1998).…”

Bacterial Flora of the Anopheles fluviatilis S. L., the Vector of Malaria in Southern Iran for Proper Candidate Paraterasgenesis

“…This is a new method to control vector borne diseases is paratransgenesis, which could reduce or stop the pathogen transmission in a vector (Chavshin et al, 2013) . This method introduced successfully for some vectors such Rhodnius prolixus vectors of Trypanosoma cruzi and vector of Chagas disease (Beard et al, 2001), Triatominae spp and also Glossina morsitans, vector of African sleeping sickness (Aksoy et al, 2008, Pontes andDale, 2011). High diversity of the intestinal bacterial microbiota was registered using culture dependent in relation to different populations of Phelebotomus argentipes and Lutzomyia longipalpis.…”

“…This is a new method to control vector borne diseases is called paratransgenesis reducing pathogen transmission in an insect (Chavshin et al, 2013). This method introduced successfully for some vectors such Rhodnius prolixus vectors of Trypanosoma cruzi and vector of Chagas disease, Triatominae spp and also Glossina morsitans, vector of African sleeping sickness (Aksoy et al, 2008;Pontes and Dale, 2011). This is a new method to control vector borne diseases is paratransgenesis, which could reduce or stop the pathogen transmission in a vector (Chavshin et al, 2013) .…”