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Steinernema entomopathogenic nematodes form specific, obligate symbiotic associations with gram-negative, gammaproteobacteria members of the Xenorhabdus genus. Together, the nematodes and symbiotic bacteria infect and kill insects, utilize the nutrient-rich cadaver for reproduction, and then reassociate, the bacteria colonizing the nematodes’ anterior intestines before the nematodes leave the cadaver to search for new prey. In addition to their use in biocontrol of insect pests, these nematode-bacteria pairs are highly tractable experimental laboratory models for animal-microbe symbiosis and parasitism research. One advantageous feature of entomopathogenic nematode model systems is that the nematodes are optically transparent, which facilitates direct observation of nematode-associated bacteria throughout the lifecycle. In this work, green- and red-fluorescently labeled X. griffiniae HGB2511 bacteria were created and associated with their S. hermaphroditum symbiotic nematode partners and observed using fluorescence microscopy. As expected, the fluorescent bacteria were visible as a colonizing cluster in the lumen of the anterior intestinal caecum of the infective stage of the nematode. These tools allow detailed observations of X. griffiniae localization and interactions with its nematode and insect host tissues throughout their lifecycles.

Symbiosis, the beneficial interactions between two organisms, is a ubiquitous feature of all life on Earth, including associations between animals and bacteria. However, the specific molecular and cellular mechanisms which underlie the diverse partnerships formed between animals and bacteria are still being explored. Entomopathogenic nematodes transport bacteria between insect hosts, together they kill the insect, and the bacteria consume the insect and serve as food source for the nematodes. These nematodes, including those in the Steinernema genus, are effective laboratory models for studying the molecular mechanisms of symbiosis because of the natural partnership they form with Xenorhabdus bacteria and their straightforward husbandry. Steinernema hermaphroditum nematodes and their Xenorhabdus griffiniae symbiotic bacteria are being developed as a genetic model pair for studying symbiosis. Our goal in this project was to begin to identify bacterial genes that may be important for symbiotic interactions with the nematode host. Towards this end, we adapted and optimized a protocol for delivery and insertion of a lacZ-promoter-probe transposon for use in the S. hermaphroditum symbiont, X. griffiniae HGB2511 (Cao et al., 2022). We assessed the frequencies at which we obtained exconjugants, metabolic auxotrophic mutants, and active promoter-lacZ fusions. Our data indicate that the Tn10 transposon inserted relatively randomly based on the finding that 4.7% of the mutants exhibited an auxotrophic phenotype. Promoter-fusions with the transposon-encoded lacZ, which resulted in expression of β-galactosidase activity, occurred in 47% of the strains. To our knowledge, this is the first mutagenesis protocol generated for this bacterial species, and will facilitate the implementation of large scale screens for symbiosis and other phenotypes of interest in X. griffiniae.