Genomic Tools for Steinernema hermaphroditum
This project is a collaboration of the Goodrich-Blair, Sternberg, Dillman and Schwarz laboratories
Our overall objective is to develop Steinernema nematodes and their Xenorhabdus bacterial symbionts as a favored genetic model system for studying the molecular, evolutionary, genomic, and behavioral mechanisms of mutualism and parasitism. To this end, we are sequencing genomes of and developing genetic tools for the mutualistic nematode-bacterium symbiont pair: S. hermaphroditum and X. griffiniae. We are conducting proof‑of‑concept experiments with the genetic tools we develop, to investigate the mutualistic and parasitic behaviors of our animal-bacterium symbiont pair. We have published a few papers on these species.
We sequenced the Steinernema hermaphroditum genome.
We analyzed S. hermaphroditum chromosomes. We assembled long, short, and Hi-C reads into five chromosomes of 16.7 to 18.4 Mb, totalling 87.9 Mb; we used conserved synteny (Nigon groups, shown here) to identify four autosomes and one X chromosome; and we predicted 19,628 genes encoding 43,884 proteins with a BUSCO completeness of 89.0%.
We developed molecular genetics. We demonstrated classical mutagenesis and genetics to define loci by mapping and complementation. We used the genome sequence to find the gene mutated in multiple alleles of a locus controlling Steinernema body size. We developed CRISPR-Cas9 gene knockout and used it to confirm this identification.
We have three high quality X. griffiniae bacterial genomes. Our comparative sequence analyses are yielding high resolution information on X. griffiniae relatedness and genomic variation. The X. griffiniae symbiont of S. hermaphroditum is genetically tractable; X. griffiniae can accept exogenous DNA by conjugation and it can be genetically modified by homologous recombination and transposon-based methods, used to create X. griffiniae:
- strains with stable in-genome, neutral site insertions of genes encoding fluorescent proteins. Fluorescent X. griffiniae associated with S. hermaphroditum can be visualized by microscopy throughout the lifecycle.
- strains with allelic exchange deletion mutations in three genes predicted to encode colonization and/or virulence factors. We currently are complementing these mutants, and testing their host association phenotypes.
- RbTnseq mutant library. Quality control testing of this library revealed that insertions were non-random and enriched around the origin of replication. Modified protocols are being developed to increase randomness of insertion and chromosome coverage. However, the library has yielded X. griffiniae mutants defective in essential metabolic biosynthetic pathways.
Cao M, Schwartz HT, Tan CH, Sternberg PW. The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis. Genetics. 2022 Jan 4;220(1):iyab170. doi: 10.1093/genetics/iyab170. PMID: 34791196; PMCID: PMC8733455.
Alani OS, Cao M, Goodrich-Blair H, Heppert JK. Conjugation and transposon mutagenesis of Xenorhabdus griffiniae HGB2511, the bacterial symbiont of the nematode Steinernema hermaphroditum (India). MicroPubl Biol. 2023 Apr 25;2023:10.17912/micropub.biology.000772. doi: 10.17912/micropub.biology.000772. PMID: 37179970; PMCID: PMC10170317.
Garg P, Tan CH, Sternberg PW. DiI staining of sensory neurons in the entomopathogenic nematode Steinernema hermaphroditum. MicroPubl Biol. 2022 Feb 24;2022:10.17912/micropub.biology.000516. doi: 10.17912/micropub.biology.000516. PMID: 35224464; PMCID: PMC8874337.
Bhat AH, Chaubey AK, Shokoohi E, William Mashela P. Study of Steinernema hermaphroditum (Nematoda, Rhabditida), from the West Uttar Pradesh, India. Acta Parasitol. 2019 Dec;64(4):720-737. doi: 10.2478/s11686-019-00061-9. Epub 2019 May 10. PMID: 31077031.