Date of Completion

8-9-2019

Embargo Period

8-8-2020

Keywords

Microbiology, Symbiosis, Euprymna scolopes, metagenomics, metatranscriptomics

Major Advisor

Dr. Spencer Nyholm

Associate Advisor

Dr. Joerg Graf

Associate Advisor

Dr. Marcy Balunas

Associate Advisor

Dr. Jonathan Klassen

Associate Advisor

Dr. Nichole Broderick

Field of Study

Molecular and Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The Hawaiian bobtail squid, Euprymna scolopes, maintains a diverse bacterial consortium in epithelium lined tubules in a female reproductive organ, the accessory nidamental gland (ANG). During egg laying, bacteria from the ANG are deposited into a jelly coat (JC) that surrounds the yolk sac. The ANG/JC communities are composed of Alphaproteobacteria, Verrucomicrobia, Flavobacteriia, and Gammaproteobacteria. Removing or altering the JC bacterial community leaves eggs vulnerable to biofouling and infections. To understand the metabolism of this community and look for potential mechanisms of egg defense, the antimicrobial activity of ANG/JC symbionts was tested, 4 ANG metagenomes were sequenced, and metatranscriptomes were sequenced from 6 ANGs, 6 freshly laid egg JCs, 5 JCs from eggs midway through development, and 5 JCs that were challenged with a fungal pathogen for half of egg development. Inhibition against Gram-negative and/or Gram-positive marine bacteria was seen in 20% of tested isolates, and strong to moderate activity against the fungal pathogen, Fusarium keratoplasticum, was seen in 33% of tested isolates in vitro. Metagenomic sequencing of the ANG resulted in recovery of 9 new high quality genomes from Verrucomicrobia, Alterythrobacter, Mesorhizobium, and Vibrio strains. Mapping metatranscriptomic reads to one Verrucomicrobia genome revealed utilization of many nutrients in the ANG and JCs, including fucose, sulfate, and sialic acid, likely originating from host-produced glycoproteins. Analysis of the ANG metagenome also predicted 255 secondary metabolite biosynthetic gene clusters, which may produce or regulate some antimicrobial compounds that could act in egg defense. Of these clusters, genes for synthesis of nonribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs), terpenes, bacteriocins, thiopeptides, homoserine lactones, siderophores, and aryl polyenes were expressed at similar levels between all ANG and JC samples. The antimicrobial compounds, indigoidine and bromoalterochromides, were described in two JC isolates, Leisingera sp. JC1 and Pseudoalteromonas sp. JC28. Expression of secondary metabolite genes in the ANG and eggs demonstrated the potential for other defensive bioactive compounds to be produced and provides a baseline to further explore the mechanisms of bacterial egg defense in E. scolopes.

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