A modular plasmid toolkit applied in marine Proteobacteria reveals functional insights during bacteria-stimulated metamorphosis

Abstract

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacteriumPseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm,Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for rapidly creating and iteratively testing genetic tractability by modifying marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts enabled the successful transformation of twelve marine strains across two Proteobacteria classes, four orders and ten genera. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with broader implications for environmental restoration and biotechnology.

Future research directions of the model marine tubeworm Hydroides elegans and synthesis of developmental staging of the complete life cycle

“…Improvements in husbandry and long‐term culture will facilitate generation of animal lines in Hydroides , enabling Hydroides to keep pace with advancements in other marine invertebrate larvae 51‐56 . In parallel, emerging synthetic biology tools in established model microbes also hold promise for the manipulation of more diverse marine bacteria that promote Hydroides development 57 . These new tools compliment powerful classical bacterial genetics approaches which have already helped reveal key aspects of Hydroides interaction with stimulatory microbes 39,40 …”

“…[51][52][53][54][55][56] In parallel, emerging synthetic biology tools in established model microbes also hold promise for the manipulation of more diverse marine bacteria that promote Hydroides development. 57 These new tools compliment powerful classical bacterial genetics approaches which have already helped reveal key aspects of Hydroides interaction with stimulatory microbes. 39,40 Future work leveraging Hydroides holds promise for promoting biotechnology in the contexts of evolution, the environment, and human health.…”

Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis

“…We determined that Pseudoalteromonas sp. PS5 is amenable to genetic manipulation by conjugation using a broad-host-range gfp reporter plasmid from the Marine Modification Kit (MMK) plasmid system [ 11 ]. We generated a Pseudoalteromonas sp.…”

“…PS5 (Fig. 1B ) [ 11 ]. Luminescence was measured during each growth phase with the highest activity measured in late stationary and biofilm phases (Fig.…”

“…We therefore developed an integrative Tn10 transposon for use in Pseudoalteromonas sp. PS5, which is compatible with existing modular genetic toolkit parts [ 11 , 14 , 15 ] (Supplementary Fig. S2 ).…”