In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Diverse microorganisms use motility and chemotaxis to exploit patchy nutrient environments, find hosts, or avoid deleterious conditions^1,2,3. These microbial behaviours can in turn influence rates of chemical transformation⁴ and promote symbiotic partnerships across terrestrial, freshwater, and marine ecosystems^2,5.

Chemotaxis has been extensively studied under laboratory conditions for the past 60 years⁶. The first quantitative method to stud....

We recommend executing section 1 prior to field experiments to optimize results.

1. Laboratory optimization

NOTE: The volumes described in the optimization procedure are sufficient for a single ISCA (composed of 20 wells).

1. Preparation of the chemical of interest
   NOTE: The optimal concentration for each chemoattractant often needs to be determined under laboratory conditions prior to field deployments. The chemical concentration field will decrease.......

This section presents laboratory results using the ISCA to test the chemotactic response of marine microbes to a concentration range of glutamine, an amino acid known to attract soil bacteria¹⁴. The concentration of glutamine that elicited the strongest chemotactic response in the laboratory tests was used to perform a chemotaxis assay in the marine environment.

To perform the laboratory tests, seawater communities sampled from coastal water in Sydney, Australia, were e.......

At the scale of aquatic microorganisms, the environment is far from homogenous and is often characterized by physical/chemical gradients that structure microbial communities^1,15. The capacity of motile microorganisms to use behavior (i.e., chemotaxis) facilitates foraging within these heterogeneous microenvironments¹. Studying chemotaxis directly in the environment has the potential to identify important interspecific interactions and chem.......

This research was funded in part by the Gordon and Betty Moore Foundation Marine Microbiology Initiative, through grant GBMF3801 to J.R.S. and R.S., and an Investigator Award (GBMF3783) to R.S., as well as an Australian Research Council Fellowship (DE160100636) to J.B.R., an award from the Simons Foundation to B.S.L. (594111), and a grant from the Simons Foundation (542395) to R.S. as part of the Principles of Microbial Ecosystems (PriME) Collaborative.