In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Summary

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.

Abstract

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.

Introduction

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 study chemotaxis, the capillary assay, involves a capillary tube filled with a putative chemoattractant immersed in a suspension of bacteria⁶. Diffusion of the chemical out of the tube creates a chemical gradient, and chemotactic bacteria respond to this gradient by migrating into the tube⁷. Since the development of the capillary assay, still widely used today, many other techniques have been developed to study chemotaxis under increasingly controlled physical/chemical conditions, with the most recent involving the use of microfluidics^8,9,10.

Microfluidics, together with high-speed video microscopy, enables tracking of the behavior of single cells in response to carefully controlled gradients. Although these techniques have vastly improved our understanding of chemotaxis, they have been restricted to laboratory use and do not translate easily to field deployment in environmental systems. As a consequence, the capacity of natural communities of bacteria to use chemotaxis within natural ecosystems has not been examined; thus, current understanding of the potential ecological importance of chemotaxis is biased toward artificial laboratory conditions and a limited number of laboratory-cultured bacterial isolates. The recently developed ISCA overcomes these limitations¹¹.

The ISCA builds on the general principle of the capillary assay; however, it makes use of modern microfabrication techniques to deliver a highly replicated, easily deployable experimental platform for the quantification of chemotaxis toward compounds of interest in the natural environment. It also allows identification and characterization of chemotactic microorganisms by direct isolation or molecular techniques. While the first working device was self-fabricated and constructed of glass and PDMS¹¹, the latest injection-molded version is composed of polycarbonate, using a highly standardized fabrication procedure (for interest in the latest version of the device, the corresponding authors can be contacted).

The ISCA is credit card-sized and consists of 20 wells distributed in a 5 x 4 well array, each linked to the external aquatic environment by a small port (800 µm in diameter; Figure 1). Putative chemoattractants loaded into the wells diffuse into the environment via the port, and chemotactic microbes respond by swimming through the port into the well. As many factors can influence the outcome of an ISCA experiment in the natural environment, this step-by-step protocol will help new users overcome potential hurdles and facilitate effective deployments.

Protocol

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). 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…

Representative Results

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…

Discussion

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…

Materials

Acrylic glue	Evonik	1133	Acrifix 1S 0116
Acrylic sheet	McMaster-Carr	8505K725	Or different company
Adhesive tape	Scotch	3M 810	Scotch Magic tape
Autoclave	Systec	D-200	Or different company
Benchtop centrifuge	Fisher Scientific	75002451	Or different company
Bungee cord	Paracord Planet	667569184000	Or different company
Centrifuge tube – 2 mL	Sigma Aldrich	BR780546-500EA	Eppendorf tube
Conical centrifuge tube – 15 mL	Fisher Scientific	11507411	Falcon tube
Conical centrifuge tube – 50 mL	Fisher Scientific	10788561	Falcon tube
Deployment arm	Irwin	1964719	Or different company
Deployment enclosure plug	Fisher Scientific	21-236-4	See alternatives in manuscript
Disposable wipers	Kimtech – Fisher Scientific	06-666	Kimwipes
Flow cytometer	Beckman	C09756	CYTOFlex
Glutaraldehyde 25%	Sigma Aldrich	G5882	Or different company
Green fluorescent dye	Sigma Aldrich	S9430	SYBR Green I – 1:10,000 final dilution
Hydrophilic GP filter cartridge – 0.2 µm	Merck	C3235	Sterivex filter
In Situ Chemotaxis Assay (ISCA)	–	–	Contact corresponding authors
Laser cutter	Epilog Laser	Fusion pro 32	Or different company
Luria Bertani Broth	Sigma Aldrich	L3022	Or different company
Marine Broth 2216	VWR	90004-006	Difco
Nylon slotted flat head screws	McMaster-Carr	92929A243	M 2 × 4 × 8 mm
Pipette set	Fisher Scientific	05-403-151	Or different company
Pipette tips – 1 mL	Fisher Scientific	21-236-2A	Or different company
Pipette tips – 20 µL	Fisher Scientific	21-236-4	Or different company
Pipette tips – 200 µL	Fisher Scientific	21-236-1	Or different company
Sea salt	Sigma Aldrich	S9883	For artificial seawater
Serological pipette – 50 mL	Sigma Aldrich	SIAL1490-100EA	Or different company
Syringe filter – 0.02 µm	Whatman	WHA68091002	Anatop filter
Syringe filter – 0.2 µm	Fisher Scientific	10695211	Or different company
Syringe needle 27G	Henke Sass Wolf	4710004020	0.4 × 12 mm
Syringes – 1 mL	Codau	329650	Insulin Luer U-100
Syringes – 10 mL	BD	303134	Or different company
Syringes – 50 mL	BD	15899152	Or different company
Tube rack – 15 mL	Thomas Scientific	1159V80	Or different company
Tube rack – 50 mL	Thomas Scientific	1159V80	Or different company
Uncoated High-Speed Steel General Purpose Tap	McMaster-Carr	8305A77	Or different company
Vacuum filter – 0.2 µm	Merck	SCGPS05RE	Steritop filter