This data set summarises the chemotactic response of a model marine bacteria (Vibrio alginolyticus YM4 wild-type) to filtered exudates of the cyanobacteria Synechococcus sp WH8102. Two filtrate sets were collected, each spanning 6 time points (named T1 -> T6), with the initial assays split into 4 biological replicates (named A,B,C,D). The two treatments were: 1) A control treatment (named "Control", or shortened to "C") 2) A phage-infected treatment (named "Phage", or shortened to "P"), where host Synechococcus were infected with the T-4 like Myovirus S-SSM5, with data collected over the pre-lysis cycle. These treatments are fully described in: https://doi.org/10.1038/s43705-022-00169-6. The filtrates were kept frozen at -80C in 1ml aliquots, and only thawed to room temperature immediately prior to experimental use. For each time point (T1-6), and replicate (A-D), three sets of chemotaxis assays were collected: Phage vs ASW, Control vs ASW, Phage vs Control. (Each with three biological replicates with fresh cell suspensions, with three physical replicates per sample, totaling nine samples at each time point/sample replicate). The chemotactic preference of Vibrio alginolyticus was measured by analyzing the cell distribution over time, and comparing the cell populations on opposite sides of the channel. Upon completion of the assays, the data shows that the bacteria will preferentially chemotax towards the phage-infected cells, with the strongest response occurring immediately in the infection cycle (T1), contrary to the assumed belief that it is post-lysis behavior that drives microbial chemotaxis in the oceans.

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

The data in this data set is organized as follows:

Accumulation Curves: The final processed data for each time point (T1-6), each replicate (A-D) with the average and SEM values tabled. 

Filtrates_T#R: Where # is the time point number (1-6) and R the replicate (A-D). Each folder contains:

1) Three Excel sheets corresponding to the raw cell counts for each biological replicate (each with 3 repeats)

2) A supplemental folder containing a cell array with the full bacteria positions for each time point for each replicate (.mat format)

The chemotaxis data was collected by Dr. Richard J. Henshaw of Tufts University (Boston, MA, USA) from 3rd December 2020 to 8th October 2021. Queries about this dataset should be directed to rhenshaw@ethz.ch.

Methods & Sampling

Vibrio alginolyticus (YM4; wild-type) from -80℃ stock were grown overnight in Marine 2216 media (Difco) by incubating at 30℃ and shaking at 600 revolutions per minute (RPM). The overnight culture was diluted 100-fold into fresh pre-warmed 2216 media and grown for three hours (30℃, shaking at 600 RPM) to O.D. ≈ 0.2. 2 ml of culture was then washed and resuspended (1,500 RCF for 5 min) in 0.5ml of artificial seawater (ASW).

Artificial seawater (ASW) was prepared following the NCMA ESAW Medium recipe, which was adapted from Harrison et al.and modified by Berges, and filtered through a 0.2um filter immediately prior to use.

A simple three-inlet gradient generation microfluidic device was used to produce the chemical gradients.  Microfluidic devices were made using standard soft lithography techniques. Polydimethylsiloxane (Dow Corning SYLGARD 184) channels were cast on photoresist (Microchem) molds fabricated via photolithography and plasma bonded to standard glass slides. Gradient generation channels were designed with three inlets (width 0.5mm) carrying the chemostimulus solution, cell suspension and ASW media, respectively. Prior to use, the chambers were pretreated with a 0.5% BSA solution to mitigate cell adhesion. The three solutions were flow stratified for a minimum of 2min using a syringe pump(Harvard Apparatus), whereby flow rates were adjusted to maintain a 4:1:4 ratio of the stream widths. Upon halting the flow, a monotonic chemotaxis profile was established through diffusion. A chemostimulus gradient develops in the channel via diffusion, and the chemotactic response of the cell population was observed over time. Imaging was performed with phasecontrast microscopy (4×, 0.13 NA objective; Nikon Ti-E) at 1 fps over the course of ~10 min using a CMOS camera (Blackfly S, Teledyne FLIR). An example of the gradient generator used can be found in http://dx.doi.org/10.1038/s41567-021-01247-7

This data set summarises the chemotactic response of a model marine bacteria (Vibrio alginolyticus YM4 wild-type) to filtered exudates of the cyanobacteria Synechococcus sp WH8102. Two filtrate sets were collected, each spanning 6 time points (named T1 -> T6), with the inital assays split into 4 biological replicates (named A, B, C, D). The two treatments were:

1). A control treatment (named "Control", or shortened to "C")

2). A phage-infected treatment (named "Phage", or shortened to "P"), where host Synechococcus were infected with the T-4 like Myovirus S-SSM5, with data collected over the pre-lysis cycle.

These treatments are fully described in: https://doi.org/10.1038/s43705-022-00169-6. The filtrates were kept frozen at -80C in 1ml aliquots, and only thawed to room temperature immediately prior to experimental use.

For each time point (T1-6), and replicate (A-D), three sets of chemotaxis assays were collected: Phage vs ASW, Control vs ASW, and Phage vs Control. (Each with three biological replicates with fresh cell suspensions, with three physical replicates per sample, totaling nine samples at each time point/sample replicate). The chemotactic preference of Vibrio alginolyticus was measured by analyzing the cell distribution over time, and comparing the cell populations on opposite sides of the channel.

All data were processed using custom MATLAB scripts (version 2021a). The processing summary is:

1) Locate particles using a bandpass filter/peak finding algorithm (available: https://site.physics.georgetown.edu/matlab/)

2) Bin particles according to x-position to examine distribution across the field of view

An example of the analysis script is included in the dataset.

Coverage: Culture-based work

NSF Award Abstract:
Drifting photosynthetic microbes in surface ocean waters carry out nearly half of global carbon (C) fixation, both supporting the marine food web and reducing atmospheric carbon dioxide (CO2) levels. The fate of C in ocean ecosystems is controlled by myriad individual interactions within a highly interconnected planktonic food web, the sheer complexity of which has hindered predictive understanding of global C cycling. Chemical cues govern microbial interactions, and during infection, marine viruses manipulate the metabolism of phytoplankton and bacteria, facilitating the release of dissolved organic matter from infected cells. This research aims to determine how viral metabolic reprogramming of and organic matter release from intact, infected phytoplankton influences microbial interactions and C cycling. The interdisciplinary, collaborative nature of the project will enable direct training of two postdoctoral researchers, one graduate student, and undergraduate students in viral ecology, microfluidics, and metabolomics. An educational outreach program that engages middle school students in hands-on, high speed imaging of microbes will be expanded, and the project will culminate in a three-day workshop to advance the application of microfluidic devices and mass spectrometry analyses in microbial ecology.

The overarching hypothesis behind this research is that viral infection alters the chemical landscape of intact, infected picophytoplankton cells, attracting neighboring chemotactic bacteria and protistan zooplankton, and altering C flux pathways. To test this idea, a series of linked multi-scale laboratory-based experiments will be run to 1) Characterize the response of diverse model marine microbes to dissolved organic matter (DOM) released from intact, virus-infected picophytoplankton using microfluidics-based chemotaxis assays, 2) Identify key viral-derived DOM compounds eliciting chemotactic responses using stable isotope labeling, metabolomics analyses, and chemotaxis assays, and 3) Quantify micron-scale cross-trophic encounter dynamics and evaluate their impact on bulk-scale C cycling using liter-scale measurements of C dynamics linked to high spatiotemporal resolution live imaging of microbial food webs. The ultimate goal of the project is to develop a mechanistic understanding of the role of intact, virus-infected cells in oceanic C cycling.