Bacterial strains, media and batch cultures
We used the wildtype strain Vibrio natriegens ATCC 14048 and Alteromonas macleodii sp. 4B03 (non-clumping variant) isolated from marine particles [8]. Strains were cultured in Marine Broth (MB, Difco 2216) and grown overnight at 25 °C. In total, 1 ml of cell culture was centrifuged (13,000 rpm for 2 min) in a 1.5 ml microfuge tube. After discarding the supernatant, the cells were washed with 1 ml of MBL minimal medium medium without carbon source. Cells were centrifuged again and the cell pellet was resuspended in 1 ml of MBL (marine minimal medium) [30, 34] adjusted to an 0.002 OD600. Cells from these cultures were used for experiments in MBL minimal medium containing 0.1% (weight/volume) Pentaacetyl-Chitopentaose (Megazyme, Ireland). The carbon source was added to the MBL minimum medium and filter sterilized using 0.22 μm Surfactant-Free Cellulose Acetate filters (Corning, USA). A total of 500 µl of the prepared cultures (250 µl + 250 µl for co-cultures) were added to 9.5 ml of MBL + 0.1 % chitopentaose (v/w) in serum flasks. This resulted in a starting OD of 0.0001. The flasks included a stirrer and were sealed with a rubber seal. Serum flasks were stored on a bench top magnetic stirrer (500 rpm) and connected to the microfluidics setup via Hamilton NDL NO HUB needles (ga21/135 mm/pst 2).
Microfluidics
Microfluidics experiments were performed as described previously [35,36,37,38]. Cell growth was imaged within mother machine channels of 25 × 1.4 × 1.26 μm (length × width × height). Within these channels, cells could experience the batch culture medium that diffused through the main flow channels. The microfluidic device consisted of a PDMS flow cell (50 µm/23 µm). The PDMS flow cell was fabricated by mixing the SYLGARD 184 Silicone Elastomer Kit chemicals 10:1 (w/v), pouring the mix on a master waver and hardening it at 80 °C for 1 h. The solid PDMS flow cell was cut out of the master waver and holes were pierced at both ends of each flow channel prior to binding it to a cover glass (Ø 50 mm) by applying the “high” setting for 30 s on the PDC-32G Plasma Cleaner by Harrick Plasma. The flow cell was connected via 40 mm Adtech PTFE tubing (0.3 mm ID × 0.76 mm OD) to a Ismatech 10 K Pump with 40 mm of Ismatech tubing (ID 0.25 mm, OD 0.90 mm) which again was connected via 80 mm Adtech PTFE tubing (0.3 mm ID × 0.76 mm OD) via a 5 mm short Cole-Parmer Tygon microbore tubing (EW-06418-03) (ID 0.762 mm OD 2.286 mm) connector tubing to a Hamilton NDL NO HUB needle (ga21/135 mm/pst 2) that was inserted into the feeding culture. During the whole experiment the pump flow was set to 1.67 µl/min (0.1 ml/h).
Time-lapse microscopy
Microscopy imaging was done using fully automated Olympus IX81 or IX83 inverted microscope systems (Olympus, Japan), equipped with a ×100 NA1.3 oil immersion, phase contrast objective, an ORCA-flash 4.0 v2 sCMOS camera (Hamamatsu, Japan), an automated stage controller (Marzhauser Wetzlar, Germany), shutter, and laser-based autofocus system (Olympus ZDC 1 and 2). Detailed information about the microscopy setup has been described by D’Souza et al. [39]. Channels on the same PDMS Chip were imaged in parallel, and phase-contrast images of each position were taken every 5 min. The microscopy units and PDMS chip were maintained at room temperature. All experiments were run at a flow rate of 0.1 ml h−1, which ensures nutrients enter the chamber through diffusion. Four biological replicates were performed. These replicates consist of four independent microfluidics channels (two for each of the strains). These channels were connected to one of two independent batch cultures.
The microscopy dataset consists of 200 mother machine channels; 49 channels for the degrader on co-culture, 51 for the degrader on mono-culture, 40 for the cross-feeder on mono-culture and 60 for the cross-feeder on co-culture.
Image analysis
Image processing was performed using a modified version of the Vanellus image analysis software (Daan Kiviet, https://github.com/daankiviet/vanellus), together with Ilastik [40] and custom written Matlab scripts.
Movies were registered to compensate for stage movement and cropped to the region of growth channels. Subsequently, segmentation was done on the phase contrast images using Ilastik’s supervised pixel classification workflow and cell tracking was done using the Vanellus build-in tracking algorithm.
After visual curation of segmentation and tracking for each mother machine and at every frame growth parameters were calculated using custom written matlab scripts [36]. Lengths of individual cells were estimated by finding the cell center line by fitting a third-degree polynomial to the cell mask; then the cell length was calculated as the length of the center line between the automatically detected cell pole positions (see Kiviet et al. [33] for details).
We quantified cell growth by calculating single-cell elongation rates r from measured cell length trajectories: L(t) = L(0)∙e^(r ∙ t). Cell lengths and growth rates varied drastically over the time course of the experiment; we thus developed a robust procedure that can reliably estimate elongation rates both for large fast-growing cells as well as for small non-growing cells. We first log-transformed cell lengths, which were subsequently smoothed over a moving time window with a length of 5 h (60 time points). We used a second order local regression using weighted linear least squares (rloess method of Matlab smooth function) in order to minimize noise while maintaining sensitivity to changes in elongation rates. Subsequently the instantaneous elongation rate was estimated as the slope of a linear regression over a moving time window of 30 min (7 time points). Time points for which the fit quality was bad (χ2 > 10−4) were removed from the analysis [32]. All parameters were optimized manually by visually inspecting the fitting procedure of many cell length trajectories randomly selected from across all replicates.
As cells are continuously lost from the mother machine channels it is non-trivial to calculate the total amount of biomass produced in the chip. We thus need to estimate this quantity from the observed single-cell elongation rates. Specifically, we estimated the total amount of biomass produced per individual mother machine until a given time point as:
$$B_T = e^{Delta tmathop {sum}limits_{i = 1}^T { < r_i > } }$$
Where <ri> is the average growth rate of all cells in a given replicate at time point i, and where Δt is the time interval between two timepoints. By using the average growth rate, we ignore the variation in growth rates between cells. However, it is difficult to calculate population growth when growth rates vary both with time and between cells and the current method still allows us to capture the overall effect of interactions on cell growth.
Datasets and statistical analysis
All microfluidics experiments were replicated four times. No cells were excluded from the analysis after visual curation. For V. natriegens 2227 cells were analyzed on mono-culture, and 1707 cells were analyzed on co-culture. For A. macleodii 2657 cells were analyzed on mono-culture, and 3901 cells were analyzed on co-culture. Each mother machine channel was treated as an independent sample. All statistical analysis was performed in Rstudio v1.2.5033. Percent increases were calculated using the relative differences of estimated between the corresponding values. For mixed effect models analysis the LmerTest package (Version 3.1-3) [41] with the following equation were used: y ~ Batch + (1 | Replicate) The Tukey Post hoc test was performed using the Multcomp package (Version 1.4-15) [42].
Chitinase assay
Degrader and Cross-feeder cells were cultured in Marine Broth (MB, Difco 2216) and grown overnight at 25 °C. In total, 1 ml of cell culture was centrifuged (13,000 rpm for 2 min) in a 1.5 ml microfuge tube. After discarding the supernatant the cells were washed with 1 ml of MBL minimal medium medium without carbon source. Cells were centrifuged again and the cell pellet was resuspended in 1 ml of MBL adjusted to an 0.002 OD600. A total of 10 µl of cell culture was added to 190 µl of MBL containing 0.1% Chitopentaose (w/v). Cultures were grown to exponential phase in a plate reader (Eon, BioTek) at 25 °C. Cell free supernatants were generated by sterile filtering cultures using a multi-well filter plate (AcroPrep) into a fresh 96 well plate. Chitinase activity of cell free supernatants was measured using a commercially available fluorometric chitinase assay kit (CS1030, Sigma-Aldrich) following the protocol. In short, 10 µl of sterile supernatant was added to 90 µl of the assay mix. The solution was incubated in the dark at 25 °C for 40 min before measuring fluorescence (Excitation 360 nm, Emission 450 nm) in a plate reader (Synergy MX, Biotek). Logarithmic chitinase activity per OD600 was analyzed for eight replicates.
Chitinase activity in units per ml was calculated using a standard concentration. Using the following Formula: ({{{{{rm{Units}}}}}}/{{{{{rm{ml}}}}}} = frac{{left( {{{{{{rm{FLU}}}}}} – {{{{{rm{FLUblank}}}}}}} right) times 1.9 times 0.3 times {{{{{rm{DF}}}}}}}}{{{{{{{rm{FLUstandard}}}}}} times {{{{{rm{time}}}}}} times {{{{{rm{Venz}}}}}}}})
Here, FLU indicates measured fluorescence, DF indicates the dilution factor, and V indicates the volume of the sample in ml [43].
Acetate assay
Cell cultures were prepared and grown in serum flasks as described above. At different time intervals 1 ml of culture was removed and OD600 was measured. Cultures were filter sterilized using 0.22 μm Surfactant-Free Cellulose Acetate filters (Corning, USA) into a 1.5 ml microfuge tube. Cell free supernatants were stored at −4 °C until they were used for acetate measurements. Acetate concentrations were measured using a colorimetric assay kit (MAK086, Sigma-Aldrich) following the protocol. In short, 50 µl of cell free supernatant was added to 50 µl of assay mix. The solution was incubated in the dark at 25 °C for 30 min. Acetate concentrations were measured in a plate reader (Eon, Biotek) at 450 nm [44].
Growth on spend media
Degrader and Cross-feeder cells were cultured in Marine Broth (MB, Difco 2216) and grown overnight at 25 °C. In total, 1 ml of cell culture was centrifuged (13,000 rpm for 2 min) in a 1.5 ml microfuge tube. After discarding the supernatant, the cells were washed with 1 ml of MBL minimal medium medium without carbon source. Cells were centrifuged again and the cell pellet was resuspended in 1 ml of MBL adjusted to an 0.002 OD600. A total of 10 µl of cell culture was added to the 190 µl cell free supernatant described above. Cultures were grown in a plate reader (Eon, BioTek) at 25 °C. Cell free supernatants after this growth assay were generated by sterile filtering cultures using a multi-well filter plate (AcroPrep) into a fresh 96 well plate. These supernatants were used as described above to measure acetate levels after growth on spend media.
Source: Ecology - nature.com
