Bacteriophage-mediated lysis supports robust growth of amino acid auxotrophs

Microbial communities host many auxotrophs—organisms unable to synthesize one or more metabolites required for their growth. Auxotrophy is thought to confer an evolutionary advantage, yet auxotrophs must rely on other organisms that produce the metabolites they require. The mechanisms of metabolite provisioning by “producers” remain unknown. In particular, it is unclear how metabolites such as amino acids and cofactors, which are found inside the cell, are released by producers to become available to auxotrophs. Here, we explore metabolite secretion and cell lysis as two distinct possible mechanisms that result in the release of intracellular metabolites from producer cells. We measured the extent to which secretion or lysis of Escherichia coli and Bacteroides thetaiotaomicron amino acid producers can support the growth of engineered Escherichia coli amino acid auxotrophs. We found that cell-free supernatants and mechanically lysed cells provide minimal levels of amino acids to auxotrophs. In contrast, bacteriophage lysates of the same producer bacteria can support as many as 47 auxotroph cells per lysed producer cell. Each phage lysate released distinct levels of different amino acids, suggesting that in a microbial community the collective lysis of many different hosts by multiple phages could contribute to the availability of an array of intracellular metabolites for use by auxotrophs. Based on these results, we speculate that viral lysis could be a dominant mechanism of provisioning of intracellular metabolites that shapes microbial community structure.

phage adsorption and then transferred to 37 C.At each time point, 0.4 ml was removed.Half of the sample was added to a few drops of chloroform, vortexed for 10 seconds, and placed at room temperature until the chloroform settled.The remaining 0.2 ml was stored on ice and processed during the experiment.These samples were serially diluted up to 10 -8 , and spot plated onto BPRM top agar overlays (0.35% w/v) prepared with B. thetaioaomicron host cells to measure infective centers and free phage, and plated onto BPRM agar (1.5% w/v) to measure cell survival following phage addition.Three biological replicates were measured for each growth curve.At the end of the experiment, chloroform-treated samples were serially diluted up to 10 -8 , spot plated onto BPRM top agar overlays, and grown overnight in an anaerobic chamber.One-step growth curves for T4rI and vir were performed similarly, except that E. coli cultures were grown aerobically in H1 glycerol minimal medium at 37C.For PFU calculations, samples were spot plated on LB top agar overlays prepared with E. coli host cells (0.7% w/v) and CFU measurements were spot plated on LB agar (1.5% w/v).

Generating Phage Lysates for Auxotroph Assay
To generate E. coli phage lysates for the auxotroph assay, saturated cultures of E. coli MG1655 were diluted 1:100 in fresh H1 glycerol minimal medium.Once cells reached an O.D.600 of approximately 0.3, T4rI phage was added at an MOI of 3 for 100 minutes.Each phage lysate treatment had a parallel mock treatment that served as a control, prepared by passing phage stocks twice through 100 kDa centrifugal filters (Amicon).After 100 minutes, the phage-treated culture and the mock treatment were collected by centrifugation at 4,427 rcf for 10 minutes, and bacterial cells were removed by passing through a 0.2 um filter.The number of new phage particles generated during incubation was calculated as the difference between the titer measured at the beginning and end of the incubation period.This value was divided by the burst sizes calculated from the one-step growth curves to estimate the number of cells lysed by the phage.The samples were further processed by passing through a 100 kDa centrifugal filter to remove phage particles.Samples were stored at -80 C.Phage lysates for vir were prepared similarly except the incubation time was 150 minutes.Phage lysates from B. thetaiotaomicron were similarly processed, except the bacteria were grown anaerobically at 37C in defined minimal medium, and B. thetaiotaomicron was incubated with SJC12 for 4 hours.All lysates were generated in triplicate.

E. coli Auxotroph Assay
E. coli amino acid auxotrophs were pre-cultured in biological triplicate in H1 glycerol minimal medium at 37C with their required amino acid for 18-24 hours until saturation, and subsequently washed three times in H1 glycerol minimal medium to remove amino acids supplemented during the pre-culturing step.The cultures were then diluted to an O.D.600 of 0.02 in H1 glycerol minimal medium, and 100 µl of culture was dispensed into wells of a 96-well plate (Corning ®).One hundred microliters of phage lysate was then added to each well, bringing the final O.D.600 to 0.01 and the final glycerol concentration to 0.2%.The plates were shaken at 1200 rpm for 24 hours at 37C in a Southwest Science heated plate shaker.Absorbance measurements were measured using a multi-well plate reader (Tecan Spark).Colony forming units (CFU) were calculated after 24 hours of growth by spot plating 10-fold serial dilutions onto LB agar plates.

Phage Lysate Preparation and Titering
E. coli MG1655 was used as the host for the propagation of T4rI (Carolina Biological Supply Company).High-titer stocks of E. coli phages were generated by growing cultures at 37 C with aeration in either LB or H1 glycerol minimal medium to an O.D.600 of 0.2-0.3, and adding phage to an MOI of 0.01.After about 7 hours, chloroform was added to lyse remaining cells.After the chloroform settled, the sample was centrifuged at 6,026 rcf and the lysate was passed through a 0.2 um filter to remove bacterial cells.Phage stocks were titered by spot plating onto soft agar overlays.High titers for vir were generated similarly.
B. thetaiotaomicron was used as the host for the propagation of SJC12 phage.SJC12 was a gift from Andrew Hryckowian and Bryan Merrill.High-titer stocks of SJC12 were generated by a soft agar overlay method similar to [25].Briefly, 0.5 ml of saturated B. thetaiotaomicron culture was combined with 1-10 µl of high-titer SJC12 stock for 20 minutes to allow for phage adsorption.Then 4.5 mL of molten BPRM top agar (0.35%) prepared with B. thetaiotaomicon host cells added was poured onto BPRM agar (1.5 %) and incubated anaerobically overnight at 37C.Top agar overlays that showed a "lacy" pattern (confluent lysis) were flooded with sterile phage buffer (an autoclaved solution of 5 mL of 1 M Tris pH 7.5, 2 g NaCl, 5 mL of 1 M MgSO4, in 500 mL of ddH2O) and incubated at room temperature for at least 2 hours to suspend the phage.The phage buffer was removed, passed through a 0.2 µm filter and stored at 4 C for future experiments.For phage lysates used to generate lysate treatments for the auxotroph assays, defined minimal medium top agar was used instead of BPRM top agar.

Calculating Amino Acid Auxotrophs per Lysed Cell
To calculate the number of auxotrophs supported per lysed cell, the difference in CFU/ml between the treatment (addition of supernatant, French pressed lysate, or phage lysate) and its control (no addition control, French press control, or mock phage) was divided by the calculated number of lysed cells.

Figure S1 .
Figure S1.The growth yield of each amino acid auxotroph was measured after treatment with lysates and supernatants from either stationary or exponential phase E. coli cultures.Growth yield was calculated as the O.D.600 of cultures grown with the indicated supplements as a percentage of the O.D.600 of cultures grown with a saturating concentration of the required amino acid.Significance was determined by a one-way ANOVA with Tukey's multiple comparison test (p<0.0001= ****; p<0.0002= ***, p<0.0021=**, p<0.0332= *)