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Bacteria produce extracellular enzymes to obtain resources from complex chemical substrates, but this strategy is vulnerable to cheating by cells that take up reaction products without paying the cost of enzyme production. We hypothesized that cheating would suppress enzyme production in co-cultures of cheater and producer bacteria, particularly under well-mixed conditions. To test this hypothesis, we monitored protease expression and frequencies of
The breakdown of complex organic matter is a fundamental biogeochemical process that is largely controlled by microbes. Living biomass contains a wide range of polymeric macromolecules, including proteins, nucleic acids, and structural carbohydrates (
Microbes can benefit from enzyme production by accessing energy and nutrients in complex compounds, but this strategy also requires a substantial resource investment in enzyme synthesis and excretion (
Extracellular enzymes belong to a class of chemical compounds known as “public goods” because they are costly to individual microbes but increase resource availability for other organisms. Public goods include compounds like signaling molecules, antibiotics, siderophores, and secreted proteins (
Theoretical studies suggest that cheaters can suppress enzyme producers and reduce substrate degradation rates in model systems (
Similar patterns have been observed with other public goods. For instance, colicin-producing, colicin-resistant, and colicin-susceptible strains of
Although some empirical studies are consistent with cheating in extracellular enzyme systems, few studies have confirmed that reductions in enzymatic function are due to cheaters. For example,
The predicted negative impact of cheaters on enzyme producers has been demonstrated via controlled competition experiments with genetically manipulated microbes.
The objective of our study was to analyze the effects of diffusion, cheating, and evolution in extracellular enzyme systems. Although spatial structure is known to promote diversity in many microbial systems, theoretical predictions about the effect of diffusion on enzyme cheating have not been confirmed. Based on findings with similar public goods (
We obtained strains of
Strains were grown on either M63 medium or casein medium. The base M63 medium (pH 7.0) contained 2.0 g/l (NH4)2SO4, 13.6 g/l KH2PO4, 0.5 mg/l FeSO4[H2O]7, and 0.2% (w/v) glucose. Casein medium contained 0.4% (w/v) casein in diluted M63 medium (0.25 × in deionized water). Base and casein media were supplemented with 1 mM MgSO4, 0.35 mM CaCl2, and 50 μg/ml ampicillin. Solid media for plate experiments contained 1.5% agar.
For experiments in shaking flasks, cells were grown overnight in M63 medium to an OD600 of ~1 and inoculated into triplicate 50 ml flasks containing 10 ml casein medium. One set of triplicates was inoculated with overnight culture containing only cheater cells. We added 5 mg/l proteinase K to these flasks to hydrolyze casein and allow for cheater growth. This concentration of proteinase was equivalent to the protease present in producer cultures at stationary phase. These flasks served as a control for cheater growth and allowed us to confirm that the Tn5 insertion was maintained in the absence of kan for the duration of the experiment. Another set of triplicate flasks contained casein medium and was inoculated with a mixture of 50% cheater cells and 50% producer cells to examine competition between the strains. A final set of triplicate flasks also contained casein medium but was inoculated with only producer cells. This treatment was designed to test whether protease function was maintained in the absence of inoculated cheaters. For all three experiments, starting OD600 in each of the triplicate flasks was 0.01.
All flasks were maintained at 28°C in a dark incubator with orbital shaking at 200 rpm. After the cultures reached stationary phase, 100 μl culture fluid was transferred to a new flask containing 10 ml fresh medium. For trial 1 of this experiment, transfers were done every 1 or 2 days for 23 days depending on the availability of laboratory staff. For the second trial, transfers were standardized to occur every 48 hours for 30 days. Optical densities at stationary phase were ~1.0, which corresponds to ~3 × 109 cells/ml (
For the agar plate experiments, triplicate plates were inoculated with ~6.6 × 106 producer cells or a 50–50 mixture of cheaters and producers, as in the flask experiment. Because growth was slower on agar, transfers occurred every 3 days for 81 days. Cells were transferred to new agar medium by contact plating. Our shaking flask experiment showed that after a period of time, protease phenotype did not necessarily correspond to kan resistance phenotype. Therefore, we assayed protease expression directly on clones isolated from agar plates to determine the frequencies of cheaters and producers in this experiment.
For the shaking flask experiment, we used the colorimetric azocasein method (
To identify possible genetic variation underlying changes in protease phenotype, we extracted and sequenced genomic DNA from all replicates of trial 2 (day 30) in the shaking flask experiment. We also extracted and sequenced the genomic DNA of the ancestral strain
Optical densities were compared across inoculation treatments using analysis of covariance with time as the covariate. We used Tukey tests to determine if changes in optical densities over time were significantly different across treatments. For the agar plate experiment, we calculated the frequencies of cheaters versus producers based on relative enzyme expression of isolated clones. Background fluorescence of the substrate and medium was subtracted from the fluorescence of each clone to calculate net fluorescence. Relative expression values were calculated by dividing the net fluorescence of each clone by the maximum net fluorescence observed at a given transfer. For the agar plate experiment, clones with relative expression values >0.56 were considered producers whereas clones below this value were classified as cheaters. We also calculated the coefficient of variation in relative protease expression across all clones at each time point and used linear regression to determine if there was a significant increase in variation over time.
In trial 1 with shaking flasks, we observed significant (
Concurrent with the decline in optical density, protease concentration and kan sensitivity also declined during trial 1 in cultures growing on casein medium. Protease was initially higher in producer cultures than in mixed cultures, but neither culture showed detectable protease by day 20 of the trial (
The second mixed flask trial was similar to the first in overall patterns of optical density (
We did not find evidence for genetic changes in the protease operon or surrounding regions that could account for observed differences in protease phenotype. Although we detected multiple genetic variants with frequencies >10% in each of the mapped genomic libraries from trial 2, these variants were also present in the ancestral ON2 strain. Our genetic analyses were clearly capable of detecting genetic variation because sequences containing Tn5 genetic material were found in all libraries that contained the ON2-pd5 cheater strain.
To assess the effect of spatial structure and restricted diffusion on enzyme production, we monitored the frequency of cheater and producer phenotypes in cultures maintained on agar plates. When starting with 50% cheater and 50% producer genotypes, cultures on plates were initially dominated by producer phenotypes (>80%), but cheater phenotypes became dominant after 30 days and remained dominant through the end of the experiment (
Data from both trials under shaking conditions support the hypothesis that cheater phenotypes have a selective advantage. In the first trial, a loss of protease function over 23 days was accompanied by a loss of the kan sensitive genotype characteristic of our producer strain. Under shaking conditions, protease-negative mutants likely have a selective advantage because they do not pay the costs of enzyme production, yet they can access casein hydrolysis products (
Even if no cheater knockouts were inoculated into the initial culture, we still observed loss of protease function over time (
Our second trial under shaking conditions suggests that cheaters may arise by multiple evolutionary pathways with different fitness consequences. In this trial, we observed a decline in protease expression in the 50–50 mixed culture, but there was no associated loss of kan sensitivity. This pattern suggests that
It is somewhat surprising that we observed distinct evolutionary outcomes in trial 1 versus trial 2. Cheaters derived from the wild-type swept all three replicate flasks in the 50–50 mixture of Trial 2, but the original cheater knockout (or its descendants) swept the populations in Trial 1. With only three replicates, we cannot rule out that the different outcomes were due to chance (
The results from the liquid producer culture in trial 2 suggest that the loss of protease function is reversible. Protease loss during the first 25 days of the trial reduced the availability of casein hydrolysis products. Under these conditions, protease-producing variants are favored because proteolysis increases resource availability for growth while cheater densities are relatively low. The protease-positive phenotype that emerged after 25 days could have resulted from compensatory mutations or positive selection on rare producer genotypes that survived the population bottleneck on day 22.
Alternatively, loss and recovery of protease function could have resulted from changes in gene expression. However, this mechanism is unlikely given that most clones isolated on days 12 and 24 from producer flasks in trial 2 did not express protease even after plating on agar and regrowth on casein medium. Furthermore, the complete loss of protease function and crash of producer populations probably would not have occurred if gene expression were flexible.
The recovery of protease expression is indicative of frequency-dependent selection, whereby producers are favored at low population densities and cheaters are favored at high densities (
Results from maintaining cultures on agar plates support the hypothesis that limitations on diffusion enhance the coexistence of cheater and producer strains. Frequency variations from the 50–50 mixture are suggestive of oscillations predicted by theoretical models under intermediate diffusion levels (
As in the shaken flask experiment, the agar plate experiment showed evidence for evolution of phenotypes with reduced protease expression. However, the shaking flasks were subject to sweeps by cheaters with undetectable levels of protease expression, whereas the agar plates hosted strains with a continuum of enzyme production levels that we arbitrarily classified into cheaters versus producers. This diversity is evident from the significant increase in the coefficient of variation in protease expression across randomly selected isolates (
Reduced diffusion and increased spatial structure likely both contributed to increased diversity on agar plates. Limits on diffusion allow cheaters and cooperators to coexist because cooperators gain preferential access to the benefits of public goods production (
Our results have implications for the maintenance of microbial diversity and ecological function in environmental settings. Few natural environments are completely well-mixed, and therefore the selective sweeps that may have occurred in our shaking flasks are probably uncommon. Even in aquatic and marine ecosystems, particulate organic matter generates spatial heterogeneity that could select for a range of enzyme production strategies (
Although we identified important mechanisms for maintaining diversity and function despite the potential for cheating, our results also suggest that cheaters play a role in many microbial systems. At least in aquatic and saturated systems most similar to our experimental conditions, cheaters are probably always present to some degree. Broadly defined, cheating need not involve a complete loss of enzyme expression or other function. A reduction in the amount of enzyme production relative to neighboring cells is also a form of cheating if the enzyme benefits are distributed equally but the costs are unequal.
Regardless of its form and magnitude, cheating can impact ecological processes, such as hydrolysis rates for biomolecules. In the case of enzyme production strategies, greater diversity and overall microbial growth may not translate into maximal rates of hydrolysis (
Steven D. Allison conceived the project, designed experiments, analyzed data, and wrote the manuscript. Lucy Lu conducted the experiments, analyzed data, and helped write the manuscript. Alyssa G. Kent and Adam C. Martiny conducted the genetic analyses and helped write the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This research was supported by NSF programs in Population and Evolutionary Processes and Advancing Theory in Biology. Alyssa G. Kent was supported by NSF GRFP DGE-1321846. Ole Nybroe provided