Exclusion zones are formed by stressed A. graevenitziimicrocolonies in nutrient competition with S. aureus
Exclusion zones could be easily visualized in co-cultures of A.
graevenitzii and GFP-expressing S. aureus (Fig. 3B), allowing us
to measure multiple aspects of the co-culture that we thought might
influence exclusion zone formation.
Given that exclusion zones formed around each individual A.
graevenitzii microcolony, we hypothesized that co-cultures containing
increasing numbers of A. graevenitzii microcolonies would have
decreasing amounts of S. aureus growth. Using fluorescence
microscopy, we measured the percent confluence of S. aureusgrowth based of the area of GFP fluorescence within each co-culture
chamber as a fraction of the total area of the chamber. S. aureusconfluence ranged from 19-96%, depending on number of A.
graevenitzii colonies in the co-culture. As expected, a significant
correlation (r2 = 0.1355, ***p<0.0001) was
observed between the final number of A. graevenitziimicrocolonies and observed suppression of S. aureus growth (Fig.
3D). Interestingly, we did not find a significant correlation
(r2 = 4.4x10-5, p = 0.9297) between
the initial species:species ratio of bacteria loaded and later S.
aureus growth (Fig. 3E), likely because the rapid doubling time ofS. aureus overcomes difference in initial bacteria ratio. Thus,
the key factor influencing the outcome appears to be the number ofA. graevenitzii microcolonies present in the co-culture.
In monoculture, A. graevenitzii exhibited extensive filamentous
growth, formation of new microcolonies and grew to effectively fill the
chamber. In contrast, co-culture of A. graevenitzii with S.
aureus resulted in formation of smaller microcolonies with optically
dense “core” region bordered by a radial array of relatively short
filaments extending outwards into the environment (Fig. 4A,B). These
stunted colonies rarely produced secondary colonies (data not shown). We
compared the size of exclusion zones formed around microcolonies to the
size of the colony “core” and the total colony diameter, which largely
reflected the length of the radial filaments extending outwards. These
measurements revealed that significantly larger exclusion zones were
generated around microcolonies with larger “core” regions
(r2 = 0.5575, ***p < 0.0001) (Fig. 4C),
while colonies with more extensive radial filamentous growth exhibited
significantly smaller exclusion zones (r2 = 0.2141,
***p < 0.0001) (Fig. 4D). It is likley that the A.
graevenitzii microcolony morphology observed in co-culture may reflect
a state of stress for the bacterium, which might also be related to the
formation of exclusion zones. Microcolonies under less stress might
exhibit more extensive radial filamentous growth (as observed in
monoculture), while colonies under more stress might exhibit restricted
filamentous growth, resulting in formation of a larger dense “core”
region.
To test whether making more nutrients available affected exclusion zone
formation, we compared co-cultures performed in 50 versus 100 µm tall
chambers. Relative to S. aureus monocultures in each condition,
we observed less restriction of S. aureus growth in co-culture
with A. graevenitzii in 100 µm tall (9.8%) chambers compared to
50 µm tall (21.6%) chambers (Fig. S1F). This observation supports our
hypothesis that exclusion zone formation occurs in response to
competition for nutrients.