Invasions
Pseudomonas fluorescens with a lacZ marker was used as the
invader; the lacZ marker makes it visually distinguishable from
the wildtype on agar containing X-gal
(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) due to a blue colour
change (Zhang & Rainey 2007). Although originally reported as a neutral
marker (Zhang & Rainey 2007), the lacZ insert has previously
been found to offer a fitness advantage to invaders (Lear et al.2020). The strain was left to diversify for five days in static KB
before being plated and a single smooth (SM) and wrinkly spreader (WS)
colony was selected, grown and stored in 25% glycerol solution at
-80oC. SM morphotypes (our ‘coloniser’ invader)
inhabit the broth where they grow rapidly, whereas WS (our ‘competitor’
invader) form biofilms at the air-broth interface: biofilm formation
increases competitive ability for oxygen but at a cost to growth rate
(Hall et al. 2012). Before each invasion, these freezer stocks
were used to grow each morphotype overnight in shaken KB as described
above; these cultures were diluted to 1% with M9 salt buffer before
use. All microcosms were invaded every four days with 60µL of either SM
or WS invader (total colony forming units (CFU) added over the three
events: SM = 8.1x106; WS = 6.6x106).
If a microcosm had been disturbed, invasion would occur
post-disturbance. This resulted in 6 replicates for each disturbance by
resource abundance combination per invader morphotype.
Experiments finished on day 16, when all microcosms were homogenised and
a 900µL sample was frozen in 25% glycerol at -80ºC. After plating on KB
agar containing 100µg/L of X-gal, wildtype and invader SM, WS and fuzzy
spreader (FS; a rarer bottom-dwelling morph (Rainey & Travisano 1998))
colonies were counted.