G. diazotrophicus colonies were raised on solid LGI-P medium (Cavalcante 1988) for five days under ambient (21 kPa) and low (2 kPa) partial pressures of atmospheric oxygen. Whole colonies were observed live using laser scanning confocal microscopy to quantify differences in cell positioning or freeze-substituted, fixed, embedded and sectioned for light microscopy. We found that the uppermost bacterial cells were located at a greater depth below the surface of the colony mucilage under the ambient 02 treatment (85-110 pm) than in the low 02 treatment (45-60 pm).
Colonies raised at 21 kPa 02 for 5 days were assessed for nitrogenase activity (measured by H2 evolution) then gently spread over the surface of the agar with a bent glass rod. After disruption the nitrogenase activity of the colonies was re-assessed. This showed that disruption of the colony structure resulted in a decrease in H2 evolution to 3.5% that of the intact colonies.
As G. diazotrophicus colonies age, their structure begins to break down; the mucilage slumps to one side of the colony. By the 8th day post-inoculation, 75% of the colonies on a petri plate are collapsed. Nitrogenase activity of the colonies was tracked from day 3 to day 8 post-inoculation to determine the effect of colony structure disruption on nitrogenase activity. As the colonies increased in size and then collapsed over the time period, nitrogenase activity of G. diazotrophicus peaked at day 6, then decreased to 76% of maximum at day 8.
These data support a role for colony structure in the protection of nitrogenase by G. diazotrophicus.
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