Evidence of rapid non-targeted effects of cycloheximide on soil bacteria using 13C-PLFA analysis

Abstract

Stable isotope probing of phospholipid fatty acids (PLFA-SIP) is useful when studying bacterial contributions to soil processes, and it is an effective way to separate fungal and bacterial activity by linking 13C enrichment to specific PLFAs. Distinguishing bacterial contributions to soil processes often employs selective inhibitors; however, studies demonstrating their efficacy when using PLFA-SIP are less common. Here, we determined the effect of the fungal inhibitor cycloheximide (4.8 mg g−1 dry soil) and the bacterial inhibitor bronopol (0.48 mg g−1 dry soil) on microbial communities white spruce [Picea glauca (Moench) Voss] forest floor by measuring the uptake of 13C-enriched glucose (2 mg g−1 dry soil) in microbial PLFAs. We targeted [13C]glucose uptake by the bacterial community conditioned to a stable soil environment of 23 °C for over 2 wk rather than new bacteria generated from active colony growth caused by glucose addition. Nearly all bacterial PLFAs exhibited pronounced inhibition of 13C enrichment in the presence of bronopol. Limited inhibition of 13C enrichment in the presence of cycloheximide was observed as bacterial PLFA affected by cycloheximide had roughly one third less 13C enrichment than samples emended with [13C]glucose alone. Inhibitory effects only reduced 13C enrichment and did not affect total PLFA concentrations, implying that the inhibitors in the concentrations applied were impeding bacterial activity without causing cell death. Based on this work, we conclude that bronopol is an effective inhibitor for bacteria. Additionally, non-targeted effects of cycloheximide on soil bacteria must be accounted for when it is used in soil incubations.