Kellermann et al 2022_Preparation for denitrification at the cusp of anoxia
Supporting information to Kellermann et al, submitted to mBio January 2022; five separate files named supplemental item 1-5. Paper abstract: Adaptation to anoxia by synthesizing a denitrification proteome costs metabolic energy, and the anaerobic respiration conserves less energy per electron than aerobic respiration. This implies a selective advantage of the stringent O2- repression of denitrification gene transcription, which is found in most denitrifying bacteria. In some bacteria, the metabolic burden of adaptation can be minimized further by phenotypic diversification, colloquially termed bet-hedging, where all cells synthesize the N2O reductase (NosZ) but only a minority synthesizes nitrite reductase (NirS), as demonstrated for the model strain Paracoccus denitrificans. We hypothesized that the cells lacking NirS would be entrapped in anoxia, but with the possibility of escape if supplied with O2 or N2O. To test this, cells were exposed to gradual O2-depletion or sudden anoxia, and subsequent spikes of O2 and N2O. The synthesis of NirS in single cells was monitored by using an mCherry-nirS fusion replacing the native nirS, and their growth was detected as dilution of green, fluorescent FITC-stain. We demonstrate anoxic entrapment due to e--acceptor deprivation and show that O2-spiking leads to bet-hedging, while N2O-spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the N2O-spike had much lower respiration rates than those rescued by the O2-spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O2. Our results bring into relief a fitness advantage of pairing restrictive nirS expression with universal NosZ synthesis in energy limited systems. Supplemental item 1 is a description and qualification of the glucose oxidase-catalase (GOX) approach used for the removal of residual oxygen in experimental vials before inoculation. In supplemental item 2, we display the gas- and flow cytometry data in the O2 spiking experiment, including the data shown in Figure 2 and 3 in the paper. In supplemental item 3, we show the gas kinetics and microscopy analyses from the N2O spiking experiment, which in the paper is summarized in Fig 4. In supplemental item 4, we describe the steps taken to estimate apparent specific growth rates in single vials and cell yield per mol electron to N-oxides. In supplemental item 5, we describe a simple experiment where nitrite reducing cultures of Paracoccus denitrificans was spiked with N2O and O2 and the subsequent rate of nitrite reduction was assessed.