The ammonium transporter AmtB is dispensable for the uptake of ammonium in the phototrophic diazotroph Rhodopseudomonas palustris

Ammonium (NH 4 + ) transport across cell membranes plays an important role in assimilation or removal of the environmental nitrogen. The membrane protein AmtB has been considered to be the NH 4 + transporter that is responsible for the NH 4 + uptake. The phototrophic diazotroph Rhodopseudomonas palustris harboring two amtB genes has been widely used in wastewater treatment and bioremediation. However, the role of AmtB in NH 4 + uptake remains unclear in R. palustris . Here, we employed an innovative approach combining stable isotope probing (SIP) with Raman spectroscopy to determine the physiological functions of AmtB1 and AmtB2 in R. palustris. This powerful technique allowed us to investigate NH 4 + uptake at the single-cell level. The generated R. palustris Δ amtB1 Δ amtB2 mutant lacking AmtB1 and AmtB2 proteins was still capable of utilizing 15 NH 4 + even the 15 NH 4 + concentration was as low as 5 μM. These data demonstrate that both of the AmtB proteins are not essential for R. palustris to take up NH 4 + regardless of environmental NH 4 + levels. However, both AmtB1 and AmtB2 can contribute to NH 4 + uptake under nitrogen-limiting conditions. Given that R. palustris primarily expresses AmtB2 in these conditions, AmtB2 plays a more important role in NH 4 + uptake compared to AmtB1. In addition, transcriptomic analysis showed that the deletion of the amtB1 and amtB2 genes resulted in the upregulation of many transporter genes, providing potential targets for future investigation of alternative NH 4 + uptake systems. • The removal of environmental ammonium can benefit from rapid ammonium uptake. • AmtB2 plays a major role in ammonium acquisition under nitrogen-fixing conditions. • AmtB is dispensable for the ammonium uptake in the phototrophic Rhodopseudomonas . • Ammonium utilization was determined by Raman spectroscopy at the single-cell level. • The deletion of the amtB genes caused the upregulation of other transporter genes.