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

Ammonium (NH₄⁺) 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 NH₄⁺ transporter that is responsible for the NH₄⁺ 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 NH₄⁺ 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 NH₄⁺ uptake at the single-cell level. The generated R. palustris ΔamtB1 ΔamtB2 mutant lacking AmtB1 and AmtB2 proteins was still capable of utilizing ¹⁵NH₄⁺ even the ¹⁵NH₄⁺ 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 NH₄⁺ regardless of environmental NH₄⁺ levels. However, both AmtB1 and AmtB2 can contribute to NH₄⁺ uptake under nitrogen-limiting conditions. Given that R. palustris primarily expresses AmtB2 in these conditions, AmtB2 plays a more important role in NH₄⁺ 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 NH₄⁺ uptake systems.