PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

Abstract

Maintenance of cellular phosphate homeostasis is essential for cellular life. The PhoU protein has emerged as a key regulator of this process in bacteria, and it is suggested to modulate phosphate import by PstSCAB and control activation of the phosphate limitation response by the PhoR-PhoB two-component system. However, a proper understanding of PhoU has remained elusive due to numerous complications of mutating phoU, including loss of viability and the genetic instability of the mutants. Here, we developed two sets of strains of Sinorhizobium meliloti that overcame these limitations and allowed a more detailed and comprehensive analysis of the biological and molecular activities of PhoU. The data showed that phoU cannot be deleted in the presence of phosphate unless PstSCAB is inactivated also. However, phoU deletions were readily recovered in phosphatefree media, and characterization of these mutants revealed that addition of phosphate to the environment resulted in toxic levels of PstSCAB-mediated phosphate accumulation. Phosphate uptake experiments indicated that PhoU significantly decreased the PstSCAB transport rate specifically in phosphate-replete cells but not in phosphate-starved cells and that PhoU could rapidly respond to elevated environmental phosphate concentrations and decrease the PstSCAB transport rate. Sitedirected mutagenesis results suggested that the ability of PhoU to respond to phosphate levels was independent of the conformation of the PstSCAB transporter. Additionally, PhoU-PhoU and PhoU-PhoR interactions were detected using a bacterial two-hybrid screen. We propose that PhoU modulates PstSCAB and PhoR-PhoB in response to local, internal fluctuations in phosphate concentrations resulting from PstSCAB-mediated phosphate import.IMPORTANCE Correct maintenance of cellular phosphate homeostasis is critical in all kingdoms of life and in bacteria involves the PhoU protein. This work provides novel insights into the role of the Sinorhizobium meliloti PhoU protein, which plays a key role in rapid adaptation to elevated phosphate concentrations. It is shown that PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death. Additionally, a new model for phosphate sensing in bacterial species which involves the PhoR-PhoB two-component system is presented. This work provides new insights into the bacterial response to changing environmental conditions and into regulation of the phosphate limitation response that influences numerous bacterial processes, including antibiotic production and virulence.KEYWORDS Pho regulon, PhoU, PstSCAB, Sinorhizobium meliloti, environmental adaptation, modulation of TCS, phosphate homeostasis, phosphate sensing, phosphate transport, transport modulation

Phosphate starvation response controls genes required to synthesize the phosphate analog arsenate

“…In all Gram-negative bacteria thus far studied, the PSR is regulated via a two-component signal transduction system comprised of the sensor histidine kinase PhoR and its cognate response regulator PhoB. PhoU and PstS are also involved, with the most recent evidence suggesting they interact with PhoR and are involved in de-activating the PSR Hsieh and Wanner, 2010;diCenzo et al, 2017). In A. tumefaciens, induction of an aioB::lacZ reporter is either undetectable or the kinetics significantly disrupted in DphoB1, DphoU1 and DpstS1 mutants (Kang et al, 2012a).…”

Limitation of phosphate assimilation maintains cytoplasmic magnesium homeostasis

“…Finally, we establish that even at physiological levels of cytoplasmic Mg 2+ , Pi exerts its toxicity following its incorporation into ATP and subsequent disruption of Mg 2+ -dependent processes. While providing a conceptual framework to understand the underlying basis of Pi cytotoxicity, a phenomenon observed in both bacteria and eukaryotes (3)(4)(5)(6)(7)(33)(34)(35)(36), our results uncover a regulatory logic employed by bacterial cells for the global control of P assimilation.…”

“…Furthermore, they provide a framework to understand the molecular basis of Pi cytotoxicity and reveal a regulatory logic employed by bacterial cells to control P assimilation. Interestingly, while the assimilation of P is essential, excessive cytoplasmic Pi is toxic (2)(3)(4)(5)(6)(7). This implies that cells must tightly regulate Pi acquisition and utilization to avoid self-poisoning.…”

Molecular Mechanism Associated With the Impact of Methane/Oxygen Gas Supply Ratios on Cell Growth of Methylomicrobium buryatense 5GB1 Through RNA-Seq

“…It was proposed that there was a dynamic equilibrium between active and inactive PstSCAB in response to local internal fluctuations in phosphate concentrations, which was modulated by PhoU (diCenzo et al, 2017). PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death (diCenzo et al, 2017). It has been known that phosphate is an indispensable compound for cell growth, whereas high concentration phosphate can still be toxic to cell.…”