PII signal transduction superfamily acts as a valve plug to control bicarbonate and ammonia homeostasis among different bacterial phyla

Abstract

Life on Earth relies on carbon and nitrogen assimilation by RubisCO and GS-GOGAT enzymes, respectively, whose activities depend on a constant supply of inorganic carbon (Ci) and nitrogen (N). Members of the PII signal transduction superfamily are among the most ancient and widespread cell signaling proteins in nature. One of their most highly conserved functions is controlling Ci- and N-transporters, a feature found in different phyla of the Archaea and in both Gram-positive and Gram-negative bacteria. Recently, we identified the PII-like protein SbtB as Ci-sensing module, mainly controlling the HCO3- transporter SbtA in cyanobacteria. Similar to canonical PII proteins, SbtB is able to bind the adenine nucleotides ATP and ADP. Unlike those, it also binds AMP and preferentially binds the second messenger cAMP and c-di-AMP. The functional significance of the binding of different adenyl-nucleotides to SbtB has remained elusive, particularly in the context of the interaction of SbtB with SbtA. By a combination of structural, biochemical and physiological analysis, we revealed that by binding to SbtA, SbtB acts as unidirectional valve, preventing the reverse transport of intracellular enriched bicarbonate. This mechanistic principle holds true for the PII protein from Bacillus acting on the ammonium transporter AmtB, suggesting an evolutionary conserved role for PII superfamily proteins in controlling unidirectional flow of different transporters.

The redox-sensitive R-loop of the carbon control protein SbtB contributes to the regulation of the cyanobacterial CCM

“…The experiment revealed, as seen before [19], that the absence of SbtB causes a signi cantly higher amount of bicarbonate to be leaked during the dark incubation. The C105A-C110A mutation did not affect the leakage, whereas truncation of the R-loop in the Δ104 mutant caused a signi cantly increased leakage, albeit still almost two-fold inferior to what the knocking-out of sbtB caused.…”

“…In agreement, we found diminished amounts of membranebound SbtB after treatment with reducing agents, especially TrxA that very e ciently can convert the oxidized R-loop in the open reduced conformation in vitro. Thereafter, the role of the R-loop on the activity of SbtB as a plug for SbtA to prevent back-ow of bicarbonate outside the cells [19] was investigated. Contrary to expectations, the open reduced R-loop conformation in mutant C105A-C110A did not seem to in uence the leakage of bicarbonate, but its truncation caused more bicarbonate to ow out of the cells possibly due to its smaller plug size.…”

“…Truncation of R-loop affects the function of SbtB as a plug for SbtA Finally, as highlighted by Haffner et al, 2023 [19], the interaction of SbtB with SbtA is not limited to the regulation of SbtA-mediated bicarbonate uptake, but presents more complex regulatory functions, such as plugging SbtA to prevent the leakage of bicarbonate when the CCM is not active. After discovering that mutations in the R-loop impact the a nity to adenyl nucleotides and/or the association of SbtB to the membrane, a C i leakage experiment using 14 C-labelled bicarbonate was performed (Fig.…”

“…The binding of AMP or ADP promote a strong interaction between SbtB and SbtA, while ATP, cAMP or c-di-AMP hinder or prevent the binding of SbtB on SbtA [11,13,16,17]. In such a manner, SbtB can regulate SbtA activity by modulating bicarbonate transport when not needed by the cell [18] and to function as a plug to prevent the back-ow of bicarbonate outside of the cells during darkness, to avoid waste of energy [19].…”

The second messenger c-di-AMP controls natural competence via ComFB signaling protein

“…Binding of recombinantly produced ComFB to c-di-AMP was analysed in vitro by isothermal titration calorimetry (ITC), thermal shift assay (TSA), and differential scanning fluorimetry (nanoDSF), as described previously (Lapina et al 2018; Haffner et al 2023a; Mantovani et al 2023b). For ITC and TSA, both ComFB and c-di-AMP were dissolved in binding buffer (50 mM Tris/HCl, pH 8.0, 300 mM NaCl, 0.55 mM EDTA).…”