Energetics and mechanisms of phosphate transport in Acinetobacter johnsonii

The biological removal of phosphorus from waste water is an attractive method to control eutrophication of surface waters. The process is currently perceived to depend on the provision of alternate stages in which the activated sludge is subjected to anaerobic and aerobic conditions. A characteristic feature of such plant is that P i , after being released from biomass in the anaerobic stage, is reincorporated into biomass during aeration, together with part or all of the influent P i . Analysis of the population structure of activated sludge has focussed attention on the strictly aerobic, gram-negative genus Acinetobacter as being one of the important genera in enhanced biological phosphorus removal. However, due to the lack of insight into the relevant physiological processes in these microorganisms our understanding of the mechanisms of enhanced biological phosphorus removal is only superficial.The project of this thesis was initiated to study the mechanisms and regulation of P i uptake and efflux in the polyphosphate-accumulating Acinetobacter johnsonii 210A. The nature of polyphosphates and the enzymology of their metabolism have been a subject of previous studies with A.johnsonii 210A and other Acinetobacter spp. Chapter 1 presents a review of these investigations and those concerning the molecular mechanisms of P i transport in prokaryotes. The results described in this thesis show that A.johnsonii 210A is well adapted to the environmental conditions encountered in activated sludge plants through (i) the efficient acquisition of the predominant P i species from its habitat, and (ii) the ability to survive prolonged periods of anaerobiosis, by using polyphosphate as a source of metabolic energy when oxidative phosphorylation is impaired.P i is taken up in A.johnsonii 210A against a concentration gradient by energydependent, carrier-mediated processes (Chapter 2). Kinetic analysis of P i uptake in cells grown under P, limitation, revealed the presence of two P i transport systems with an apparent Kt for P i of 0.7 and 9 μM. The high-affinity permease could be classified as an ATP- and periplasmic binding protein-dependent P i uptake system. Induction of this system under P i limitation, and the ability to maintain a low internal P i by the synthesis of polyphosphate enable the organism to reduce the P i concentration in the environment to micromolar levels. The low-affinity system is a constitutive secondary P i transport system involved in P i uptake and efflux.P i transport via the secondary transport system was studied in membrane vesicles and proteoliposomes in which the carrier protein was successfully reconstituted (Chapter 3). These model systems allow detailed studies on the mechanism of P i transport without the interference of polyphosphate metabolism or other cellular processes. P i uptake is strongly dependent on the presence of divalent metal ions, such as Mg 2+, Ca 2+, Mn 2+, or Co 2+. These cations form a MeHPO 4 complex with up to 87% of the P i present in the incubation mixtures, suggesting that divalent cations and P i are cotransported via aMeHPO 4 complex. MeHPO 4 uptake is driven by the proton motive force with an mechanistic MeHPO 4 /H +stoichiometry of one. The pH dependence of various modes of facilitated diffusion processes, such as efflux, exchange, and counterflow catalyzed by the MeHPO 4 carrier suggests that H +and MeHPO4 binding and release to and from the carrier protein occur via an ordered mechanism.In view of the similarities between P i transport in cells of A.johnsonii 210A and Escherichia coli, a more extensively studied organism (Chapter 2), the mechanism and energetics of the phosphate inorganic transport (Pit) system of E. coli were investigated (Chapter 4). P i and metal transport studies in proteoliposomes containing reconstituted Pit protein identified Pit as a MeHPO 4 /H +symport system. The effects of pH and the proton motive force on the different modes of MeHPO 4 transport are consistent with the ordered binding model proposed for the MeHPO 4 transporter in A. johnsonii 210A.Chapter 5 describes the substrate specificity of the two P i transport systems in A. johnsonii 210A in relation to P i speciation in the aquatic environment. In natural waters and domestic waste water in which divalent metal ions are present in excess of P i , the species H 2 PO4-, HPO42-and MeHPO 4 prevail at physiological pH values for Acinetobacter (pH 5.5 to 8.0). The transport of MeHPO 4 by the secondary P i transport system is demonstrated in proteoliposomes by the (i) divalent cation dependent uptake and efflux of P i , (ii) P i -dependent uptake of Ca 2+and Mg 2+, (iii) equimolar transport of P i and Ca 2+, and (iv) inhibition by Mg 2+of Ca 2+uptake in the presence of P i , but not of P i uptake in the presence of Ca 2+.The transport of MeHPO 4 is closely related to the metabolism of cytoplasmic polyphosphate granules in which P i and divalent cations are accumulated. H 2 PO4-and HPO42-are translocated by the primary P i uptake system. P i uptake, but not MeHPO 4 uptake, was stimulated in cells under P i limitation. The periplasmic P i -binding protein showed affinity for H 2 PO4-and HPO42-, but not for MeHPO 4 .Chapter 6 demonstrates the presence of high-affinity secondary transport systems for L-lysine, L-alanine and L-proline in A. johnsonii 210A. The lysine and alanine carriers translocate their solute in symport with one proton. In contrast, the proline carrier is strictly dependent on the presence of Na +ions and mediates Na +/proline symport. The low internal Na +concentration, necessary for optimal proline uptake, is achieved by a sodium/proton antiporter. High-affinity systems will enable the organism to scavenge the environment for traces of metabolizable substrates and to recapture endogenous compounds leaking out of the cell.Retention of metabolites will become particularly important for survival when oxidative phosphorylation is impaired in A.johnsonii 210A. In Chapter 7, evidence is presented for the ability of the organism (i) to use polyphosphate as a source of metabolic energy during anaerobiosis, (ii) to maintain a considerable, outwardly directed MeHPO4 gradient across the cytoplasmic membrane during the degradation of polyphosphate, and (iii) to generate a proton motive force by the excretion of MeHPO 4 and H +via the MeHPO 4 carrier. This MeHPO 4 efflux-induced proton motive force can drive energy- requiring processes such as the accumulation of lysine and proline, and the synthesis of ATP. Conservation of metabolic energy from polyphosphate degradation may enable A. johnsonii 210A to survive alternating aerobic/anaerobic conditions as encountered in natural habitats and wastewater treatment plants.The significance of the here described findings for the cotransport of P i and divalent metal ions across biomembranes and the recycling of metabolic energy in microorganisms by the excretion of inorganic endproducts is discussed in Chapter 8.