Energetics and mechanisms of phosphate transport in Acinetobacter johnsonii

H.W. van Veen

Research output: Thesisinternal PhD, WU


<p>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 <sub><font size="-2">i</font></sub> , after being released from biomass in the anaerobic stage, is reincorporated into biomass during aeration, together with part or all of the influent P <sub><font size="-2">i</font></sub> . Analysis of the population structure of activated sludge has focussed attention on the strictly aerobic, gram-negative genus <em>Acinetobacter</em> 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.<p>The project of this thesis was initiated to study the mechanisms and regulation of P <sub><font size="-2">i</font></sub> uptake and efflux in the polyphosphate-accumulating <em>Acinetobacter johnsonii</em> 210A. The nature of polyphosphates and the enzymology of their metabolism have been a subject of previous studies with <em>A.</em><em>johnsonii</em> 210A and other <em>Acinetobacter</em> spp. Chapter 1 presents a review of these investigations and those concerning the molecular mechanisms of P <sub><font size="-2">i</font></sub> transport in prokaryotes. The results described in this thesis show that <em>A.</em><em>johnsonii</em> 210A is well adapted to the environmental conditions encountered in activated sludge plants through (i) the efficient acquisition of the predominant P <sub><font size="-2">i</font></sub> 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>P <sub><font size="-2">i</font></sub> is taken up in <em>A.</em><em>johnsonii 210A</em> against a concentration gradient by energydependent, carrier-mediated processes (Chapter <em>2).</em> Kinetic analysis of P <sub><font size="-2">i</font></sub> uptake in cells grown under P, limitation, revealed the presence of two P <sub><font size="-2">i</font></sub> transport systems with an apparent <em>K</em><sub><font size="-2">t</font></sub> for P <sub><font size="-2">i</font></sub> of 0.7 and 9 μM. The high-affinity permease could be classified as an ATP- and periplasmic binding protein-dependent P <sub><font size="-2">i</font></sub> uptake system. Induction of this system under P <sub><font size="-2">i</font></sub> limitation, and the ability to maintain a low internal P <sub><font size="-2">i</font></sub> by the synthesis of polyphosphate enable the organism to reduce the P <sub><font size="-2">i</font></sub> concentration in the environment to micromolar levels. The low-affinity system is a constitutive secondary P <sub><font size="-2">i</font></sub> transport system involved in P <sub><font size="-2">i</font></sub> uptake and efflux.<p>P <sub><font size="-2">i</font></sub> 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 <sub><font size="-2">i</font></sub> transport without the interference of polyphosphate metabolism or other cellular processes. P <sub><font size="-2">i</font></sub> uptake is strongly dependent on the presence of divalent metal ions, such as Mg <sup><font size="-2">2+</font></SUP>, Ca <sup><font size="-2">2+</font></SUP>, Mn <sup><font size="-2">2+</font></SUP>, or Co <sup><font size="-2">2+</font></SUP>. These cations form a MeHPO <sub><font size="-2">4</font></sub> complex with up to 87% of the P <sub><font size="-2">i</font></sub> present in the incubation mixtures, suggesting that divalent cations and P <sub><font size="-2">i</font></sub> are cotransported via aMeHPO <sub><font size="-2">4</font></sub> complex. MeHPO <sub><font size="-2">4</font></sub> uptake is driven by the proton motive force with an mechanistic MeHPO <sub><font size="-2">4</font></sub> /H <sup><font size="-2">+</font></SUP>stoichiometry of one. The pH dependence of various modes of facilitated diffusion processes, such as efflux, exchange, and counterflow catalyzed by the MeHPO <sub><font size="-2">4</font></sub> carrier suggests that H <sup><font size="-2">+</font></SUP>and MeHPO4 binding and release to and from the carrier protein occur via an ordered mechanism.<p>In view of the similarities between P <sub><font size="-2">i</font></sub> transport in cells of <em>A.</em><em>johnsonii</em> 210A <em></em> and <em>Escherichia coli,</em> a more extensively studied organism (Chapter <em>2),</em> the mechanism and energetics of the phosphate inorganic transport (Pit) system of <em>E. coli</em> were investigated (Chapter 4). P <sub><font size="-2">i</font></sub> and metal transport studies in proteoliposomes containing reconstituted Pit protein identified Pit as a MeHPO <sub><font size="-2">4</font></sub> /H <sup><font size="-2">+</font></SUP>symport system. The effects of pH and the proton motive force on the different modes of MeHPO <sub><font size="-2">4</font></sub> transport are consistent with the ordered binding model proposed for the MeHPO <sub><font size="-2">4</font></sub> transporter in A. <em>johnsonii</em> 210A.<p>Chapter 5 describes the substrate specificity of the two P <sub><font size="-2">i</font></sub> transport systems in <em>A. johnsonii</em> 210A <em></em> in relation to P <sub><font size="-2">i</font></sub> speciation in the aquatic environment. In natural waters and domestic waste water in which divalent metal ions are present in excess of P <sub><font size="-2">i</font></sub> , the species H <sub><font size="-2">2</font></sub> PO<font size="-2"><sub>4</sub><sup>-</SUP></font>, HPO<font size="-2"><sub>4</sub><sup>2-</SUP></font>and MeHPO <sub><font size="-2">4</font></sub> prevail at physiological pH values for <em>Acinetobacter</em> (pH 5.5 to 8.0). The transport of MeHPO <sub><font size="-2">4</font></sub> by the secondary P <sub><font size="-2">i</font></sub> transport system is demonstrated in proteoliposomes by the (i) divalent cation dependent uptake and efflux of P <sub><font size="-2">i</font></sub> , (ii) P <sub><font size="-2">i</font></sub> -dependent uptake of Ca <sup><font size="-2">2+</font></SUP>and Mg <sup><font size="-2">2+</font></SUP>, (iii) equimolar transport of P <sub><font size="-2">i</font></sub> and Ca <sup><font size="-2">2+</font></SUP>, and (iv) inhibition by Mg <sup><font size="-2">2+</font></SUP>of Ca <sup><font size="-2">2+</font></SUP>uptake in the presence of P <sub><font size="-2">i</font></sub> , but not of P <sub><font size="-2">i</font></sub> uptake in the presence of Ca <sup><font size="-2">2+</font></SUP>.The transport of MeHPO <sub><font size="-2">4</font></sub> is closely related to the metabolism of cytoplasmic polyphosphate granules in which P <sub><font size="-2">i</font></sub> and divalent cations are accumulated. H <sub><font size="-2">2</font></sub> PO<font size="-2"><sub>4</sub><sup>-</SUP></font>and HPO<font size="-2"><sub>4</sub><sup>2-</SUP></font>are translocated by the primary P <sub><font size="-2">i</font></sub> uptake system. P <sub><font size="-2">i</font></sub> uptake, but not MeHPO <sub><font size="-2">4</font></sub> uptake, was stimulated in cells under P <sub><font size="-2">i</font></sub> limitation. The periplasmic P <sub><font size="-2">i</font></sub> -binding protein showed affinity for H <sub><font size="-2">2</font></sub> PO<font size="-2"><sub>4</sub><sup>-</SUP></font>and HPO<font size="-2"><sub>4</sub><sup>2-</SUP></font>, but not for MeHPO <sub><font size="-2">4</font></sub> .<p>Chapter 6 demonstrates the presence of high-affinity secondary transport systems for L-lysine, L-alanine and L-proline in <em>A. johnsonii</em> 210A. <em></em> 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 <sup><font size="-2">+</font></SUP>ions and mediates Na <sup><font size="-2">+</font></SUP>/proline symport. The low internal Na <sup><font size="-2">+</font></SUP>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.<p>Retention of metabolites will become particularly important for survival when oxidative phosphorylation is impaired in <em>A.</em><em>johnsonii</em> 210A. <em></em> 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 <sub><font size="-2">4</font></sub> and H <sup><font size="-2">+</font></SUP>via the MeHPO <sub><font size="-2">4</font></sub> carrier. This MeHPO <sub><font size="-2">4</font></sub> efflux-induced proton motive force can drive energy- requiring processes such as the accumulation of lysine and proline, and the synthesis of ATP. <em></em> Conservation of metabolic energy from polyphosphate degradation may enable <em>A. johnsonii</em> 210A <em></em> to survive alternating aerobic/anaerobic conditions as encountered in natural habitats and wastewater treatment plants.<p>The significance of the here described findings for the cotransport of P <sub><font size="-2">i</font></sub> 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.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Zehnder, A.J.B., Promotor
  • Konings, W.N., Promotor, External person
  • Kortstee, G.J.J., Promotor, External person
Award date13 Jun 1994
Place of PublicationS.l.
Publication statusPublished - 1994


  • gram negative bacteria
  • microorganisms
  • biochemistry
  • metabolism
  • synthesis
  • phosphates
  • phosphorus pentoxide
  • derivatives
  • cum laude

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