Polyphosphate metabolism in Acinetobacter johnsonii 210A

Since the seventies, there is a growing interest in the process of biological phosphate removal in which microorganisms able to accumulate large amounts of phosphate play a central role. Over the years many bacteria have been isolated from sludge systems showing enhanced biological phosphate removal, either to investigate the bacterial population or to obtain pure cultures which were used as model organisms in studies on enhanced biological phosphate removal. Often, bacteria of the genus Acinetobacter were found. Therefore, it was suggested that these organisms probably play an important role in biological phosphate removal. In 1980, Acinetobacter strain 210A was isolated from sludge of a wastewater treatment plant in Renkum. This organism was able to accumulate large amounts of phosphorus in the form of polyphosphate (Van Groenestijn, 1988). The aim of this study was to examine various aspects of the metabolism of polyphosphate in this organism. This thesis describes next to the identification of Acinetobacter strain 210A, properties of bacterial polyphosphate, degradation and in vivo synthesis of polyphosphate and the regulation of polyphosphate metabolism by external phosphate.Acinetobacter strain 210A was identified as Acinetobacter johnsonii by using a combination of biochemical and genetic properties. The organism was able to synthesize two polymers, polyphosphate and poly-β-hydroxybutyric acid. Polyphosphate was formed during growth at excess phosphate whereas poly-β-hydroxybutyric acid was synthesized when phosphate was the limiting nutrient. Intact cells were able to oxidize a variety of monosaccharides in the presence of PQQ (pyrollo-quinoline quinone). Ibis ability was also found in other Acinetobacter strains and was used in the examination of the bacterial population of two activated sludge types showing enhanced biological phosphate removal. It was found that the bacterial community structure of these sludges differed strongly. Therefore, it was suggested that the presence of Acinetobacter sp. in activated sludge systems showing enhanced biological phosphate removal depends on process design and influent composition of the treatment system (Chapter 2).Polyphosphate synthesized by Acinetobacter johnsonii 210A was localized in the cytoplasm mostly complexed in one or two large and several small granules. Electron microscopy and energy dispersive X-ray micro-analysis were used to examine the elemental composition of the large polyphosphate granules in unfixed and unstained intact cells of A. johnsonii 210A. When the organism was grown in standard medium, the granules were composed of phosphorus, magnesium and potassium. By modifying the amount of Ca 2+and Me 2+in the medium, the intracellular concentration of Ca 2+and Mg 2+as well as the elemental composition of the polyphosphate granules could be changed. A high Mg/Ca-ratio in the medium resulted in a high Mg/Ca-ratio in the cytoplasm and in the presence of Mg as counterion in the polyphosphate granule. Ca became the major cation in the polyphosphate bodies during growth in a medium with a low Mg/Ca-ratio. These results were at variance with previous studies dealing with the elemental composition of large polyphosphate granules which revealed Ca as the dominant counterion in the large polyphosphate bodies (Buchan 1981; Buchan 1983). This discrepancy could be ascribed to fixation and embedding procedures. Fixation of cells in glutaraldehyde and embedding in EPON have a profound effect on the elemental composition of polyphosphate granules (Chapter 3). Polyphosphate accumulated by A.johnsonii 210A consisted of about 700 Presidues as determined by gelelectrophoresis. In vivo31P-NMR was used to follow polyphosphate formation in intact cells. The amount of polyphosphate synthesized, correlated positively with the intracellular ATP concentration, suggesting an involvement of ATP in polyphosphate formation. In contrast to polyphosphate synthesis, polyphosphate degradation is a slow process (Chapter 4). Two polyphosphate degrading enzymes, polyphosphate:AMP phosphotransferase and polyphosphatase, have been found in A. johnsonii 210APolyphosphate: AMP phosphotransferase phosphorylates AMP to ADP with polyphosphate: PP n (polyphosphate) + AMP _>PP n-1 + ADP. The enzyme was purified more than 1,500-fold from A.johnsonii 210A by streptomycin sulfate precipitation, and by Mono- Q Phenyl Superose, and Superose column chromatography. Kinetic studies showed apparent K m values of 0.26 mM for AMP and 0. 8 μM for polyphosphate with an average chain length of 35 P-groups. The highest activities were found with polyphosphate molecules of 18 to 44 phosphate residues. The polyphosphate chain was degraded completely to ADP via a processive mechanism. No activity was obtained with ortho-, pyro-, tri-, and tetraphosphate. The enzyme was inhibited by pyro-, tri-, and tetraphosphate (Chapter 5).Chapter 6 describes the purification and characterization of polyphosphatase of A. johnsonii 210A This enzyme hydrolyzes polyphosphate to P i : PP n + H 2 O _>PP n-1 + P i . It was purified 77-fold by Q-Sepharose, hydroxylapatite and mono-Q column chromatography. The enzyme was specific for high polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. Analysis of kinetic properties revealed an apparent K m -value for polyphosphate with an average chain length of 64 residues of 30 ing polyphosphate per liter and for tetraphosphate of 1.2 mM. Polyphosphate chains were degraded to short chain polymers via a processive mechanism. The enzyme was inhibited by iodoacetamide and, in the presence of high Mg 2+-concentrations, by pyro-and triphosphate. The activating effect of Mg 2+on polyphosphatase was enhanced by K +and NH4+.Pyro-and triphosphate were hydrolyzed by pyrophosphatase, an enzyme which is in contrast to the polyphosphate degrading enzymes, widely distributed in nature. It was purified from A.johnsonii 210A and showed except against pyroand triphosphate no activity towards polyphosphates and a wide variety of organic phosphate esters. The enzyme is composed of 6 identical subunits of 23 kDa, giving a molecular mass of 141 kDa for the native enzyme. Mg 2+was required for activity. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The apparent K m value for pyrophosphate was estimated to be 0.26 mM (Chapter 7).The effect of varying phosphate concentrations on biomass, cellular composition, phosphate uptake rate and activities of enzymes involved in (poly)phosphate metabolism of A.johnsonii 210A was investigated in P-or C-limited chemostat cultures. The organism accumulated poly-β-hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed.When shifting a culture from P-to C-limitation, phosphate was accumulated as polyphosphate. No poly-β-hydroxybutyric acid could be detected in these cells. As soon as polyphosphate synthesis was possible, the specific activities of polyphosphate:AMP phosphotransferase and polyphosphatase increased about four fold. The specific activities of alkaline phosphatase and the P-uptake system were induced at residual phosphate concentrations below the detection limit. The effect of phosphate on the cellular polyphosphate content and on the P-uptake rate showed a hysteresis behaviour. When chemostat cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 ing P per gram of dry weight at a phosphate influent concentration of 2.5 mM. In the reverse case (high to low), polyphosphate did never exceed 45 mg P per gram of dry weight (Chapter 8).To optimize the process of biological phosphate removal, a thorough understanding of all processes involved in polyphosphate metabolism of bacteria present inactivated sludge is needed. These processes comprise: polyphosphate synthesis, polyphosphate degradation, and phosphate transport. In this thesis, properties of polyphosphate and polyphosphate degradation in A. johnsonii 210A were examined extensively. However, polyphosphate synthesis and phosphate transport were not clarified. From the positive correlation between intracellular ATP levels and polyphosphate synthesis, it was suggested that polyphosphate kinase may be involved in the process of polyphosphate formation. However, in cell extracts of A. johnsonii 210A, no polyphosphate kinase activity could be detected. This discrepancy can be interpreted in two ways. Either the polyphosphate kinase enzyme looses its activity during extract preparation or other polyphosphate synthesizing enzymes are involved in the formation of polyphosphate in A.johnsonii 210A. Two enzyme systems different from the polyphosphate kinase can be hypothesized: (1) a system in which a phosphorylated compound other than ATP is the P-donor or (2) a membrane bound proton translocating enzyme system. However, no positive indications for either of these two systems were found. Recently, the transport of phosphate in A. johnsonii 210A was characterized. Two transport systems could be demonstrated, (i) an inducible, ATP-driven, binding protein-dependent system, and (ii) a constitutive, low-affinity uptake system. It was suggested that the low- affinity system may be involved in the anaerobic metabolism of A.johnsonii 210A. Metabolic energy could be conserved by the generation of an electrochemical gradient across the cytoplasmic membrane, when phosphate is excreted together with ions (Van Veen et al. 1993). A more detailed knowledge of polyphosphate synthesis and phosphate transport would not only enlarge the insight in polyphosphate metabolism of phosphate accumulating bacteria, but would be of major importance for a better understanding of the process of biological phosphate removal. A better understanding would allow the optimization of its application based on information on physiological and biochemical mechanisms

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii210A was purified 200-fold to apparent homogeneity. The enzyme catalyzedthe hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate.No activity was observed with other polyphosphates and a wide variety oforganic phosphate esters. The molecular mass of the enzyme was estimatedto be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis indicated a subunit composition of six identical polypeptideswith a molecular mass of 23 kDa. The cation Mg2 was required foractivity, the activity with Mn2, Co2 and Zn2 was 48, 48 and 182% of the activity observed with Mg2, respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent Km for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate

Polyphosphate formation by Acinetobacter johnsonii 210A: effect of cellular energy status and phosphate-specific transport system

In acetate-limited chemostat cultures of Acinetobacter johnsonii 210A at a dilution rate of 0.1 h-1 the polyphosphate content of the cells increased from 13% to 24% of the biomass dry weight by glucose (100 mM), which was only oxidized to gluconic acid. At this dilution rate, only about 17% of the energy from glucose oxidation was calculated to be used for polyphosphate synthesis, the remaining 83% being used for biomass formation. Suspensions of non-growing, phosphate-deficient cells had a six- to tenfold increased uptake rate of phosphate and accumulated polyphosphate aerobically up to 53% of the biomass dry weight when supplied with only orthophosphate and Mg2+. The initial polyphosphate synthesis rate was 98 ± 17 nmol phosphate min-1 mg protein-1. Intracellular poly-β-hydroxybutyrate and lipids served as energy sources for the active uptake of phosphate and its subsequent sequestration to polyphosphate. The H+-ATPase inhibitor N,N'- dicyclohexylcarbodiimide caused low ATP levels and a severe inhibition of polyphosphate formation, suggesting the involvement of polyphosphate kinase in polyphosphate synthesis. It is concluded that, in A. johnsonii 210A, (i) polyphosphate is accumulated as the energy supply is in excess of that required for biosynthesis, (ii) not only intracellular poly-β- hydroxybutyrate but also neutral lipids can serve as an energy source for polyphosphate-kinase-mediated polyphosphate formation, (iii) phosphate- deficient cells may accumulate as much polyphosphate as activated sludges and recombinants of Escherichia coli designed for polyphosphate accumulation

Polyphosphate formation by Acinetobacter johnsonii 210A: effect of cellular energy status and phosphate-specific transport system

In acetate-limited chemostat cultures of Acinetobacter johnsonii 210A at a dilution rate of 0.1 h-1 the polyphosphate content of the cells increased from 13% to 24% of the biomass dry weight by glucose (100 mM), which was only oxidized to gluconic acid. At this dilution rate, only about 17% of the energy from glucose oxidation was calculated to be used for polyphosphate synthesis, the remaining 83% being used for biomass formation. Suspensions of non-growing, phosphate-deficient cells had a six- to tenfold increased uptake rate of phosphate and accumulated polyphosphate aerobically up to 53% of the biomass dry weight when supplied with only orthophosphate and Mg2+. The initial polyphosphate synthesis rate was 98 ± 17 nmol phosphate min-1 mg protein-1. Intracellular poly-β-hydroxybutyrate and lipids served as energy sources for the active uptake of phosphate and its subsequent sequestration to polyphosphate. The H+-ATPase inhibitor N,N'- dicyclohexylcarbodiimide caused low ATP levels and a severe inhibition of polyphosphate formation, suggesting the involvement of polyphosphate kinase in polyphosphate synthesis. It is concluded that, in A. johnsonii 210A, (i) polyphosphate is accumulated as the energy supply is in excess of that required for biosynthesis, (ii) not only intracellular poly-β- hydroxybutyrate but also neutral lipids can serve as an energy source for polyphosphate-kinase-mediated polyphosphate formation, (iii) phosphate- deficient cells may accumulate as much polyphosphate as activated sludges and recombinants of Escherichia coli designed for polyphosphate accumulation