Polyphosphates and Enzymes of Polyphosphate Metabolism in Escherichia Coli

Biochemistry (Moscow), Vol. 65, No. 3, 2000, pp. 309-314. Translated from Biokhimiya, Vol. 65, No. 3, 2000, pp. 368-374. Original Russian Text Copyright © 2000 by Nesmeyanova. MINI-REVIEW Polyphosphates and Enzymes of Polyphosphate Metabolism in Escherichia coli M. A. Nesmeyanova Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 923-3602; E-mail: [email protected] Received December 15, 1999 AbstractThis review summarizes the results of our study of polyphosphate and enzymes of polyphosphate metabolism in E. coli and their regulation by exogenous orthophosphate and other physiological and genetic factors. Key words: E. coli, polyphosphate, enzymes, regulation, orthophosphate Most microorganisms synthesize different reserve mined. Our study of polyP and enzymes of polyP polymers with polyphosphate (polyP) being of special metabolism in Escherichia coli resulted in the following interest. This could be anticipated, since polyP con- conclusions. tains phosphorus, one of the most important vital ele- ments that is a constituent of all major subunits of life: amino acids, nucleotides, sugars, phospholipids, and ACCUMULATION OF POLYPHOSPHATE recently has been quite obviously shown to be a mol- IN E. coli CELLS IS A FUNCTION ecule for many reasons of living cells [1]. The accu- OF CULTURE GROWTH STAGE mulation of polyP in bacteria is not only a natural AND REGULATED BY EXOGENOUS BUT NOT phenomenon, but also a finely regulated process. It INTRACELLULAR ORTHOPHOSPHATE can be used as an energy source, a phosphate donor for sugars, and a chelator for divalent cations. The earliest works with bacteria showed that polyP Moreover, related molecules (e.g., guanosine tetra- occurs in them in extremely low amounts, if at all, and is phosphate) can be involved as regulatory molecules in present in bacterial cells not continually and often under the functioning of global regulatory systems. The bac- specific conditions of bacterial growth, usually under teria have been known to accumulate polyP for a com- growth limitation by some nutrient sources [8]. Indeed, paratively long time [2, 3]. However the regular our study of the dynamics of polyP accumulation during absence of this polymer in exponentially growing bac- the growth of the wild-type strain E. coli K12 on a min- terial cultures resulted in a wrong notion of its absence eral medium supported such a suggestion, simultaneous- in some bacteria. The gram-negative bacterium E. coli ly closing the question of polyP occurrence in E. coli [9]. was also attributed to the latter [4]. Meanwhile, it is We found in this bacterium a high-molecular-weight just the cells of E. coli where the most important acid-insoluble polyP that was identified by a specific enzyme of polyP synthesisATP-polyphosphate product of its partial acidic hydrolysis, trimethophos- phosphotransferase, or polyphosphate kinase [5, 6] phate, using the method of Thilo and Wieker [10]. Much was first found and identified, and recently purified to later, direct methods confirmed that polyP in E. coli may a homogenous state [7]. have a length of up to 1000 phosphate groups [11]. We This circumstance determined our search of polyP showed that the highest amount of this high-polymer in this bacterium, which was interesting first of all due polyP, reaching 0.2-0.4% of dry bacterial weight (in yeast to a very intensive investigation of its phosphorus its amount may reach 20%), occurs in the cells of this metabolism both on the biochemical and genetic levels bacterium only in the end of latent and beginning of log- as a complex two-component regulatory system (Pho arithmic growth phase. When the culture passes to expo- regulon). Analogs of such a system were later revealed nential growth, the level of intracellular polyP dramati- in many metabolic pathways. However the place of this cally decreases 5-10-fold (Fig. 1). Thus, the accumulation regulatory system in polyP metabolism was not deter- of polyP precedes intensive culture growth, and then it is 0006-2979/00/6503-0309$25.00 ©2000 MAIK Nauka/Interperiodica 310 NESMEYANOVA a Important observations of the dynamics of polyP 1 accumulation in bacteria described in other works [15] 1.0 are concerned with certain conditions of bacterial growth limitations, e.g., nitrogen or sulfur deficiency or 2 unfavorable pH conditions. As a rule, the accumulation 4 0.6 of polyP under the above limitations was due to the 1 excess of energy sources and the availability of phos- 2 phate, as well as K and Mg ions. In essence, the polyP 3 0.2 Orthophosphate, µg/mg biosynthesis responds to different external factors, mg biomass/ml medium whether they are components of the nutrient medium, or 10 b its physical and chemical conditions. However, polyP was utilized in most cases when bacterial cells were placed in a medium enabling cell growth. This supports 6 polyP functioning as a phosphogen or an energy source. We showed that polyP was utilized at the same rate 6 when cells were subjected to phosphate starvation. In 2 5 this case, the level of intracellular orthophosphate 7 remained actually the same, again supporting the regulatory role of polyP in the maintenance of the phosphate c level in the cell (Fig. 2). 0.2 Enzyme activities The best known exclusion from the described con- 8 ditions of polyP accumulation in bacteria appeared to be 0.1 for several strains of Micrococcus lysodeikticus, where polyP granules accumulated during exponential growth and disappeared only in the stationary phase [16]. On 9 the contrary, some Acinetobacter strains are able to 120 240 360 480 500 accumulate polyP in the end of log phase or even in the Time, min stationary phase [17], and in mycobacteria the rate of polyP accumulation was the highest in the stationary Fig. 1. PolyP and enzymes of polyP metabolism in the process of E. coli growth. 1) Ñulture growth (mg dry biomass/ml phase [18]. medium); 2) polyP (µg Pi/mg dry biomass); 3) intracellular orthophosphate (µg Pi/mg dry biomass); 4) orthophosphate of the medium (µg Pi/ml medium); 5) polyphosphatase (mU/mg dry biomass, the same as 6-9); 6) tripolyphosphatase; 7) alkaline phosphatase; 8) polyphosphate kinase; 9) 1,3- diphosphoglycerate polyphosphate phosphotransferase. a b 1 utilized by growing cells [9]. The above peculiarity 2.1 explains unsuccessful attempts to reveal polyP in E. coli, 40 though it was observed earlier in other bacteria as well 3 PolyP and intracell- 1.7 ular orthophosphate [12]. The synchronization of bacterial cultures, display- 30 ing noticeable fluctuations in the polyP content during its 2 4 growth, showed [13, 14] that its highest level is observed 1.3 1 20 immediately before cell division, which seems to be very 1 1.1 2 important. The content of DNA increased in the course OD of biomass 3 2 10 of the whole cycle after cell division. The dynamics of 0.7 3 accumulation and utilization of polyP undoubtedly indi- 246 24 6 Orthophosphate in the medium cates first of all the phosphogenic and probably energy Time, h source function of polyP in bacteria. It should be men- tioned that in our experiments intracellular orthophosphate was accumulated less intensively than polyP under Fig. 2. The effect of exogenous orthophosphate on the level of cell growth with an excess of exogenous orthophosphate. intracellular orthophosphate and polyP in E. coli under Besides, orthophosphate remained actually on the same orthophosphate starvation (a) and orthophosphate excess (b): 1) cell growth; 2) polyP content (µg P /mg dry biomass); 3) level, which also favored the involvement of polyP in the i intracellular orthophosphate (µg Pi/mg dry biomass); 4) maintenance of its constant intracellular level. orthophosphate in the medium (µg Pi/ml medium). BIOCHEMISTRY (Moscow) Vol. 65 No. 3 2000 POLYPHOSPHATE METABOLISM IN Escherichia coli 311 THE ACTIVITY OF ENZYMES resulting acute need of orthophosphate and(or) energy. OF POLYPHOSPHATE METABOLISM IS ALSO In this case, the factor regulating the activity and even A FUNCTION OF CULTURE GROWTH STAGE the direction of polyphosphatase functioning in a bacterial cell will probably be a ratio of orthophosphate to The accumulation of polyP in cells results from the polyP in the cell and the size of polyP. two processes: synthesis and utilization of polyP. The dynamics of the known polyP-synthesizing enzymes, polyphosphate kinase [6] and 1,3-diphosphoglycerol- E. coli POLYPHOSPHATASES polyphosphate phosphotransferase [19], analyzed in our ARE CO-REGULATED ON THE METABOLIC experiments showed that these enzyme activities weakly AND GENETIC LEVELS WITH ALKALINE correlate with the dynamics of polyP accumulation (Fig. PHOSPHATASE 1). The main peak of polyphosphate kinase activity was observed, for instance, during the stationary culture As Fig. 1 shows, polyphosphatase activities correlate growth, when the content of polyP in cells significantly during the culture growth with the activity of alkaline decreased. On the contrary, the activity of enzymes of phosphatasea nonspecific phosphomonoesterase polyP degradation, polyphosphatase and tripolyphos- whose function is to supply the cell with orthophosphate phatase in E. coli, directly correlated with polyP accu- from all available exogenous sources. This enzyme is reg- mulation and with the activity of alkaline phosphatase ulated by orthophosphate, which represses its synthesis (nonspecific phosphohydrolase) of E. coli. The polyP when occurring in the medium in a high concentration. phosphohydrolases studied here were first found in E. Under phosphate starvation, the enzyme synthesis is dere- coli in our experiments, and the dynamics of their activ- pressed, and it is accumulated in high quantities. The ity appeared to be very interesting from the standpoint polyphosphatase function may be similar to the function of possible participation of the enzymes of polyP metab- of alkaline phosphatase, which provides the cell with olism in the catalysis of both forward and back reac- orthophosphate.

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