H. Rosenberg

The inducible citrate-dependent iron transport system in Escherichia coli K12.

Abstract A citrate-dependent system of iron uptake was found in wild-type cells of Escherichia coli K12, and in mutants which were unable, for a variety of reasons, to make use of the enterochelin system of iron transport. A mutant strain which did not show a growth response to citrate, was shown to lack the citrate-dependent iron uptake system. Identification of this citrate-dependent system was facilitated in the presence of nitrilotriacetate, which abolished a low-affinity iron uptake observed in the absence of added chelators. In growing cells, induction of the citrate-dependent system occurred within 20 min of exposure to citrate, and required new protein synthesis. The citrate-dependent system has a lower initial rate of iron uptake and a slightly lower affinity for iron (Km 0.2 μM) that the enterochelin-mediated system (Km 0.1 μM). In cells with both systems operating optimally, the overall initial rate of iron uptake was equal to the sum of the uptakes contributed by the two individual system. The inducible citrate-dependent system is thus entirely separate from the respressible enterochelin-mediated system, and the two systems can operate simultaneously in wild-type cells.

The enzymic cleavage of the carbon-phosphorus bond: Purification and properties of phosphonatase

Abstract 1. 1. Bacillus cereus degrades 2-aminoethylphosphonate by the following pathway: 2. 2. Reaction II above involves the enzymic cleavage of the carbon-phosphorus bond and has not been described previously. The enzyme responsible for this reaction has now been purified and studied; we have suggested that it be named “2-phosphonoacetaldehyde phosphonohydrolase”, and that the trivial name be “phosphonatase”. 3. 3. Phosphonatase shows optimal activity between pH 8 and 9, and has a K m for 2-phosphonoacetaldehyde of 40 μM. Apartfrom 2-phosphonoacetaldehyde, phosphonate shows virtually no activity with any of a number of phosphonates and phosphate esters tested. 4. 4. The active enzyme is composed of two similar subunits, each of an approximate molecular weight of 33 000–37 000. Mg2+ is required for activity, but this probably helps to maintain the enzyme in its aggregated state rather than play a catalytic role at the active site; no other metals have been shown to be necessary for activity. 5. 5. Phosphonatase is inhibited by CN−, S2− and SO32−, which suggests that disulphide bridges may be necessary in maintaining the tertiary structure of the activeform of the enzyme. 6. 6. Orthophosphite, which has been shown previously to abolish the growth of B. cereus on 2-aminoethylphosphonate, inhibits the activity of phosphonatase, but only in the presence of 2-phosphonoacetaldehyde or acetaldehyde. Experiments with [32P]orthophosphite showed that, although the inhibitor was bound tightly to phosphonatase, it did not form a covalent bond with the enzyme.

The phosphate-binding protein of Escherichia coli

Abstract 1. 1. Escherichia coli contains a phosphate-binding protein, in amounts exceeding 2 · 104 molecules per cell. Over 80% of this protein is released by osmotic shock. 2. 2. The phosphate-binding protein has been purified and found to have a molecular weight of 42 000. There is no evidence of subunits or aggregation. 3. 3. Each molecule of the protein binds one molecule of phosphate. This activity is inhibited by a specific rabbit antiserum. 4. 4. Cold-shocked E. coli lose the ability to transport phosphate. The purified binding protein when added to cold-shocked E. coli stimulates phosphate uptake. This effect is abolished by the specific antiserum. 5. 5. The phosphate-binding protein also stimulates phosphate uptake in a mutant of E. coli with impaired phosphate transport and which also lacks this protein. It does not have any effect on phosphate uptake in another mutant which has the binding protein but is deficient in phosphate uptake through another lesion.

Phosphate transport in Escherichia coli

Abstract 1. 1. Escherichia coli accumulates phosphate against a concentration gradient by an energy-dependent process with an activation energy of over 12 kcal. 2. 2. When cells grown in ample phosphate are starved for phosphorus, or alternately, when cells are grown in phosphate-limiting medium, a specific phosphate pool of the cells is depleted. When phosphate is again provided, this pool is rapidly filled. From then on, phosphate is taken up at a slower rate, sufficient to replenish pool phosphate used up predominantly for nucleic acid synthesis. Isotope displacement experiments show that all phosphate taken up passes through the pool. 3. 3. The uptake of phosphate occurs by two kinetically distinct systems, a high-affinity component and a low-affinity component. The two components function simultaneously during the filling of the pool, but thereafter uptake takes place only by the low-affinity component. The phosphate-binding protein appears to function within the high affinity component. Either of the two components can be selectively abolished by a variety of treatments. 4. 4. A number of arsenate-resistant mutants were shown to be deficient in phosphate uptake. One of these mutants, however, took up and metabolized phosphate at an abnormally high rate, but only over a period of time which did not exceed division time.

The biosynthesis of the phosphonic analogue of cephalin in tetrahymena

Abstract The presence of glyceryl-aminoethylphosphonic acid has been demonstrated in the products of alkaline hydrolysis of Tetrahymena lipid and a pure cephalin fraction, consisting of phosphatidylethanolamine and its phosphonic analogue, in the molecular ratio of approx. 13:1, has been isolated from this species. Extracts of Tetrahymena pyriformis have been shown to incorporate radioactive aminoethyl-phosphonic acid (AEP) into a nucleotide-bound form. The reaction has a specific requirement for cytidine nucleotide and the product was identified as cytidinemonophosphate-aminoethylphosphonate. The synthesis in vitro of a phosphonate-containing glycerophosphatide from cytidinemonophosphate-aminoethylphosphonate and dipalmitin by a Tetrahymena cell-free preparation has also been shown. The reactions are considered to be a salvage mechanism for free AEP or a result of lack of specificity for base in the normal phospholipid-synthesizing systems m Tetrahymena.

The identification of 2-phosphonoacetaldehyde as an intermediate in the degradation of 2-aminoethylphosphonate by Bacillus cereus☆

Abstract 1. 1. Cell-free preparations from a strain of Bacillus cereus isolated in our laboratory release orthophosphate (P i ) from 2-aminoethylphosphonate (AEP). The overall reaction is dependent on the presence of pyridoxal phosphate and pyruvate in the reaction mixture. 2. 2. An intermediate has been isolated from the reaction mixture and identified as 2-phosphonoacetaldehyde on a number of criteria, including comparison with the authentic compound. 3. 3. Synthetic 2-phosphonoacetaldehyde is degraded by the cell-free preparations to acetaldehyde and P i . This reaction is inhibited by orthosphosphite, and is not dependent on the presence of pyridoxal phosphate and pyruvate. 4. 4. This work establishes the following pathway for the breakdown of AEP in B. cereus :

Phosphate transport in Bacillus cereus

Abstract 1. 1. Bacillus cereus accumulates phosphate against a concentration gradient by a process which is energy-dependent and exhibits a high temperature coefficient with an activation energy of over 12 kcal. 2. 2. The rate of uptake is doubled in cells which had been deprived of phosphorus for two hours. In such cells, a ‘primary’ pool of phosphate must be filled before esterification of phosphate begins, and the fast rate of uptake persists throughout this period. The rate of uptake falls to about one-half the fast value once the primary pool is filled. Isotope displacement experiments show that phosphate, taken up from that point in time, still passes through that pool. 3. 3. Arsenate and pyrophosphate which share the phosphate transport system in Bacillus cereus , can also fill the primary pool. 4. 4. The primary pool appears to be closely related to the external phosphate and to its transport, since it does not interact with phosphate liberated within the cell from α-glycerophosphate.

Phosphate Transport in Prokaryotes

Publisher Summary This chapter discusses phosphate transport in prokaryotes. In terms of quantitative requirements and disregarding the elements of water, phosphorus ranks third, after carbon and nitrogen, among the nutrients required for bacterial growth. For maximal growth yields, Escherichia coli requires a phosphorus supplement of 0.4-0.6 mM, and the phosphorus content of the grown cells ranges around 20 mg P/g dry weight. When grown in the laboratory, bacteria are usually provided with orthophosphate (Pi) as the source of phosphorus, in concentrations ranging from minimal requirements to 0.1 M. The existence of specific phosphate transport systems in bacteria was demonstrated by the isolation of mutants of Streptococcus faecalis and Bacillus cereus unable to transport phosphate. As phosphate is accumulated against an electrochemical gradient, energy of some form must be coupled to the process. Three types of energy coupling to metabolite transport have been recognized in bacteria: (1) group translocation, (2) the proton-motive force, and (3) coupling to the high-energy phosphate bond.

On the deposition and utilization of inorganic pyrophosphate in Tetrahymena pyriformis

Abstract 1. 1. Tetrahymena pyriformis deposit calcium magnesium pyrophosphate in spherical granules which appear to be surrounded by membranes. 2. 2. The deposition of granules occurs in the stationary phase and is greatly augmented by elevated levels of phosphate, Ca 2+ and Mg 2+ in the medium; the presence of two divalent cations is essential for this process. The granules disappear rapidly during phosphate deprivation. 3. 3. Associated with the granules is a pyrophosphatase activity which hydrolyses specifically the pyrophosphate of the granules. 4. 4. Phosphotransferase activity involving pyrophosphate could not be demonstrated in Tetrahymena and we conclude that the function of these deposits is that of phosphate storage.

Transport phenomena associated with the deposition and disappearance of pyrophosphate granules in Tetrahymena pyriformis.

Abstract 1. 1. Tetrahymena pyriformis rapidly accumulate Mg 2+ and Ca 2+ when both cations, as well as phosphate, are present in the medium. The uptake of the ions is closely followed by the deposition of magnesium-calcium pyrophosphate granules. We believe that this process, through complexing the intracellular Mg 2+ and Ca 2+ , facilitates their uptake by the cells. 2. 2. The process is reveresed as soon as the cells are placed in a phosphate-free medium. The granules disappear within 6–8 h, but there is a distinct difference in the transport of Ca 2+ and Mg 2+ out of the cells. Ca 2+ is removed from the cells by an active process (against a concentration gradient) from the moment the cells are placed in the phosphate-free medium. Ca 2+ efflux continues at a steady rate until the granules are depleted. No Mg 2+ leaves the cells until this time, and its efflux starts when all of the granules (and presumably Ca 2+ ) have been removed. 3. 3. The maintenance of the balance of intracellular Ca 2+ and Mg 2+ may be a part of the control mechanism governing the deposition and utilization of pyrophosphate. This may involve, on one hand, the ability of the two cations to complex pyrophosphate and, on the other hand, the activation by Mg 2+ , and inhibition by Ca 2+ , of inorganic pyrophosphatase.