Page 70 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
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Localization of polyphosphates in cells
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Chapter 2), and they strongly absorb electrons, in exactly the same way as PolyPs (Drews,
1960a,b). Secondly, the accumulation of volutin granules almost always correlated well with
the accumulation of specific PolyP fractions (Belousova and Popova, 1961). Bacterial mu-
tants, which are unable to accumulate PolyPs, have no volutin granules in their cells (Harold
and Harold, 1963, 1965). Thirdly, the utilization of PolyPs present in cells of certain bacte-
ria by polyphosphate glucokinase is accompanied by the disappearance of volutin granules
(Szymona and Szymona, 1961; Szymona, 1962). There is no doubt that PolyP-containing
granules are actually present in cells and are not artifacts formed during the fixation and
staining of cells by particular dyes. This may be concluded from the fact that they are readily
visible without staining in living cells of microorganisms by phase-contrast microscopy.
It has already been pointed out that it is not only PolyPs but other anionic polymers,
such as poly-β-hydroxybutyrate, which may form metachromatic granules in the cells of
prokaryotes. However, recently a number of methods for differential staining of PolyPs and
polyhydroxyalkanoate-containing granules in cells have been developed (see the review of
Serafim et al., 2002).
Various cytochemical methods were elaborated to detect volutin-like granules in differ-
ent microorganisms (Keck and Stich, 1957; Ebel and Muller, 1958; Ebel et al., 1958a,b;
Singh, 1959; Serafim et al., 2002). Cytological methods for detecting polyphosphate gran-
ules were boosted by the use of the electron microscope (Niklowitz and Drews, 1955;
Ebel et al., 1958a,b; Drews, 1960a; Voelz et al., 1966; Jensen, 1968, 1969; Friedberg and
Avigad, 1968; Jensen and Sicko, 1974; Jensen et al., 1977). The most comprehensive data on
volutin granules in cyanobacteria were obtained by Jensen and co-workers (Jensen, 1968,
1969; Jensen and Sicko, 1974; Sicko-Goad et al., 1975; Sicko-Goad and Jensen, 1976;
Jensen et al., 1977; Lawry and Jensen, 1979; Baxter and Jensen, 1980a,b). Using electron
microscopy, Jensen and his colleagues investigated the accumulation of PolyP granules un-
der various cultivation conditions in the cyanobacteria Nostoc pumiforme (Jensen, 1968),
Plectonema boryanum (Jensen, 1969; Jensen and Sicko, 1974; Sicko-Goad and Jensen,
1976; Baxter and Jensen, 1980a,b; Jensen et al., 1982) and Anacystis nidulans (Lawry and
Jensen, 1979). Under normal growth conditions, PolyP granules were found mainly near
the DNA region and in a zone enriched with ribosomes. Under conditions of ‘phosphate
overplus’, PolyP granules appeared in the polyhedral bodies involved in the dark reac-
tions of photosynthesis in cyanobacteria (Stewart and Godd, 1975). In certain cells, PolyP
granules formed close to thylakoids, which in these organisms perform phosphorylation
reactions. Electron microscopy established that in cyanobacteria PolyP granules are local-
ized in most cases in the region of nucleoid and sub-cellular structures participating in
photosynthesis (Vaillancourt el al., 1978; Barlow et al., 1979). The data obtained, at least
those on localization of polyphosphate granules in the vicinity of the bacterial nucleoid,
correlated well with the previous findings using the same method on heterotrophic prokary-
otes (Drews, 1958a, 1960a, Voelz et al, 1966; Friedberg and and Avigad, 1968; Kulaev and
Vagabov, 1983).
Volutin granules were isolated from cells of Agrobacterium tumefaciens (Seufferheld
et al., 2003) by gradient centrifugation. The volutin granule fraction contained 20 % of
the total amount of PP i and short-chain PolyPs and more than 35 % of total amount
of P i and long-chain PolyPs. The total extract of Agrobacterium tumefaciens contained
∼ 315 nmol (mg protein) −1 of PolyP with a chain length less than 50 residues, and 217 nmol
(mg protein) −1 of PolyP of about 700–800 P i residues. X-ray microanalysis showed that the