Page 153 - The Biochemistry of Inorganic Polyphosphates

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Azotobacter 137
Harold and Harold (1963, 1965) also obtained mutants with defects in the regulation
of phosphorus metabolism. One of them could not de-repress the PolyP-metabolizing en-
zymes by P i deprivation and did not display phosphate overplus, while the other had these
enzymes constitutively de-repressed and accumulated PolyP during the exponential phase
(Harold and Harold, 1965). Later it was established that there is a putative Pho box in
the promoter region of the K. aerogenes ppk-ppx operon (Kato et al., 1993b; Kuroda and
Ohtake, 2000). Unlike the E. coli ppk–ppx operon, the K. aerogenes ppk–ppx operon seems
to be under a stronger control of the PhoB and PhoR proteins (Kato et al., 1993b). The
existence of the PolyP operon in this bacterium indicates that both increased PolyP synthe-
sis and decreased PolyP degradation are responsible for regulation of the PolyP content in
K. aerogenes.

8.5 Azotobacter

PolyP metabolism in several species of Azotobacter has been investigated by Zaitseva and
co-workers(ZaitsevaandBelozersky,1958,1960;ZaitsevaandLiTszyun-in,1961;Zaitseva
and Frolova, 1961; Zaitseva et al., 1959, 1960a,b, 1961). These investigations were initially
concerned with the changes in PolyP content during the development of cultures of various
species of Azotobacter. It was shown that, as in E. coli, acid-insoluble PolyPs accumu-
lated during the latent phase of development and was subsequently utilized actively during
the exponential growth of the culture. In the case of Azotobacter agile, it disappeared com-
pletely at this growth stage, reappearing only in the stationary phase. Acid-insoluble PolyPs
accumulated in the early stationary phase, followed later by accumulation of acid-soluble
PolyPs (Zaitseva and Belozersky, 1958; Zaitseva et al., 1959, 1960a,b). The behaviour of
PolyPs in a synchronized culture of Azotobacter was also studied (Zaitseva et al., 1961). It
was revealed that acid-insoluble PolyP attained its highest level in synchronously growing
cells immediately prior to the onset of cell division, and during cell division it degraded to
acid-soluble PolyP and then to P i . These results indicate possible participation of PolyPs in
cell-cycle regulation.
The dependence of PolyP metabolism in Azotobacter on the nutrient medium compo-
sition was investigated. The greatest interest was connected with possible participation of
PolyPs in nitrogen fixation. However, no specific features were observed, which could in-
dicate a direct participation of PolyPs in this process. In a medium containing ammonium
salts, PolyPs accumulated in larger amounts than under nitrogen-fixation conditions or in
the absence of a nitrogen source in the medium. The inhibitory effect of Ca 2+ ions on both
nitrogen fixation and PolyP accumulation was established (Esposito and Wilson, 1956;
Zaitseva et al., 1960b). However, Zaitzeva et al. (1960b) showed that these two phenomena
were not directly related and that PolyP did not provide the source of phosphorus and energy
in nitrogen fixation. It was revealed that a high Ca 2+ concentration had an inhibitory effect
on glycolitic phosphorylation, which was the common energy source for both processes in
Azotobacter.
In Azotobacter, the main enzyme of PolyP metabolism was shown to be polyphosphate
kinase (Zaitseva and Belozersky, 1958, 1960), which was capable of PolyP synthesis and of
a reverse reaction. This enzyme was isolated and purified to a considerable extent (Zaitseva
and Belozersky, 1960).