Page 146 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-08
Peculiarities of polyphosphate metabolism
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in exopolyphosphatase (ppx) has more PolyP than the parent strain (Keasling and Hupf,
1996).
The ppk1 gene has many pleiotrophic effects on E. coli viability and cell functions
(Kornberg, 1995, 1999; Kornberg et al., 1999), which were briefly described earlier in
Chapter 7.
In E. coli, a Pho regulon controls the biosynthesis of a number of enzymes participating
in phosphorus metabolism and other proteins (Torriani-Gorini, 1994; Wanner, 1994). A
very low level of P i in the medium induces the Pho regulon, comprising the Pst-pathway
of using P i and the PhoB regulator of response, which induces the genes of proteins of this
regulon also participating in phosphorus metabolism.
One of the first attempts to elucidate the interrelations of the Pho regulon and PolyP
metabolism was made by Nesmeyanova et al. (1975a). The effect of mutation of the regu-
latory gene phoR, resulting in a non-inducible synthesis of proteins of the Pho regulon, was
studied. Neither alkaline phosphatase nor exopolyphosphatase were induced under phos-
phate starvation in this mutant strain, indicating that exopolyphosphatase was co-regulated
with alkaline phosphatase under the control of the same regulatory system.
The phoU mutant is constitutive for alkaline phosphatase and is able to synthesize five
times more PolyP than the parent strain under anaerobiosis in a rich medium (Rao et al.,
1985). Later, the accumulation of a high level of PolyP in the phoU mutant (∼ 100-fold
higher than in the parent strain) was confirmed (Morohoshi et al., 2002). This mutant was
able to remove fourfold more P i from the medium than the parent strain. By using this mutant
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andacombinedmethodofchemicalextractionand PNMRspectroscopy,theanaerobiosis-
induced PolyP accumulation in E. coli was studied (Rao et al., 1985). Under these condi-
tions, the total PolyP amount was maximal at the early stationary phase of growth. Both
trichloroacetic acid- and NAOH-soluble PolyPs were found in the cells. The acid-soluble
fraction contained polymer of about 20 ± 5 phosphate residues, whereas the alkali-soluble
31
fraction had a higher chain length. The P NMR spectroscopic analysis revealed PolyP of
more than 200 residues (Rao et al., 1985). It was observed that under these experimental
conditions E. coli cells accumulate at first acid-soluble low molecular-weight PolyP, and
high-molecular-weight PolyP is synthesized once the growth has ceased (Rao et al., 1985).
High levels of PolyP accumulation were obtained by increasing the dosage of E. coli
genes encoding polyphosphate kinase (ppk1), acetate kinase and phosphate-inducible trans-
port systems (PSTS, PSTC, PSTA, and PSTB), and by genetic inactivation of ppx encoding
exopolyphosphatase (Kato et al., 1993a; Hardoyo et al., 1994; Ohtake et al., 1994). All these
data support the idea that the massive accumulation of PolyP in E. coli may be obtained by
genetic modification in the regulatory systems, which provides P i uptake and its regulation
in this bacterium.
PhoB, the response regulator, turns on several genes, among them alkaline phosphatase
and the proteins involved in P i uptake. Pho-regulon mutants affected in PhoB synthesis
were tested for PolyP accumulation in a minimal medium containing low levels of P i (0.1
−1
mM) and amino acids (2 µgml ) (Rao and Kornberg, 1999). A large amount of PolyP
(48 nmol per mg of protein) accumulated in wild-type cells under these conditions. The
mutants lacking PhoB accumulated low levels of PolyP (0.3–1.9 nmol per mg of protein).
Inactivation of the protein kinases PhoR and CreC, which activate PhoB (Wanner, 1995),
led to a lower level of PolyP (0.1 nmol per mg of protein). The mutants with constitutive
expression of the Pho regulon or phoB mutants with multicopy phoB plasmid accumulated
PolyPs to a level comparable with those in wild-type cells (Rao and Kornberg, 1999).