Journal of Environmental Biotechnology
Vol. 6, No. 1, 41–46, 2006

Original paper (regular paper)

Polyamines Aﬀect Polyphosphate Accumulation in Escherichia coli
KEI MOTOMURA1, NOBORU TAKIGUCHI1, HISAO OHTAKE2 and AKIO KURODA1,3*
1

Department of Molecular Biotechnology, Hiroshima University, Hiroshima 739-8530, Japan2
Department of Biotechnology, Osaka University, Osaka 565-0871, Japan
3
SORST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan* TEL: 082–424–7758 FAX: 082–424–7047* E-mail: akuroda@hiroshima-u.ac.jp(Received; 4 April, 2006/Accepted; 1 May, 2006)

In the current studies, we showed that a speABC and cadA mutant of Escherichia coli, which fails to produce polyaminessuch as putrescine, spermidine, and cadaverine, accumulates reduced levels of polyphosphate (polyP) under conditions ofamino acid starvation. This was reversed by supplementation of the culture medium with polyamines. In addition, a speG
mutant, which accumulates polyamines, increased levels of polyP, suggesting that polyamines aﬀect polyP accumulation. Although polyP granules are often found in cellular inclusions containing calcium, those isolated from E. coli did not containcalcium. We found that these polyP granules soon disappear when they are placed in a buﬀer but that they are stabilized by
the presence of polyamines. We concluded that polyamines interact with polyP and aﬀect polyP accumulation.
Key words: Polyphosphate, polyamine, Escherichia coli, polyphosphate granule1.

Introduction

Increased input of inorganic phosphate (Pi) into lakes,
bays, and other surface waters causes the nuisance growthof phytoplankton, called an algal bloom6). Hence, considerable attention has been paid to eﬃcient removal of Pi fromwastewater 6,28). In traditional sewage works, 20% to 40% of
Pi in wastewater is typically removed in the sludge by biological conversion to organic phosphate such as DNA, RNA,and phospholipid. Improvement of the ability to accumulate
phosphorus compounds in the sludge should therefore help
in the removal of Pi from wastewater.
Inorganic polyphosphate (polyP) is a linear polymer ofmany tens or hundreds of Pi molecules connected by highenergy bonds14,15). PolyP is found in a wide range of prokaryotes and eukaryotes14,15). PolyP has various biological
functions, for example, as a Pi reservoir14,15), an alternative
source of high-energy bonds14,15), as a buﬀer against alkalineconditions and metal accumulation23), and as a regulator fortranscription14) and proteolysis18). PolyP has an osmotic
advantage over free Pi for accumulation of phosphorus incells. Therefore, enhanced biological phosphorus removal isbased on the use of bacteria capable of accumulating polyP.
PolyP kinase (PPK), a tetramer of 80-kDa subunits, is
responsible for the processive synthesis of long-chain polyP(750 Pi residues) in Escherichia coli14). PPK catalyzes thereversible reaction of the γ-terminal Pi of ATP to polyP14).
So far, phoU, spoT, and relA genes have been identified as
regulators of polyP accumulation16,21). A phoU mutant,which derepresses the phosphate regulon, accumulates

polyP21). Constitutive expression of the PstSCAB Pi uptake
system in the phoU mutants is responsible for the elevatedlevels of polyP21). On the other hand, the level of polyP in E.
coli is very low during the exponential phase but increases
up to 1000-fold in response to amino acid starvation16). An
E. coli relA spoT mutant that fails to produce guanosine
penta- and tetra-phosphate (pppGpp and ppGpp) is deficient in the accumulation of polyP in response to aminoacid starvation16). It was found that the stringent factors
pppGpp and ppGpp strongly inhibit polyphosphatase
PPX16). PPK and PPX activities are constitutively expressed,
but when pppGpp and ppGpp build up in the cells during
amino acid starvation, polyP may accumulate due to inhibition of PPX.
Polyamines, polycationic compounds present in all livingorganisms, have been implicated in a wide variety of biological reactions, including nucleic acid and protein synthesis3,10).
Polyamines are thought to participate in these cellular processes through binding to DNA and RNA10,11). Polyaminecontent is regulated by its biosynthesis, degradation, uptake,and excretion9,22). A polyamine-deficient strain of E. coli has
been obtained as a result of deletions in speA (arginine
decarboxylase), speB (agmatine ureohydrolase), speC
(ornithine decarboxylase), and cadA (cadavarine biosynthesis)13,29). Although this strain lacks putrescine, spermidine,and cadaverine, it is still able to slowly but indefinitely growon polyamine-deficient media13,29). In the presence of polyamines in the medium, an speABC and cadA mutant cantake up polyamines and grow well. On the other hand, aspermidine acetyltransferase gene (speG) mutant that fails

MOTOMURA et al.

42

to degrade polyamine accumulates approximately 3-foldhigher levels of spermidine when grown in a medium containing spermidine5). We speculated that polyamines interact
not only with DNA and RNA but also polyP, and we demonstrated that polyamines aﬀect polyP accumulation underconditions of amino acid starvation.

2.
2.1.

Materials and Methods

Plasmid construction

A 2.6-kb KpnI fragment containing ppk of pBC10 was
inserted into the KpnI site of pMW119. The orientationof the ppk insertion in the resulting plasmid pMWppk was
opposite the lac promoter of pMW11917).
2.2.

Bacterial strains and growth conditions

E. coli speABC, cadA, and speG mutants were constructed according to the one-step gene inactivation method
described by Datsenko and Wanner4). First, DNA fragments
containing target and antibiotic genes were prepared bypolymerase chain reaction using the primers listed in Table 1.
These DNA fragments were then introduced into E. coli
and replaced with the chromosomal genes. The speABC
cadA mutant was grown in a 2xYT rich medium (1.6%
peptone, 1.0% yeast extract, and 0.5% NaCl) with shaking
at 37°C for 12–18 h. This culture was diluted in a
MOPS-minimal medium (22.2 mM glucose, 40 mM potassium morpholinopropane sulfonate [pH 7.2], 50 mM NaCl,
9.52 mM NH4Cl, 4 mM Tricine, 2 mM K2HPO4, 0.52 mM
MgCl2, 0.28 mM K2SO4, 0.01 mM FeSO4, 0.0005 mM
CaCl2, and trace metals)17) containing 2% casamino acidand 0.4 M sorbitol and then cultivated overnight. To deplete
endogenous polyamines, this cultivation was repeated, andthe culture was inoculated into the MOPS-minimal medium
containing 2% casamino acid and 0.4M sorbitol. The fresh
medium was supplemented with 1 mM putrescine and spermidine as necessary. After a 6-h incubation, the E. coli speABC cadA mutant was collected by centrifugation. The cell

Table 1.

pellet was washed once with the MOPS-minimal medium
containing 0.4 M sorbitol and resuspended in the same
medium (amino acid-starved condition). The E. coli speG
mutant was grown to mid-log phase in the 2xYT medium
with shaking at 37°C and shifted to the MOPS-minimal
medium. PolyP granules were observed under fluorescent
microscopy (BX-50, Olympus) after staining with 4',6-diamidino-2-phenylindole (DAPI). PolyP was recovered withsilicate glass from cells lysed with guanidine isothiocyanateand determined by a two-enzyme assay1).
2.3.

Preparation of polyP granules from E. coli

The E. coli phoU mutant was grown to mid-log phase in
the 2xYT medium with shaking at 37°C and shifted to
the MOPS-minimal medium for 2 h to cause the accumulation of a large amount of polyP-containing granules21).
PolyP granules were prepared essentially as described by
Jacobson 12). Freeze-dried cells (0.1 g), zirconia beads(diameter 2 mm; 3.5 g), and carbon tetrachloride (400 µl)
were mixed in a 2-ml tube. The suspension was homogenized twice for 30 s using a Micro Smash (Tomy, Tokyo,
Japan) at a speed of 4000 rpm in a cold room. PolyP granules were precipitated by centrifugation at 13,000×g for
10 min. The cell debris was not precipitated under this condition. The pellet containing polyP granules was washed several times with carbon tetrachloride and dried. PolyP granules were observed by scanning electron microscopy, andtheir chemical composition was analyzed by electron microscopy coupled with energy-dispersive X-ray spectroscopy(EDAX; Nihon Denshi).
2.4.

In vitro preparation of polyP granules

PolyP granules were formed by mixing 50 mM polyP(average chain-length=65; Sigma), 57 mM KCl, and 39 mM
MgCl2. PolyP granules were collected by centrifugation at
13,000×g for 5 min and washed five times with a solution
containing 57 mM KCl and 39 mM MgCl2.

Primers used for gene disruption.

Gene

DNA sequence

speAB

ACTGTTTTACACTTAATAAAATAATTTGAGGTTCGCTATGATTCCGGGGATCCGTCGACC (forward)
TGCGCATCGCATCTGGTGCTTACTCGCCCTTTTTCGCCGCTGTAGGCTGGAGCTGCTTCG (reverse)
GGCGGTCGGAGCTGGTGACCAGTTTGACCCATATCTCATGATTCCGGGGATCCGTCGACC (forward)
TGACCCGTTTTTTTTATTCTTACTTCAACACATAACCGTATGTAGGCTGGAGCTGCTTCG (reverse)
GTGTTGGGAGGGGCCTTTTTTACCTGGAGATATGACTATGATTCCGGGGATCCGTCGACC (forward)
CTTCCCTTGTACGAGCTAATTATTTTTTGCTTTCTTCTTTTGTAGGCTGGAGCTGCTTCG (reverse)
TAACCTGTTATTGATTTAAGGAATGTAAGGACACGTTATGATTCCGGGGATCCGTCGACC (forward)
CGATCGATTATTATTAATGCTATTGTGCGGTCGGCTTCAGTGTAGGCTGGAGCTGCTTCG (reverse)

speC

cadA

speG

Polyamine and Polyphosphate Accumulation

2.5.

43

Stability of polyP granules

PolyP granules prepared in vitro were dispersed in
100 mM sodium hydroxyetylpiperazine ethanesulphonate(HEPES-NaOH, pH 7.2) containing each polyamine (putrescine, spermidine, cadaverine), or ammonium chloride atroom temperature. After the indicated time, polyP granules
were precipitated at 13,000×g for 3 min. PolyP was determined as Pi by the ascorbic acid method after hydrolysis in
1 N HCl at 100°C for 7 min2,7).

3.
3.1.

Results and Discussion

Polyamines aﬀect polyP accumulation in E. coli

PolyP accumulated in response to amino acid starvationin E. coli (Fig. 1A). PolyP is a Pi polymer with some similarity to RNA and DNA, and, therefore, it is reasonable
that polyP readily interacts with polycationic compounds
including polyamines. To determine whether polyamines
aﬀect polyP accumulation under conditions of amino acidstarvation, we constructed a speABC and cadA mutant thatfails to produce polyamines13,29). The intracellular levels ofpolyamines in this mutant can be restored by the additionof polyamines to the medium. The levels of polyP in thismutant were also partially restored by addition of polyamines to the medium under conditions of amino acid starvation (Fig. 1A). In addition, introduction of a low-copy
plasmid encoding ppk (pMWppk) increased the levelsof polyP. Under conditions of amino acid starvation, an
speABC and cadA mutant carrying pMWppk accumulated
more polyP in the presence than in the absence of polyamines in the medium (Fig. 1B). On the other hand, accumulation of polyamines by a speG mutant was enhanced
three-fold by the addition of polyamines to the medium.

Fig. 2. PolyP accumulation in a speG mutant and the wild typeunder conditions of amino acid starvation in the presence ofpolyamines.
Intracellular polyP concentrations were measured for E. coli
MG1655 (open circles) and a speG mutant (closed circles) at
the indicated times after shifting to MOPS medium containing
polyamines (1 mM putrescine and 1 mM spermidine).

This mutant also accumulated more polyP than the wild
type (Fig. 2). After 3 h, the wild type restarted to grow under this condition and its polyP accumulation reduced (Fig.
2). However, this mutant could not adapt to amino acidstarvation and thus polyP accumulation might prolong evenafter 3 h. These results suggested that polyamines aﬀect
polyP accumulation.
3.2. Analysis of polyP granules prepared from E. coli
PolyP is often found in cellular inclusions known as polyP granules or volutin granules19,25,26,27). Here, we isolated
polyP granules from an E. coli phoU mutant that accumulates 1000-fold higher levels of polyP than the wild type.

Fig. 1. PolyP accumulation in a speABC cadA mutant under conditions of amino acid starvation and in the presence and absence ofpolyamines.
(A) Intracellular polyP concentrations were measured in the E. coli wild type at the indicated times after shifting to amino acid-starved
conditions (open triangles). The polyP concentrations were also measured in an E. coli speABC cadA mutant at the indicated times after
shifting to amino acid-starved conditions with (closed squares) or without (open squares) added polyamines (1 mM putrescine and 1 mM
spermidine). (B) The same experiment was performed in the E. coli speABC cadA mutant carrying pMWppk in the presence (closed
circular) or absence (open circular) of polyamines.

44

MOTOMURA et al.

Fig. 4. Microscopic analysis of synthesized polyP granules.
The synthesized polyP granules were observed under phasecontrast microscopy.

Fig 3. Scanning electron microscopy (A) and EDAX spectra (B)of polyP granules prepared from E. coli cells.
(A) PolyP granules extracted from E. coli with carbon tetrachloride were evaporated on the stage, coated with carbon,
and then observed by scanning electron microscopy.
Bar=2 µm. (B) The elemental composition of polyP granules
was analyzed by EDAX.

We examined the polyP granules by microscopy andby scanning electron microscopy with EDAX (Fig. 3).
EDAX analysis revealed that polyP granules contained
phosphorus, magnesium, and potassium in a ratio of
1.0:0.31:0.19. Although polyP granules are often found incellular inclusions containing calcium14), those isolated from
E. coli did not contain calcium.

We prepared polyP granules in vitro by mixing polyP,
KCl, and MgCl2 as to approximately the same elemental
ratio as the polyP granules prepared from E. coli cells. The
synthetic polyP granules were spherical with diameters fromseveral hundreds of nanometers to tens of micrometers (Fig.
4). However, these synthetic polyP granules dissolved within
50 min when they were placed in HEPES buﬀer (datashown in Fig. 5B).
3.3. Polyamines protect polyP granules from dissolution
We suspected that polyamines protect polyP granules
from dissolution in E. coli. We found that the polyaminessuch as putrescine, spermidine, and cadaverine protect the
polyP granules from dissolution but that ammonium chloride does not (Fig. 5A). Igarashi and Kashiwagi predictedthat 12.5 mM putrescine and 0.26 mM spermidine exist in afree form in E. coli20). The polyP granules were completely
protected against dissolution by a combination of 12.5 mM
putrescine and 0.26 mM spermidine (Fig. 5B).

Fig. 5. Polyamines stabilize polyP granules.
(A) PolyP granules prepared in vitro (0.5 mg) were precipitated by centrifugation and placed in HEPES buﬀer containing various concentrations of putrescine (closed circular), spermidine (open circular), cadaverine (closed square), or ammonium chloride (open square).
Dissolved polyP could no longer be precipitated by centrifugation. The percentage of dissolved polyP after 10-min incubation vs. total
polyP granules was plotted as a function of the polyamine concentration. (B) PolyP granules prepared in vitro (0.5 mg) were incubated in
HEPES buﬀer in the presence (closed) and absence (open) of 12.5 mM putrescine and 0.26 mM spermidine. The percentage of dissolved
vs. total polyP granules was plotted as a function of the incubation time.

Polyamine and Polyphosphate Accumulation

We prepared polyP granules in vitro by mixing polyP,
KCl, and MgCl2 in the presence of 10 mM spermidine. The
synthetic polyP granules were very stable even when they
were placed in HEPES buﬀer in the absence of polyamine(data not shown). These results suggested that the polyamines also interact with polyP when polyP granules areformed.
PolyP was first identified in microorganisms as metachromatic granules that are stained pink with basic blue dyes,
and they were originally referred to as “volutin granule”14).
PolyP granules from unicellular eukaryotes (e.g., Chlamydomonas reinhardtii25) and Dictyostelium discoideum19)),
a number of human pathogens (e.g., malaria parasites andtrypanosomatids26)), and human platelets27) were recently
found to be surrounded by a membrane with a number ofpumps and exchangers and were named acidocalcisomes.
Although polyP granules are often found in cellular inclusions containing calcium14), those isolated from E. coli did
not contain calcium. We found that these polyP granules
soon disappeared when added to buﬀer, although they arestabilized by polyamines (putrescine, spermidine, andspermine), which are known to be present at millimolar
concentrations in both prokaryotic and eukaryotic cells3,10).
We concluded that polyamines may interact with polyP invivo and therefore aﬀect polyP accumulation.
Our findings also suggested that, conversely, polyP accumulation may aﬀect the intracellular levels of free polyamine. A decrease or increase in polyamine content greatly
diminishes cell growth in E. coli8,24), and their intracellularlevels are closely regulated by control of synthesis, degradation, uptake, and excretion9,22). In E. coli, approximately
50% of putrescine and 90% of spermidine exist as polyamine-RNA complexes, and they act at the level of translation to enhance the synthesis of OppA, Cya, RpoS, FecI,and Fis11). Further investigations are needed to clarify therole of polyP in polyamine metabolism.

Acknowledgments
This work was supported in part by the Research andDevelopment Program for New Bio-industry Initiatives of
Bio-oriented Technology Research Advancement Institution
Japan.

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