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Previous Article | Next Article 
Applied and Environmental Microbiology, October 1998, p. 3754-3758, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Enhanced Utilization of Phosphonate and
Phosphite by Klebsiella aerogenes
Kazuya
Imazu,
Shotaro
Tanaka,
Akio
Kuroda,
Yuki
Anbe,
Junichi
Kato, and
Hisao
Ohtake*
Department of Fermentation Technology,
Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan
Received 27 April 1998/Accepted 6 July 1998
 |
ABSTRACT |
Klebsiella aerogenes ATCC 9621 was able to utilize
phosphonates (Pn), including aminoethylphosphonate,
ethylphosphonate, methylphosphonate (MPn), and
phosphonoacetate, and inorganic phosphite (Pt) as sole sources of phosphorus (P). The products of the phn gene
cluster were absolutely required for Pn breakdown and
Pt oxidation to inorganic phosphate (Pi) in
this organism. To determine if K. aerogenes ATCC 9621 could
be engineered to enhance the utilization of Pn and
Pt, a multicopy plasmid, pBI05, which carried the entire phn gene cluster, was introduced into this strain. Despite
the increased dosage of the phn genes, K. aerogenes ATCC 9621(pBI05) could utilize only up to 1.1-fold more
Pn and Pt than did the control strain with the
parent vector alone. These results suggested that Pi, which
was generated from Pn and Pt, might limit
further utilization of these P compounds. Consequently, to convert the resulting Pi to polyphosphate (polyP), the plasmid pKP28,
which carried the K. aerogenes ppk gene (which encodes
polyP kinase), was introduced into K. aerogenes ATCC
9621(pBI05). Overexpression of the ppk gene in K. aerogenes ATCC 9621(pBI05, pKP28) resulted in a 2.5-fold increase
in Pt utilization over that of the control strain. This
recombinant strain also accumulated approximately sixfold more P than
did the control strain when the cells were grown with MPn
as a sole source of P.
| |
INTRODUCTION |
Phosphorus (P) compounds are major
contaminants in industrial and municipal effluents (13).
Inorganic phosphate (Pi) is recognized as one of the major
nutrients contributing to the eutrophication of lakes, bays, and other
natural bodies of water (13). Treatment of inorganic
phosphite (Pt) associated with high-tech industries is also
becoming a difficult problem (10, 15). For example, large
amounts of hypophosphite (H2PO2
)
have been used to reduce metal ions in chemical plating processes such
as those employed in compact-disk manufacturing plants (15). After metal plating, wastewaters contain high concentrations of Pt (HPO32) as well as organic
acids which were used to adjust the pH and stabilize metal ions.
Because of the very narrow market for Pt, it is necessary
to oxidize Pt to Pi because it has a wider
range of applications. As of now, however, there is no effective means of Pt oxidation in the presence of high concentrations of
organic acids (15).
Phosphonates (Pn), which are P compounds with a direct
carbon-phosphorus (C---P) bond in place of the more familiar
carbon-oxygen-phosphorus ester (C---O---P) bond, are widely used as
pesticides, detergent additives, antibiotics, and flame retardants
(4, 17). Since Pn are relatively rare in living
organisms, they are generally recalcitrant to biodegradation (17). The C---P bonds are also resistant to chemical
hydrolysis and thermal decomposition, and the fate of Pn in
the environment is not well understood (17).
Some bacteria are capable of utilizing Pn and
Pt as sole sources of P (2, 4, 17). For example,
Escherichia coli utilizes aminoethylphosphonate
(AEPn), ethylphosphonate (EPn),
methylphosphonate (MPn), and Pt as a sole
source of P when Pi is not available (2, 12). In
E. coli Pn utilization requires cleavage of the
C---P bond by C-P lyases (18), and Pt seems to
be enzymatically oxidized to Pi before being used as a P
source. The E. coli phn locus, which comprises 14 genes
(named alphabetically from phnC to phnP) in an
operon with a single promoter, is required for utilizing Pn
and Pt (2, 12). PhnC, PhnD, and PhnE probably
comprise a periplasmic binding protein-dependent Pn
transporter which also transports Pt, Pi
esters, and Pi (12). PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, and PhnM are likely to be components of a C-P lyase pathway
or C-P lyase enzyme complex which catalyzes C---P bond cleavage and
Pt oxidation (2). Two additional proteins, PhnF
and PhnO, appear to be regulatory proteins. The expression of the
E. coli phn genes is known to be activated under conditions
of Pi limitation (2, 12).
Previously, we examined a variety of bacterial species for their
ability to utilize Pn and Pt as sole sources of
P (14). After examining the bacterial growth on
Pn and Pt, we found that Klebsiella
aerogenes ATCC 9621 was able to grow well in minimal medium
containing AEPn, EPn, MPn,
phosphonoacetate (PnAc), or Pt as a sole source
of P (14). The growth rate of K. aerogenes ATCC
9621 was threefold higher than that of E. coli IFO13168 (B strain) in the medium with Pt. Therefore, for further
study, we have also cloned the entire phn gene cluster from
K. aerogenes ATCC 9621 by using E. coli phnCDE as
a DNA probe (2).
The present work was undertaken to determine if K. aerogenes
ATCC 9621 could be engineered to enhance the utilization of
Pn and Pt as sole sources of P by increasing
the dosage of the phn genes. In the course of this study, we
found that Pi, which was released from Pn and
Pt, apparently limited further utilization of these P
compounds. Therefore, to enhance the conversion of the resulting
Pi to polyP, we employed the K. aerogenes ppk
gene, which encodes polyphosphate (polyP) kinase (PPK) (9);
this enzyme catalyzes the formation of polyP from ATP.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
E. coli MV1184
(16) and K. aerogenes ATCC 9621 (14)
were grown on 2× YT medium (16). Plasmid pQF50, which
carried the promoterless lacZ gene, was described previously
(9). pKP01 is a derivative of pUC119 (Pharmacia) containing
the K. aerogenes ppk and its own promoter (9).
pBR322 (16) and pSTV28 (Takara Shuzo Co., Ltd., Kyoto,
Japan) were used as cloning and expression vectors. To construct
pBR322MC, a 56-bp EcoRI-HindIII fragment containing a multiple cloning site of pUC118 (Takara Shuzo Co., Ltd.)
was ligated to pBR322 which had been cut with EcoRI and HindIII. Plasmid pUC4K (Pharmacia) carrying a kanamycin
resistance (Kmr) gene cassette was used for disrupting the
chromosomal phn gene cluster. Antibiotics used for the
selection of transformants were 50 mg of ampicillin, 50 mg of
kanamycin, and 20 mg of chloramphenicol per liter. Standard procedures
were used for plasmid preparations, restriction enzyme digestion,
ligation, and agarose electrophoresis (16).
Disruption of the chromosomal phn gene cluster.
Plasmid pPI04, which carried a 14-kb BglII-SmaI
fragment containing the entire K. aerogenes phn gene
cluster, was described previously (14). pPI04 was digested
with EcoRI and ligated with a 1.2-kb EcoRI
site-flanked Kmr gene cassette from pUC4K to construct
pPI02.2. K. aerogenes ATCC 9621 was then transformed with
pPI02.2 by electroporation, and Kmr transformants were
selected on 2× YT medium containing kanamycin. The disruption of the
chromosomal phn gene cluster was confirmed by Southern
hybridization (16).
Pn and Pt uptake experiments.
K.
aerogenes cells were grown in 2× YT medium with shaking at 37°C
for 7 h, inoculated (1% inoculum) into T0 minimal
medium (7) containing either Pn or
Pt as a sole source of P. The cultures were then incubated
for 24 h under the same conditions, and samples were taken at
intervals for the determination of growth (measured as optical density
at 600 nm [OD600]) and P concentrations. Pn were measured by ammonium peroxidisulfate digestion (120°C, 30 min)
followed by Pi measurement (14) by an ascorbic
acid method (9). Pt was analyzed with a
high-performance liquid chromatography system with an IC-Anion-SW
column (4.6 by 50 mm; Tosoh Co., Ltd., Tokyo, Japan). The total P
content of K. aerogenes cells was determined as described
previously (9).
PolyP analysis.
PolyP was extracted from bacterial cells
with a 4 M guanidine thiocyanate solution and bound to silicate glass
powders (Gene Clean kit II; Funakoshi Co., Ltd., Tokyo, Japan). The
glass powders binding polyP were washed with an ethanol-NaCl solution,
and then polyP was recovered with distilled water from the glass powder solution (1). PolyP was determined as described by Crooke et al. (3). The ability to serve as a substrate for the
PPK-catalyzed conversion of [14C]ADP to
[14C]ATP was used to quantitate levels of polyP. The
amount of [14C]ATP was determined by thin-layer
chromatography followed by visualization by an image analyzer (BAS1000;
Fuji Co., Ltd., Tokyo, Japan).
Chemicals.
AEPn, MPn, and
PnAc were purchased from Sigma Chemical Co. (St. Louis,
Mo.). Pt was obtained from Aldrich Chemical Co. (Milwaukee, Wis.). EPn and dimethylphosphite (DMPt) were
from Nacalai Tesque, Inc. (Kyoto, Japan), and [14C]ADP
was from Dai-ichi Chemical Co. (Tokyo, Japan).
| |
RESULTS AND DISCUSSION |
Pn and Pt utilization.
There are two
Pn degradation pathways which are commonly referred to as
the phosphonatase and the C-P lyase pathways (11). They
differ in regard to their substrate specificities and mechanism of
C---P bond fission. Interestingly, E. coli degrades
Pn solely by the C-P lyase pathway, whereas
Salmonella typhimurium appears to contain genes only for the
phosphonatase pathway (5). The phn genes, whose
products are involved in the C-P lyase pathway, have been cloned from
K. aerogenes ATCC 9621 (14).
To determine if K. aerogenes ATCC 9621 possesses solely the
C-P lyase pathway for utilizing Pn and Pt, we
constructed the chromosomal phn mutant strain PHN1 using a
Kmr gene cassette from pUC4K (Fig.
1). K. aerogenes PHN1 could
not grow in T0 medium containing AEPn,
EPn, MPn, PnAc, DMPt,
or Pt as a sole source of P (Fig.
2). To further confirm the essentiality of the phn gene cluster, a 14-kb
XbaI-KpnI fragment which carried the entire
K. aerogenes phn gene cluster (14) was excised
from pPI04 and cloned into pBR322MC to construct pBI05 (Fig. 1).
Plasmid pBI05 could restore the ability of K. aerogenes PHN1
to grow on T0 medium containing either Pn or
Pt as a sole source of P (Fig. 2). These results indicate
that K. aerogenes ATCC 9621 possesses solely the C-P lyase
pathway for utilizing Pn and Pt as a sole source of P.
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FIG. 1.
Schematic representation of plasmids pKP28 and pBI05 (A)
and the phn region of K. aerogenes PHN1
chromosomal DNA (B).
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FIG. 2.
Growth of K. aerogenes strains ATCC 9621 (hatched bars), PHN1 (open bars), and PHN1(pBI05) (solid bars) in
T0 medium containing either Pn or
Pt as a sole source of P. Cultures were grown for 24 h
at 37°C with shaking, and cell growth was determined by
OD600 measurements. The initial concentrations of P
compounds in T0 medium were 0.5 mM. Error bars represent
standard errors.
|
|
Regulation of the K. aerogenes phn locus.
To
determine if K. aerogenes ATCC 9621 could be engineered to
enhance the utilization of Pn and Pt as sole
sources of P, Pn and Pt uptake experiments were
performed with K. aerogenes ATCC 9621(pBR322MC), a
control strain, and ATCC 9621(pBI05). However, despite the increased
dosage of the phn gene cluster, K. aerogenes ATCC
9621(pBI05) removed only up to 1.1-fold more Pn and
Pt from T0 medium than did ATCC
9621(pBR322MC) (data not shown). Since both Pn
breakdown and Pt oxidation could result in an elevated accumulation of cellular Pi, it is highly possible that
Pi, which was released from Pn and
Pt, limited further utilization of these P compounds. We
also observed that the presence of Pi in the growth medium
strongly inhibited the utilization of Pn and Pt
by K. aerogenes ATCC 9621 (data not shown).
We scanned the nucleotide sequence upstream from the K. aerogenes
phn gene cluster for the presence of a Pho box, the consensus sequence shared by the Pho regulon promoters, by using the consensus sequence published by Kasahara et al.
[CTGTCATA(A,T)A(A,T)CTGTCA(C,T)] (6).
Inspection of the promoter sequence revealed that the K. aerogenes phn gene cluster was preceded by a Pho box sequence (CTGTCATCAAACTGCGCT). There was a 14-of-18-bp match with the
consensus Pho box sequence. We further confirmed that the promoter of
the K. aerogenes phn gene cluster is activated under
conditions of Pi limitation. A 2.1-kb
XbaI-PstI fragment which carried the
phn promoter was excised from pBI05 (Fig. 1) and inserted
upstream from the promoterless lacZ gene (encoding
-galactosidase) in the vector pQF50 to construct pQF01. K. aerogenes ATCC 9621 was transformed with either pQF50 or pQF01,
and the -galactosidase levels were measured in K. aerogenes cells grown in T0 medium with or without
Pi. High enzyme levels were observed only with K. aerogenes ATCC 9621(pQF01) cells grown in T0 medium
without Pi (data not shown).
Conversion of Pn and Pt to polyP.
We
attempted to place the phn gene cluster under the control of
the tetracycline resistance (tet) promoter of pBR322 to
express it constitutively. However, because of the absence of
appropriate restriction sites for the phn gene cluster, this
attempt has not yet been successful. As we have previously demonstrated
(9), K. aerogenes is able to accumulate high
levels of polyP. Therefore, it may be possible that the conversion of
Pi to polyP, if enhanced, could improve the utilization of
Pn and Pt in this organism.
Previously, we have cloned ppk from K. aerogenes
ATCC 9621 (9). To enhance the conversion of Pi
to polyP, a 2.2-kb EcoRI-HindIII fragment,
which contained the K. aerogenes ppk gene and its own promoter, was excised from pKP01 (9) and cloned into a
vector pSTV28 to construct pKP28 (Fig. 1). This recombinant plasmid was then introduced into K. aerogenes ATCC 9621(pBI05) by
electroporation. K. aerogenes ATCC 9621(pBI05, pKP28) could
remove 2.5-fold more Pt from T0 medium
containing 0.5 mM Pt than did the control strain ATCC
9621(pBR322, pSTV28) (Fig. 3). Since the
growth of K. aerogenes ATCC 9621(pBI05, pKP28) was almost
equivalent to that of the control strain, this recombinant strain
accumulated approximately 2.1-fold more P (23 mg of P per g [dry
weight] of cells) than did the control strain. The levels of polyP in
K. aerogenes ATCC 9621(pBI05, pKP28) were approximately 3.5 mg of P per g [dry weight] of cells after the 24-h incubation in
T0 medium containing 0.5 mM Pt (Fig.
4). However, the time course analysis of
polyP accumulation revealed that the levels reached a maximum of 12 mg
of P per g (dry weight) of cells around 6 h after the start of
incubation (Fig. 5). No significant
amount of polyP was observed with K. aerogenes ATCC 9621(pBR322, pSTV28) and ATCC 9621(pBI05, pSTV28). K. aerogenes ATCC 9621(pBR322, pKP28) could accumulate polyP only
when Pi was available (Fig. 4). K. aerogenes
ATCC 9621(pBI05, pKP28) also accumulated a maximum of 10% of its dry
weight as polyP (33 mg of P per g [dry weight] of cells) when the
cells were grown in T0 medium containing 0.5 mM
MPn as a sole source of P (Fig. 5). In this experiment, the
total P content of K. aerogenes ATCC 9621(pBI05, pKP28)
reached a maximum of 62 mg of P per g (dry weight) of cells, or
approximately sixfold more than that of the control strain with the
parent vectors alone (data not shown).
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FIG. 3.
Time course of OD600 (circles) and
Pt concentration (squares) during growth of K. aerogenes ATCC 9621(pBR322, pSTV28) (A) and ATCC 9621(pBI05,
pKP28) (B).
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FIG. 4.
PolyP accumulation in K. aerogenes strains.
(A) Thin-layer chromatography of [14C]ATP generated by
the PPK-catalyzed conversion of [14C]ADP.
[14C]ATP and [14C]ADP were resolved with 1 M formic acid and 0.4 M LiCl as a solvent system and visualized with an
image analyzer (BAS1000; Fuji Co. Ltd.). Lanes 1 and 5, K. aerogenes ATCC 9621(pBR322, pSTV28); lanes 2 and 6, K. aerogenes ATCC 9621(pBI05, pSTV28); lanes 3 and 7, K. aerogenes ATCC 9621(pBR322, pKP28); lanes 4 and 8, K. aerogenes ATCC 9621(pBI05, pKP28). Cultures were grown in
T0 medium containing either Pi (lanes 1 to 4)
or Pt (lanes 5 to 8) as a sole source of P. PolyP levels
were determined with K. aerogenes cells sampled around
24 h after the start of incubation. (B) PolyP contents of K. aerogenes strains quantitated by the image analyzer. Error bars
represent standard errors.
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FIG. 5.
Time course of OD600 (circles) and polyP
content (squares) of K. aerogenes ATCC 9621(pBI05, pKP28)
during growth in T0 medium containing either 0.5 mM
MPn (A) or 0.5 mM Pt (B).
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We have examined, for the first time, the possibility of genetic
improvement of Pn and Pt utilization in
bacteria. The phn genes were essential for Pn
and Pt utilization in K. aerogenes ATCC 9621, although their roles in Pn breakdown and Pt
oxidation are specifically uncharacterized. In addition, it was
suggested that Pi, which was released from Pn
and Pt, appeared to limit further utilization of
Pn and Pt. In fact, we found that enhanced conversion of Pi to polyP could improve the utilization of
Pn and Pt as sole sources of P in K. aerogenes ATCC 9621. It has been known that exopolyphosphatase
plays an important role in the degradation of polyP to Pi
(3, 8). Previously, we showed that the K. aerogenes
ppx gene, encoding exopolyphosphatase, existed immediately
downstream of the ppk gene without transcriptional termination (9). The chromosomal ppk mutant,
which was constructed by inserting a Kmr gene cassette into
the wild-type gene, showed a reduced polyPase activity in K. aerogenes ATCC 9621 (data not shown). We also constructed a
ppk ppx double mutant of K. aerogenes ATCC 9621 and introduced pKP28 into this mutant by electroporation. When this
strain was grown in T0 medium with 0.5 mM Pt,
it accumulated 12-fold more polyP than did ATCC 9621(pKP28) (data not
shown). Consequently, we attempted to further improve the utilization
of Pn and Pt in K. aerogenes by
introducing both pBI05 and pKP28 into the ppk ppx double
mutant. However, simultaneous introduction of pBI05 and pKP28 appeared
to be detrimental to the ppk ppx double mutant, and this
attempt has not yet been successful.
Our previous work demonstrated that high levels of Pi
accumulation in E. coli were achieved by modifying the
genetic regulation and increasing the dosage of the E. coli
genes encoding PPK and the Pi-specific transport system
(8). When Pi is available, the E. coli recombinants accumulated as much as 16% of their dry weight
as P (48% as Pi), or approximately 10-fold more P than did
the control strain. This level of P content was surprisingly high and
even surpassed those of natural phosphorite deposits (typically 14% as
P). K. aerogenes ATCC 9621(pBI05, pKP28) could accumulate up
to sixfold more P than did the control strain when MPn was
available as a sole source of P. Further strain improvement would be
possible if the functions of the phn gene products in K. aerogenes were specifically understood.
| |
ACKNOWLEDGMENTS |
This research was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Science and Culture, Japan, and by Nihon Chemical Co.
We thank Arthur Kornberg for the gift of the purified polyP kinase.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Fermentation Technology, Hiroshima University, Higashi-Hiroshima,
Hiroshima 739-8527, Japan. Phone: 0824-24-7756. Fax: 0824-22-3758. E-mail: hohtake{at}ipc.hiroshima-u.ac.jp.
| |
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Applied and Environmental Microbiology, October 1998, p. 3754-3758, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
