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Appl Environ Microbiol, March 1998, p. 896-901, Vol. 64, No. 3
Lehrstuhl für Mikrobiologie, Institut
für Mikrobiologie, Biochemie und Genetik der
Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany
Received 30 September 1997/Accepted 9 December 1997
Polyphosphate kinase (Ppk) catalyzes the formation of polyphosphate
from ATP. We cloned the ppk gene (2,073 bp) from
Acinetobacter sp. strain ADP1; this gene encodes a putative
polypeptide of 78.6 kDa with extensive homology to polyphosphate kinase
from Escherichia coli and other bacteria. Chromosomal
disruption of ppk by inserting a transcriptionally fused
lacZ does not affect growth under conditions of phosphate
limitation or excess. Polyphosphate is a linear polymer of
orthophosphate residues linked by high-energy phosphoanhydride bonds.
It is found in a wide variety of organisms, including bacteria, fungi,
protozoa, plants, and mammals (23, 24). While this may
suggest a fundamental physiological role for polyphosphate in life, no
essential function has been identified. However, polyphosphate may have
many different functions in various organisms and under different
physiological conditions, as reviewed previously (22, 43);
e.g., this polymer may contribute to survival in stationary phase
(30), inhibition of RNA degradation (5), storage
of phosphate and energy, substitution of ATP, chelation of ions
(20), formation of cell capsule, regulation under stress
(19, 30), and formation of channels for DNA entry into
competent Escherichia coli cells (6, 32).
In E. coli, polyphosphate is remarkably uniform in length
(about 750 residues). It is built up from ATP by polyphosphate kinase (Ppk) in a reversible and highly processive reaction from a
phosphohistidyl-Ppk intermediate (25). Ppk is a
homotetrameric protein that is associated with the outer membrane
(1, 3) and is a component of the RNA degradosome
(5). Depolymerization of polyphosphate occurs either by the
reverse Ppk reaction leading to ATP formation from ADP or via
hydrolysis by an exopolyphosphatase (Ppx) (2) or pppGpp
hydrolase (GppA) (19). The ppk and ppx
genes are located in the same operon (2). Disputed
indications hint that the ppk ppx operon is regulated by the
PhoBR system (18, 40). Some strains of
Acinetobacter which occur predominantly in
wastewater are known to accumulate polyphosphate (8).
However, the microbial process of phosphate removal from wastewater is
very slow, and improvements are necessary to support industrial
application of this trait (22). Knowledge about the genes
involved in polyphosphate synthesis and their regulation may support
this task. Here we report the isolation and analysis of the
ppk gene from Acinetobacter sp.
strain ADP1 and its transcriptional induction by phosphate starvation.
Since it is not known if ADP1 accumulates polyphosphate, it serves as a
model for regulation of ppk transcription in
Acinetobacter.
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are listed in Table
1. Wild-type
Acinetobacter sp. strain ADP1 was formerly classified as Acinetobacter
calcoaceticus ADP1 and is synonymously called
Acinetobacter sp. strain BD413 (17,
38).
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transcription of ppk from Acinetobacter sp.
Strain ADP1, Encoding a Putative Polyphosphate Kinase, Is
Induced by Phosphate Starvation
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-Galactosidase activity expressed from the
single-copy ppk::lacZ fusion is
induced 5- to 15-fold by phosphate starvation. An increased amount of
ppk transcript (2.2 kb) was detected when cells were grown
at a limiting phosphate concentration. Primer extension analysis
revealed a regulated promoter located upstream of a second,
constitutive promoter. Potential similarities of this regulation with
the effects of PhoB and PhoR of E. coli are discussed.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
General methods. E. coli and Acinetobacter sp. strain ADP1 were transformed as described previously (13, 29). Total DNA was prepared as described by Ausubel et al. (4). Small-scale preparations of plasmids were made by the boiling lysis method (16); large-scale preparations were made by using the Nucleobond Kit (Macherey-Nagel, Düren, Germany). Total RNA was isolated with the RNeasy Mini Kit from Qiagen (Hilden, Germany).
Media, growth conditions, and -galactosidase assays.
E.
coli was grown at 37°C, and Acinetobacter
strains were grown at 28°C. E. coli cultures for
preparation of DNA were grown in LB medium with ampicillin (100 mg/liter) or kanamycin (30 mg/liter). Acinetobacter sp. strains WH386 and WH435 were
grown with kanamycin (10 mg/liter).
-galactosidase assays
were collected at times indicated in the respective results. Samples
representing 1 ml of culture at an OD600 of 0.52 were
centrifuged, and the cell pellets were frozen at 
20°C for up to 3 days for -galactosidase assays. The cell pellets were suspended in
1.3 ml of LB medium, the OD600 was determined, and 200 µl
was used as described by Miller (28). The data
(OD600 and Miller units) given in Fig. 3 were determined
from three independent cultures. -Galactosidase activities of frozen
cells are identical to those obtained from fresh cells.
DNA sequence analysis.
Nucleotide sequences were determined
on both strands by the dideoxy chain termination method (34)
with Sequenase (U.S. Biochemicals, Cleveland, Ohio) and
[
-32P]dATP or [-35S]dATP. The Thermo
Sequenase radiolabeled terminator sequencing kit with
[-33P]dideoxynucleoside triphosphates (Amersham,
Buckinghamshire, United Kingdom) was used for sequencing reactions
performed in connection with primer extension. Sequences were analyzed
by using the UWGCG software package (9). Database searches
were done by using the services offered by the National Center for
Biotechnology Information (http://www.ncbi.nlm.nhi.gov).
Southern hybridization. Restricted genomic DNA (8 µg) or plasmid DNA (10 ng) was run on a 1% agarose gel and blotted onto a nylon membrane (36). The probe was prepared by nick translation with biotin-7-dATP, and a Photogene kit was used for detection of signals. The membrane, nick translation, and Photogene kit were obtained from Gibco BRL (Gaithersburg, Md.).
Northern hybridization.
Total RNA (10 µg per lane) was run
on a 1.3% agarose gel containing 6% formaldehyde and blotted onto a
positively charged nylon membrane (Porablot NY Plus; Macherey-Nagel) by
capillary transfer as described by Sambrook et al. (33). The
ppk-specific probe was prepared by PCR with
[-32P]dATP amplifying a fragment from nucleotide 10775 to 11214 (see Fig. 1). The hybridization procedure was performed in
accordance with the recommendations of the membrane manufacturer.
Radioactivity was determined with a PhosphorImager (Fujifilm,
BAS-1500).
Primer extension. Primer extension reactions were performed as described previously (42). Total RNA (15 µg) was incubated for 5 min at 80°C and hybridized for 5 min with the 5'-end-labeled primer (35 fmol) at 37°C. The reaction mixtures containing 9 U of avian myeloblastosis virus reverse transcriptase (Promega, Madison, Wis.) were incubated for 45 min at 37°C. After treatment with RNase A and denaturation of the cDNA, half of the volume was loaded on the gel and analyzed with a PhosphorImager. The sequence of the ppk-specific primer is 5'-CTGGTGGTGTTGTCATCGC-3'.
Nucleotide sequence accession number. The 13,879-bp nucleotide sequence of Acinetobacter sp. strain ADP1 DNA cloned on pWH891 and pWH969 has been deposited in the EMBL database under accession no. Z46863.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cloning of ppk from
Acinetobacter sp. strain ADP1.
Recently,
we reported the cloning of a 10.8-kbp genomic BamHI fragment
from Acinetobacter sp. strain ADP1 harboring a
putative periplasmic Mn superoxide dismutase located upstream
of the rubredoxin-encoding gene rubA on plasmid pWH891
(Fig. 1) (11). For analysis of
the sequence neighboring the BamHI site downstream of
rubA, we subcloned the 1.7-kbp
EcoRV-BamHI fragment from pWH891 into pBluescript II SK+, cut with EcoRV, resulting in plasmid pWH891SK5i.
Sequence analysis revealed the 3'-terminal part of an open reading
frame (ORF) with homology to genes encoding polyphosphate kinases
(ppk) (Fig. 1). For cloning of the complete ppk
gene by a chromosome walking strategy, we used strain WH386. WH386 is a
derivative of ADP1 in which a 736-bp AflII-NdeI
fragment is replaced on the chromosome by a 4.73-kbp
lacZ-Kmr cassette from pKOK6.1 (Fig. 1). Genomic
DNA from WH386 was digested with PvuII and ligated with
pBluescript II SK+, linearized with EcoRV. The ligation
mixture was transformed to E. coli DH5, and clones were
selected on LB plates with kanamycin. The resulting plasmid was called
pWH969. It contains a 6.5-kbp PvuII insert consisting of
4.85 kbp of genomic ADP1 DNA and 1.65 kbp from pKOK6.1 including the
Kmr gene (Fig. 1). The nucleotide sequence of this insert
from BamHI to PvuII was determined.
AvaII restriction sites deduced from the sequence (Fig. 1)
were used in Southern hybridization. A 3.5-kbp AvaII
fragment hybridized to the probe in chromosomal DNA and pWH969,
indicating that the insert of pWH969 corresponds to a contiguous part
of ADP1 genomic DNA.
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).
For WH435 and WH386, the lacZ-Kmr cassette from
pKOK6.1 is inserted on the chromosome into the XcmI and
AflII-NdeI sites, respectively. Abbreviations:
Af, AflII; Av, AvaII; B, BamHI; E,
EcoRV; N, NdeI; P, PvuII; X,
XcmI; lacZ, gene encoding Sequence analysis of pWH969. The part of pWH969 from NdeI to PvuII (Fig. 1) consists of 4,872 bp with 40% GC, which is typical for Acinetobacter (15). Sequence analysis revealed the complete ORF encoding a putative polyphosphate kinase (ppk) preceded by three ORFs (mtgA, ORF2, and ORF3 [partial]) (Fig. 1). Stem-loop structures which may act as transcriptional termination sequences were found downstream of mtgA and ppk (Fig. 1; see Fig. 5B). The codon frequency in each ORF reflects the codon usage of Acinetobacter (41).
ORF2 and ORF3 show no striking correspondence to any protein of known function. The protein encoded by mtgA (224 amino acids; 26.7 kDa) is homologous to monofunctional biosynthetic peptidoglycan transglycosylases from Klebsiella pneumoniae (42% identical amino acids; GenBank accession no. Z54198), Neisseria gonorrhoeae (41% identity; GenBank accession no. U82700), Haemophilus influenzae (41% identity; SwissProt accession no. P44890), and E. coli (39% identity; SwissProt accession no. P46022). The members of this class of proteins have high similarity to the N-terminal transglycosylase domain of class A high-Mr penicillin-binding proteins (12), lacking the C-terminal transpeptidase. They are characterized by at least two positively charged amino acids near the N terminus followed by a stretch of about 20 hydrophobic amino acids but no AlaXAla site for cleavage by leader peptidase. Although no biochemical data are available, monofunctional biosynthetic peptidoglycan transglycosylases are believed to be anchored in the cytoplasmic membrane by a noncleaved signal sequence like class A high-Mr penicillin-binding proteins and to have a specific function in peptidoglycan biosynthesis (37). The ppk gene of Acinetobacter has the capacity to encode a protein of 691 amino acids with a deduced molecular size of 78.6 kDa. The sequence of the putative gene product has high similarity to those of the Ppk proteins from Klebsiella aerogenes and E. coli, which have been characterized biochemically, and to six other putative Ppk proteins. No significant matches of the ppk sequence were found in the complete genomes of Mycoplasma pneumoniae, Mycoplasma genitalium, Saccharomyces cerevisiae, and H. influenzae. The entire sequences of the Ppk variants are homologous (Fig. 2). Highly conserved regions are located between amino acids 20 and 69 (21 of 50 amino acids strictly conserved), 367 and 468 (26 of 102 strictly conserved), and 556 and 630 (25 of 75 strictly conserved). Two histidine residues necessary for ppk activity in E. coli (His442 and His461 in the Acinetobacter sequence) are strictly conserved throughout the family (Fig. 2) (25). Therefore, we suppose that ppk encodes a functional Ppk of Acinetobacter sp. strain ADP1.
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Chromosomal disruption of ppk. We inactivated the ppk gene on the chromosome by insertion of the lacZ-Kmr cassette from pKOK6.1, creating a single-copy transcriptional ppk::lacZ fusion. The promoterless lacZ gene is preceded by stop codons in all reading frames (27). pWH891SK5i was cut with XcmI, and the lacZ-Kmr cassette was excised from pKOK6.1 with BamHI. The protruding ends of both fragments were filled in with Klenow polymerase and ligated. The resulting plasmid, called pWH891SK5ippk::lacZ was cut with ApaI and SacII within the vector sequence to prevent integration of the circular plasmid, and the linear DNA was transformed into Acinetobacter sp. strain ADP1. Transformants were selected on LB plates with kanamycin (10 mg/liter), and integration of the cassette was confirmed by Southern hybridization (data not shown). The chromosomal organization of the resulting strain, called WH435, is shown in Fig. 1. No difference in growth rate between the wild type and the ppk mutant strain was found in LB or minimal medium under phosphate starvation or excess conditions (data not shown). Tolerance of Cd2+ ions was not affected by inactivation of ppk because WH435 and ADP1 tolerated 1 mM CdCl2, whereas 3 mM CdCl2 inhibited growth in the presence of 100 µM phosphate (data not shown). These results show that polyphosphate kinase is not essential in Acinetobacter sp. strain ADP1 and that it is not involved in heavy metal tolerance.
Induction of ppk transcription by phosphate
starvation.
We used the single-copy
ppk::lacZ fusion in WH435 to examine
the regulation of ppk transcription by phosphate. The growth curves and lacZ expression data are shown in Fig.
3. There was no difference between the
growth of WH435 and that of the wild type (data not shown), which grew
to OD600s of 1.8, 1.2, and 0.3 in minimal medium with 1 mM,
100 µM, and 10 µM phosphate, respectively. This shows that a
phosphate concentration of 100 µM limits growth under these
conditions. With 1 mM phosphate, lacZ is expressed in WH435
at a nearly constant low level, ranging between 9 ± 0.4 and
36 ± 0.6 Miller units (mean ± standard deviation). In
contrast, lacZ expression increased to 208 ± 6 Miller
units when strain WH435 was grown in the presence of 10 µM phosphate,
indicating a 5- to 15-fold induction from the 1 mM phosphate level. The
low level of expression in cells grown with 1 mM phosphate, which is
the same over the entire growth curve, clearly shows that expression is
not induced in stationary phase but depends specifically on phosphate
starvation. The graph of lacZ expression in cells grown in
medium with 100 µM phosphate shows a biphasic curve, with a plateau
at 150 Miller units at 6 to 9 h after inoculation and then an
increase to 373 ± 6 Miller units in stationary phase. The first
increase coincides with growth retardation and reaches the level also
observed for cells grown in the presence of 10 µM phosphate (Fig. 3).
Therefore, this increase may reflect the induction by phosphate
starvation as a consequence of phosphate consumption during exponential
growth. The second increase results in doubling of the
-galactosidase activity, which may be due to accumulation of
-galactosidase during stationary phase.
|
and 
), 100 µM (
and 
and 
) phosphate
are shown. The means and standard deviations of three independently
grown cultures are shown. Growth of ADP1 was identical to that WH435,
but no ppk constitutes a separate transcription unit.
A
putative transcriptional terminator sequence
(5'-AAAAAAGGAGTCTTTAAAGACTCCTTTTTT-3') is found downstream
of the ppk gene. Therefore, the size of the ppk
transcript should indicate whether ppk is a single
transcription unit or if it is cotranscribed in an operon together with
the preceding mtgA gene (Fig. 1). The ppk-specific probe was hybridized to 10 µg of RNA from
ADP1 cultures (Fig. 3) and to 10 µg of RNA isolated from ADP1 growing
exponentially in LB medium. A ppk-specific mRNA of 2.2 kb
was detected only in RNA from cells grown under limiting phosphate
conditions (Fig. 4). This confirms
regulation of ppk expression by phosphate in the medium, as
was also seen in the -galactosidase assays (Fig. 3). The size of the
ppk mRNA corresponds well with the size of the
ppk gene (2,073 bp), indicating the presence of a promoter in front of the gene.
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ppk is transcribed from two promoters.
The start
point of ppk transcription was determined by primer
extension with a primer hybridizing within the coding region of
ppk. The same RNA preparation was used for Northern blot
analysis (see above) and primer extension. Two major products were
detected (P1 and P2) (Fig. 5). The P1
band intensity is independent of the phosphate concentration in the
culture medium. The P2 signal, however, is visible only when cells are
grown under limiting phosphate conditions and disappears when an excess
of phosphate is present. This reconfirms the transcriptional regulation
of ppk by phosphate limitation. The deduced start points of
transcription corresponding to P1 and P2 (Fig. 5, sequence
interpretation) are preceded by potential 10 and 35 promoter
sequences with four and five mismatches to the E. coli
consensus sequence, respectively (14, 26). For nucleotides
up to 300 bp upstream of P1 and P2, no sequences displaying similarity
to 
54 promoters or pho boxes were found.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We cloned the ppk gene from Acinetobacter sp. strain ADP1 that encodes a putative protein with high similarity to the polyphosphate kinase (Ppk) of E. coli and homologous gene products of various gram-negative bacteria and Mycobacterium tuberculosis. We demonstrate that transcription of ppk is induced by phosphate starvation. This suggests an involvement of ppk in phosphorus metabolism.
In contrast to E. coli (2) and K. aerogenes (18), ppk in Acinetobacter sp. strain ADP1 is not followed by a ppx gene encoding an exopolyphosphatase. This is also the case for all the other bacteria containing a ppk gene, indicating that the organization of ppk and ppx in an operon is the exception found in E. coli and closely related organisms. Polyphosphate, on the other hand, is widely distributed in bacteria, fungi, protozoa, plants, and mammals (23, 24). However, no ppk homologous gene is found in S. cerevisiae, suggesting that polyphosphate synthesis in eucaryotes may require enzymes which are not related to the eubacterial Ppk. No ppk-homologous gene is found in the complete genomes of M. genitalium and M. pneumoniae, which may constitute exceptions because of their small size. H. influenzae does not contain ppk either, but a gene with high similarity to E. coli ppx is present (SwissProt accession no. P44828).
We inactivated ppk in Acinetobacter sp. strain ADP1 by insertion of a lacZ-Kmr cassette, disrupting the amino acid sequence between the two His residues necessary for function in E. coli (Fig. 2). This shows that the ppk gene is not essential in Acinetobacter. The ppk mutant has no growth defect in complex or minimal media. In E. coli, tolerance to Cd2+ ions had been increased in a ppk ppx mutant by overexpression of ppk and ppx from a plasmid compared to the mutant overexpressing only ppk, indicating that polyphosphate turnover is required for heavy-metal resistance (20). A. calcoaceticus WH435 (ppk::lacZ) exhibits no increase in sensitivity to heavy-metal ions compared to that of the wild type. Currently, we have no experimental indications of the function of the putative Ppk in Acinetobacter sp. strain ADP1. However, considering the inducibility of ppk transcription by phosphate starvation, it seems unlikely that Ppk forms polyphosphate serving as phosphate storage in Acinetobacter. It may be speculated that Ppk provides phosphate by degradation of polyphosphate under conditions of phosphate limitation.
Transcription of ppk in
Acinetobacter sp. strain ADP1 is induced
5- to 15-fold by phosphate starvation. Regulation by phosphate limitation is well known in E. coli, where PhoR, the
sensorkinase, phosphorylates PhoB upon phosphate starvation.
Phospho-PhoB, in turn, binds to the pho box
[consensus sequence, CTGTCATA(T/A)A(T/A)CTGT(A/C)A(C/T)], which
typically overlaps with the 35 promoter region, and induces transcription of about 30 genes of the pho regulon
(40). There are indications that ppk in K. aerogenes is regulated by the phoBR system because
expression of a ppk::lacZ fusion is
induced 10-fold under phosphate starvation in the heterologous E. coli host but not in an E. coli phoB mutant
(18). However, the significance of this observation was
called into question because of the low-level induction (threefold)
detected in K. aerogenes, the use of multicopy reporter
constructs in E. coli, and the experimental setup leading to
phosphate limitation (40).
No phoB or phoR genes have been cloned from any
Acinetobacter strain to date. However, it seems
probable that Acinetobacter contains genes with
homology to phoBR. First, a promoter of the polyhydroxyalkanoic acid biosynthetic genes from
Acinetobacter strain RA3849 was shown to be
induced by phosphate starvation. Putative pho box sequences
were found upstream of the 10 region, and heterologous regulation
occurs in wild-type E. coli but not in an E. coli
phoB mutant (35). Second, two phosphate uptake systems from Acinetobacter johnsonii 210A were
characterized biochemically (39): a constitutive,
low-affinity system and a high-affinity, phosphate-binding-protein-dependent system. The latter is induced by
phosphate starvation. This resembles the situation in E. coli, where the high-affinity system is induced by
phoBR (31). Taken together, these data suggest
that phoBR-like genes probably exist in
Acinetobacter and therefore PhoB may be a good
candidate for mediating transcriptional induction of ppk in
Acinetobacter sp. strain ADP1. We did not find a
pho box-like sequence in front of ppk, indicating
either that the putative Acinetobacter PhoB must
differ from E. coli PhoB in DNA recognition or that a
regulator without homology to PhoB may be involved.
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ACKNOWLEDGMENT |
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This work was supported by the Fonds der Chemischen Industrie.
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FOOTNOTES |
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* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany. Phone: 49 (9131) 858081. Fax: 49 (9131) 858082. E-mail: whillen{at}biologie.uni-erlangen.de.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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