Previous Article | Next Article 
Journal of Bacteriology, March 2001, p. 1784-1786, Vol. 183, No. 5
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.5.1784-1786.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The apeE Gene of Salmonella
enterica Serovar Typhimurium Is Induced by Phosphate Limitation
and Regulated by phoBR
Christopher A.
Conlin,*
Seng L.
Tan,
Huajun
Hu, and
Todd
Segar
Department of Biological Sciences, Minnesota
State University, Mankato, Minnesota 56001
Received 23 March 2000/Accepted 30 November 2000
 |
ABSTRACT |
Mutations in apeR, a regulatory locus of the outer
membrane esterase apeE from Salmonella enterica
serovar Typhimurium, were shown to be alleles of the
pstSCAB-phoU high-affinity phosphate transport operon.
Expression of apeE was induced by phosphate limitation, and
this induction required the phoBR phosphate regulatory system.
| |
TEXT |
The apeE gene of
Salmonella enterica serovar Typhimurium encodes an outer
membrane esterase (3, 4). The gene has been sequenced
(3) and is similar to LipI, a secreted lipase from the
insect pathogen Photorhabdus luminescens (15),
and to EstA an outer membrane esterase from Pseudomonas
aeruginosa (17). However, Southern analysis and
sequence comparisons have shown that apeE is not present in
Escherichia coli (3). In the initial identification of apeE Collin-Osdoby and Miller
(4) isolated mutations in an unlinked locus that resulted
in a 60-fold increase in apeE transcription. They named this
regulatory locus apeR and suggested that it was a gene for a
transcriptional repressor of apeE. No conditions were
identified which affected apeE expression (4).
In this study we cloned the apeR gene and showed that
apeR mutations are alleles of the pstSCAB-phoU
operon that encodes a high-affinity phosphate transport system. We also
showed that apeE expression is induced by phosphate
limitation and that this induction is dependent on the phoBR
regulatory system.
Cloning and identification of apeR.
In an
apeR wild-type background an
apeE::lacZYA fusion makes about two
Miller units of 
-galactosidase and consequently forms white colonies
on MacConkey agar. The apeR1 mutation increases this to 225 U, and the colonies are red. To clone the apeR gene, the
original MudI fusion in apeE was first replaced
with a more stable MudI-1734 insertion (strain CAC13).
Plasmid libraries containing random 8- to 12-kb fragments of
Salmonella chromosomal DNA in the vector pBR328
(7) were screened for plasmids that complemented the
MacConkey phenotype of an apeR1 mutant. Three different
plasmids were isolated that complemented both the apeR1 and
apeR47::Tn5 mutations.
To demonstrate that these plasmids contained apeR, they were
integrated into the chromosome of a polA strain by
recombination between the cloned DNA on the plasmid and its homologous
DNA on the Salmonella chromosome (6).
Antibiotic resistance on the integrated plasmid was 82 to 84% phage
P22 cotransducible with insertion
zic868::Tn10, which had previously been
shown to be 78% cotransducible with apeR1. This
demonstrated that the cloned DNA was from the apeR region of
the Salmonella chromosome.
Restriction enzyme analysis indicated that the three plasmids shared a
4.5-kb fragment (Fig.
1). Subcloning
showed that DNA
on both sides of an
EcoRI site in the middle
of this region was
required for complementation. Four Tn
1000
insertions were isolated
in the insert region of plasmid pAPR3. Two of
these, located 3
kb apart, eliminated complementation. DNA sequence was
obtained
from the right end of the insert in pAPR3. This sequence was
93%
identical to the last 261 bases of the
pstB gene and
the first
73 bases of the
phoU gene in the
E. coli
pstSCAB-phoU operon.
Alignment of the
E. coli sequence
with the restriction map and
the Tn
1000 insertions indicated
that the
apeR1 mutation must be
an allele of
pstC,
pstA, or
pstB. This is consistent with the
equivalent
map positions of
apeR on the
Salmonella
chromosome
(
11) and
pstSCAB-phoU operon
on the
E. coli chromosome (
1).
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 1.
apeR complementing plasmids. Bars indicate
chromosomal DNA contained on the plasmids. Restriction enzyme sites are
indicated as follows: H, HindIII; P, PvuII;
R, EcoRI; and S, SalI. Triangles indicate
Tn1000 insertions: closed-triangle insertions eliminated
complementation, open-triangle insertions did not. The E. coli
pstSCAB-phoU operon is aligned with the restriction map based on
DNA sequence determined from the right end of plasmid pAPR3.
|
|
Regulation of apeE by phosphate limitation and
phoBR.
The pstSCAB-phoU operon encodes a
high-affinity phosphate transport system. Mutations in the
pst operon often result in constitutive expression of genes
that are induced by phosphate limitation. This is particularly true of
members of the PhoBR regulon, such as the E. coli phoA gene
(16). This led us to examine the effect of phosphate
limitation on apeE induction. Strain CAC13, containing an
apeE::lacZYA fusion, was grown in
minimal morpholinepropanesulfonic acid medium with limiting phosphate
(0.1 mM) or excess phosphate (2 mM) as previously described
(8), and -galactosidase activity was measured
(10). Limiting phosphate induced apeE
expression 140-fold, from 1.5 to 210 U of -galactosidase (Table
1), explaining the effect of the
apeR (pst) mutation.
To determine whether
apeE is regulated by one of the known
phosphate limitation regulatory genes, we used phage P22 transduction
to construct isogenic strains that contained both the
apeE::
lacZYA fusion and mutations in
either
ntrA (rpoN), phoP, katF (rpoS),
or
phoBR.
-Galactosidase activity was measured for these strains
in both
limiting and excess phosphate. As seen in Table
1, only
the
phoBR deletion significantly affected phosphate induction,
completely eliminating induction by phosphate limitation, as well
as
the effect of the
apeR mutation. This indicated that
apeE is
a previously uncharacterized member of the
phoBR regulon.
Analysis of the apeE promoter region.
The
promoters of genes that are regulated by PhoB contain a PhoB binding
site (PHO box) in the 
35 region of the promoter. A consensus PHO box
consists of two 7-bp direct repeats separated by a 4-bp AT spacer
(16). Although there is no obvious consensus PHO box
upstream of the apeE coding region, by using a
less-stringent definition of the PHO box based on mutational analysis
of the PhoB binding site by Makino et al. (9) and
examination of other phoBR regulated genes
(14), we have identified three potential half PHO boxes
located 19 bp upstream of a likely 10 sequence (GATAAT)
(Fig. 2).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 2.
Phosphate regulation of apeE promoter
subclones. Plasmid strains were grown on 2 mM (solid bars) or 0.1 mM
(open bars) phosphate, and -galactosidase activity was assayed
(10). Assays were done in triplicate, and each strain was
assayed three times. The 5' ends of the subclones are shown below the
graph. Single underlines indicate PHO box half-sites, and the double
underline indicates a potential 10 region.
|
|
To begin identifying regions of the
apeE promoter that are
required for phosphate control, we performed deletion analysis
of the
promoter region. We used the PCR to generate fragments
with a constant
3' end 1413 nucleotides downstream of the start
of translation and
various 5' ends as indicated in Fig.
2. These
fragments were inserted
into the promoter detection vector pRS415
(
13), where they
control the expression of a promoter-less
lac operon.
Plasmid-containing strains of
S. enterica strain TN1379
were
grown in either 2 or 0.1 mM phosphate, and the
-galactosidase
activity was determined. As shown in Fig.
2, successive removal
of
potential PHO box half-sites reduced phosphate induction. Subclone
B,
which included all three proposed PHO box half sites, was induced
27-fold by phosphate limitation. Subclone D, which contained two
half-sites, was induced 19-fold, and subclone F, with one complete
half-site, was induced only 8.6-fold. Subclones C, G, and E containing
no intact half-sites were essentially uninducible. These results
support the identification of the putative PHO boxes in the
apeE promoter region. Further analysis, such as
site-directed mutagenesis
and DNA footprinting with PhoB protein, will
be needed to confirm
this. The differences in induction ratios between
the plasmids
and the chromosomal Mu
dI-1734 insertion were
probably caused by
the increased copy number of the plasmids and
cryptic promoter
activity in the approximately 600 bp between the
Mu
dI-1734 insertion
site and the 3' end of the
subclones.
Possible function of ApeE.
As mentioned above, apeE
is not present in E. coli, but a search of preliminary
genomic sequence data showed that it is clearly present in the S. enterica serovars Typhi and Paratyphi A, as well as S. enterica serovar Typhimurium. Although this suggests that
apeE could be involved in Salmonella virulence,
it has not been identified in screens for Salmonella
virulence factors. This is consistent with the report by Jiang et al.
that a phoBR deletion itself failed to attenuate virulence
(8), and therefore it is unlikely that PhoBR-regulated
genes would either.
We have previously reported that
apeE is required for the
utilization of the model lipid substrate Tween 80 (polyoxyethylene
sorbitan monooleate) and for the hydrolysis of methylumbelliferyl
caprylate (
3). Combined with the phosphate regulation
data,
this suggests that
apeE could play an important role
in the use
of phospholipids as phosphate sources. The products of ApeE
deacylation
of phospholipids would be either
sn-glycerol-3-phosphate or glycerophosphoryl
diesters. In
both
E. coli and serovar Typhimurium these organic
phosphate
sources are transported across the inner membrane by
the PhoB-dependent
Ugp transport system and so can be used as
sole phosphate sources
(
2,
5,
12). Whereas
E. coli, which
lacks ApeE,
uses PhoA to remove the phosphate from the phospholipid,
Salmonella spp., which lack PhoA but have ApeE, would
not need
PhoA to use phospholipids as phosphate sources since the
deacylated
products would be transported by the Ugp system. Additional
studies
are needed to confirm this
hypothesis.
| |
ACKNOWLEDGMENTS |
This work was supported by Public Health Service grant GM52697 from
the National Institutes of Health and a Faculty Research Grant from
Minnesota State University, Mankato, to C.A.C.
We thank C. G. Miller and B. L. Wanner for providing strains
and the Genome Sequencing Center, Washington University, St. Louis,
Mo., and the Sanger Center for communication of DNA sequence data prior
to publication.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Minnesota State University, Mankato, 242 Trafton Science Center S., Mankato, MN 56001. Phone: (507) 389-5737. Fax: (507)
389-2788. E-mail: christopher.conlin{at}mnsu.edu.
| |
REFERENCES |
| 1.
|
Berlyn, M. K. B.,
K. B. Low,
K. E. Rudd, and M. Singer.
1996.
Linkage map of Escherichia coli K-12, ed. 9, p. 1715-1902.
In
F. C. Neidhardt, R. I. Curtiss III, C. A. Gross, J. L. Ingraham, E. C. C. Lin, K. B. Low, Jr., B. Magasanik, W. Reznikoff, M. Schaechter, H. E. Umbarger, and M. Riley (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C.
|
| 2.
|
Brzoska, P., and W. Boos.
1988.
Characterization of a ugp-encoded and phoB-dependent glycerophosphoryl diester phosphodiesterase which is physically dependent on the ugp transport system of Escherichia coli.
J. Bacteriol.
170:4125-4235[Abstract/Free Full Text].
|
| 3.
|
Carinato, M. E.,
P. Collin-Osdoby,
X. Yang,
T. M. Knox,
C. A. Conlin, and C. G. Miller.
1998.
The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli.
J. Bacteriol.
180:3517-3521[Abstract/Free Full Text].
|
| 4.
|
Collin-Osdoby, P., and C. G. Miller.
1994.
Mutations affecting a regulated, membrane-associated esterase in Salmonella typhimurium LT2.
Mol. Gen. Genet.
243:674-680[Medline].
|
| 5.
|
Hengge, R.,
T. J. Larson, and W. Boos.
1983.
sn-Glycerol-3-phosphate transport in Salmonella typhimurium.
J. Bacteriol.
155:186-195[Abstract/Free Full Text].
|
| 6.
|
Hmiel, S. P.,
M. D. Snavely,
J. B. Florer,
M. E. Maguire, and C. G. Miller.
1989.
Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci.
J. Bacteriol.
171:4742-4751[Abstract/Free Full Text].
|
| 7.
|
Hmiel, S. P.,
M. D. Snavely,
C. G. Miller, and M. E. Maguire.
1986.
Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene.
J. Bacteriol.
168:1444-1450[Abstract/Free Full Text].
|
| 8.
|
Jiang, W.,
W. W. Metcalf,
K.-S. Lee, and B. L. Wanner.
1995.
Molecular cloning, mapping, and regulation of pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2.
J. Bacteriol.
177:6411-6421[Abstract/Free Full Text].
|
| 9.
|
Makino, K.,
M. Amemura,
T. Kawamoto,
S. Kimura,
H. Shinagawa,
A. Nakata, and M. Suzuki.
1996.
DNA binding of PhoB and its interaction with RNA polymerase.
J. Mol. Biol.
259:15-26[CrossRef][Medline].
|
| 10.
|
Miller, J. H. (ed.).
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 11.
|
Sanderson, K. E.,
A. Hessel,
S.-L. Liu, and K. Rudd.
1996.
The genetic map of Salmonella typhimurium, ed. VIII, p. 1903-1999.
In
F. C. Neidhardt, R. I. Curtiss III, C. A. Gross, J. L. Ingraham, E. C. C. Lin, K. B. Low, Jr., B. Magasanik, W. Reznikoff, M. Schaechter, H. E. Umbarger, and M. Riley (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C.
|
| 12.
|
Schweizer, H., and W. Boo.
1985.
Regulation of ugp, the sn-glycerol-3-phosphate transport system of Escherichia coli K-12 that is part of the pho regulon.
J. Bacteriol.
163:392-394[Abstract/Free Full Text].
|
| 13.
|
Simons, R. W.,
F. Houman, and N. Kleckner.
1987.
Improved single and multicopy lac-based cloning vectors for protein and operon fusions.
Gene
53:85-96[CrossRef][Medline].
|
| 14.
|
VanBogelen, R. A.,
E. R. Olson,
B. L. Wanner, and F. C. Neidhardt.
1996.
Global analysis of proteins synthesized during phosphorous restriction in Escherichia coli.
J. Bacteriol.
178:4344-4366[Abstract/Free Full Text].
|
| 15.
|
Wang, H., and B. C. A. Dowds.
1993.
Phase variation in Xenorhabdus luminescens: cloning and sequencing of the lipase gene and analysis of its expression in primary and secondary phases of the bacterium.
J. Bacteriol.
175:1665-1673[Abstract/Free Full Text].
|
| 16.
|
Wanner, B. L.
1996.
Phosphorous assimilation and control of the phosphate regulon, p. 1357-1381.
In
F. C. Neidhardt, R. I. Curtiss III, C. A. Gross, J. L. Ingraham, E. C. C. Lin, K. B. Low, Jr., B. Magasanik, W. Reznikoff, M. Schaechter, H. E. Umbarger, and M. Riley (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C.
|
| 17.
|
Wilhelm, S.,
J. Tommassen, and K.-E. Jaeger.
1999.
A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa.
J. Bacteriol.
181:6977-6986[Abstract/Free Full Text].
|
Journal of Bacteriology, March 2001, p. 1784-1786, Vol. 183, No. 5
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.5.1784-1786.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Henderson, I. R., Navarro-Garcia, F., Desvaux, M., Fernandez, R. C., Ala'Aldeen, D.
(2004). Type V Protein Secretion Pathway: the Autotransporter Story. Microbiol. Mol. Biol. Rev.
68: 692-744
[Abstract]
[Full Text]
Top
Abstract
Text
