Previous Article | Next Article 
Journal of Bacteriology, October 1998, p. 5231-5234, Vol. 180, No. 19
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cell Division Inhibition in Salmonella typhimurium
Histidine-Constitutive Strains: an ftsI-Like Defect in the
Presence of Wild-Type Penicillin-Binding Protein 3 Levels
David A.
Cano,1
Chakib
Mouslim,1
Juan A.
Ayala,2
Francisco
García-del Portillo,2 and
Josep
Casadesús1,*
Departamento de Genética, Facultad de
Biología, Universidad de Sevilla, Seville
41080,1 and
Centro de Biología
Molecular Severo Ochoa, CSIC-Universidad Autónoma de Madrid,
Cantoblanco, Madrid 28049,2 Spain
Received 6 April 1998/Accepted 11 July 1998
 |
ABSTRACT |
Histidine-constitutive (Hisc) strains of
Salmonella typhimurium undergo cell division inhibition in
the presence of high concentrations of a metabolizable carbon source.
Filaments formed by Hisc strains show constrictions and
contain evenly spaced nucleoids, suggesting a defect in septum
formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a
filamentation pattern identical to that of Hisc strains.
However, the Hisc septation defect is caused neither by
reduced PBP3 synthesis nor by reduced PBP3 activity. Gross
modifications of peptidoglycan composition are also ruled out.
D-Cycloserine, an inhibitor of the soluble pathway
producing peptidoglycan precursors, causes phenotypic suppression of
filamentation, suggesting that the septation defect of Hisc
strains may be caused by scarcity of PBP3 substrate.
| |
TEXT |
When histidine-constitutive
(Hisc) mutants of Salmonella typhimurium were
first isolated, the authors noted that high levels of histidine
biosynthetic enzymes caused wrinkled colony morphology on 2% glucose
plates (25). Wrinkledness reflects cell filamentation (12, 19), which is triggered by overproduction of
hisH and hisF gene products (5, 9,
19). A similar response has been described for Escherichia
coli (11). HisH and HisF are subunits of the
heterodimeric imidazole-glycerol-phosphate synthase (1, 34),
which catalyzes the formation of imidazole-glycerol-phosphate (IGP)
with release of the purine precursor AICAR
(5-aminoimidazole-4-carboxamide ribonucleotide) (17, 26).
However, division inhibition does not require metabolic flow through
the histidine biosynthetic pathway, suggesting that HisH and HisF
trigger filamentation through an activity unrelated to IGP synthesis
(10, 19). The involvement of AICAR has been also ruled out
(10, 11). In both S. typhimurium and E. coli, the cell division defect of Hisc strains is
unrelated to the SOS response and does not involve the cell division
inhibitor SulA (11, 12). We show below that the cell
division defect of S. typhimurium Hisc strains
is a block in septum formation, as proposed by Frandsen and D'Ari
(11). We also describe the unexpected finding that strains
that overproduce IGP synthase contain wild-type levels of active
penicillin-binding protein 3 (PBP3). These contradictory data are
tentatively reconciled by the ability of D-cycloserine to
suppress filamentation in Hisc mutants. The latter
observation suggests that HisHF overproduction may cause a
shortage in PBP3 substrate.
Filament formation by Hisc strains.
Mid-exponential-phase cultures of strains LT2
(hisO+) and TR6753 (hisO1242
[5, 16]) were observed under the microscope by using
Hiraga's fluo-phase combined method, a procedure that permits the
simultaneous observation of nucleoids and cells (15). Nucleoid staining was achieved with DAPI (4',6-diamino-2-phenylindole). The Hisc strain formed long filaments which contained
evenly spaced nucleoids, indicating that their division defect is
unrelated to DNA synthesis or chromosome partition (Fig.
1). Moreover, the presence of blunt constrictions indicates that the division block lies beyond the stage
of FtsZ action (8). The filaments are similar in morphology and length to those formed by ftsI and ftsA
mutants of E. coli (8). The ftsI gene
encodes PBP3, an essential cell division protein involved in septum
formation (27). FtsA is a membrane-bound protein that
interacts with PBP3 (22, 31). A difference is that
ftsI and ftsA mutants are conditional
(thermosensitive) lethals unable to form colonies under restrictive
conditions (3, 18), while the filaments produced by
Hisc strains of S. typhimurium in the presence
of 2% glucose give rise to colonies which are distinctly wrinkled
(9, 19, 25).
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 1.
Microscopic photographs of cells and filaments prepared
with Hiraga's fluo-phase combined method (15). (A)
Filaments formed by the Hisc strain TR6753 grown in E
medium containing 2% glucose. (B) An isogenic His+ strain
(LT2) grown under the same conditions does not form filaments. (C)
Addition of aztreonam to a culture of strain LT2 in E medium induces
filaments identical to those formed by a Hisc strain in
high-glucose medium (compare panels A and C). Bar, 5 µm.
|
|
Antibiotics that inhibit PBP3, such as aztreonam and azlocillin,
produce phenocopies of ftsI mutants in E. coli
(29). Based on this precedent, we investigated whether PBP3
inhibitors were able to reproduce the filamentation phenotype of
Hisc strains. Addition of aztreonam (1 mg/liter) to a
culture of strain LT2 triggered filament formation (Fig. 1C), and the
filaments were identical in morphology and length to those formed by
the Hisc strain TR6753 (Fig. 1A). The same effect was
induced by azlocillin (data not shown). At the concentrations used,
these antibiotics inhibit specifically PBP3 (21). These
observations suggest that Hisc strains of S. typhimurium behave as ftsI mutants.
Additional evidence against an FtsA-like defect was provided by the
failure of a plasmid carrying the
E. coli ftsA gene to
relieve cell division inhibition when introduced in strain TR6753.
If
His
c strains were
ftsA-like, a plasmid-borne
ftsA gene should restore
the FtsA/FtsZ ratio (
6),
thereby causing a certain degree of
suppression. However, the actual
result was that pMFV26, an
ftsA+ plasmid
provided by Miguel Vicente (CIB-CSIC, Madrid, Spain),
failed to relieve
septation inhibition. The conclusion that His
c strains
behave as
ftsI (rather than
ftsA) mutants
receives further
support from the ability of
D-cycloserine
to suppress filamentation
(see below).
HisHF overproduction does not cause reduced synthesis of PBP3.
The levels of PBP3 produced by HisO+ and HisOc
strains were compared by using envelope extracts from
mid-exponential-phase cultures (optical density at 600 nm, 0.5 to 0.6)
in E medium containing 2% glucose (33). Envelope proteins were
fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (8% acrylamide) and detected by immunoblotting against
a polyclonal anti-PBP3 serum (21). The levels of PBP3
protein are similar in HisO+ and HisOc strains
(Fig. 2); densitometric analysis (not
shown) confirmed the absence of differences. Thus, the cell division
defect associated with IGP synthase overproduction is not caused by
reduced synthesis of PBP3. An additional observation is that the
electrophoretic mobilities of PBP3 are similar in HisO+ and
HisOc strains, thereby eliminating the occurrence of
gross structural changes in the protein.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 2.
Immunodetection of PBP3 from the wild type (lanes A and
C) and the Hisc strain TR6753 (lanes B and D). Lanes A and
B contain membrane vesicles corresponding to 80 µg of protein in a
final reaction volume of 20 µl. Lanes C and D contain membrane
vesicles corresponding to 120 µg of protein in a final volume of 20 µl.
|
|
HisHF overproduction does not cause reduced activity of PBP3.
As an indicator of the functionality of PBP3 in membrane extracts from
HisO+ and HisOc strains, we analyzed their
capacities to bind covalently 3H-labelled benzylpenicillin
(2). Cultures were prepared as described above. Protein
separation was performed on an SDS-polyacrylamide gel (2).
Radioactivity was detected by fluorography (28, 30). Protein
quantitation was performed by the method of Bradford (4). No
differences in penicillin binding were found (Fig. 3 and densitometric data not shown).
A side observation is that the remaining high-molecular-weight
PBPs were also unaffected (Fig. 3).
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 3.
Binding of 3H-labelled benzylpenicillin to
cell envelopes from exponentially growing cells of the His+
strain LT2 (lane I) and the Hisc strain TR6753 (lane II).
PBPs are numbered on the right by standard nomenclature
(27).
|
|
Peptidoglycan composition of Hisc strains.
To
investigate the possibility that Hisc strains might
synthesize an abnormal cell wall, we prepared peptidoglycan extracts from strains LT2 and TR6753 grown in E medium containing 2% glucose. Concentrated exponential cultures containing approximately
1011 bacterial cells were cooled to 4°C, centrifuged at
12,000 × g for 15 min, and resuspended in 3 ml of
phosphate-buffered saline, pH 7.4. The suspension was mixed 1:1
(vol/vol) with a boiling solution of 8% SDS (24). The
SDS-insoluble material was washed in distilled water (13).
Peptidoglycan was digested with Cellosyl muramidase (20 µg/ml)
(Hoechst, Sommerville, N.J.); this treatment yields muropeptides of low
molecular weight (23). The reaction was stopped in a boiling
bath for 5 min. Insoluble material was removed by centrifugation
(1,000 × g, 10 min). Peptidoglycan composition was
determined by high-performance liquid chromatography analysis (13, 14). The main conclusions are that (i) abnormal
peptidoglycan muropeptides were not found and (ii) gross differences in
peptidoglycan composition or organization were not found between
HisO+ and HisOc strains (Table
1). These experiments do not rule out the
formation of an abnormal PBP3 substrate but certainly exclude the
possibility that any unusual substrate is incorporated into growing
peptidoglycan chains. This absence of differences does not eliminate
the possibility that Hisc strains may have a defect in the
reaction catalyzed by PBP3; peptidoglycan from strain LT2 treated with
the PBP3 inhibitor aztreonam showed also standard composition (Table
1). In fact, a well-known and surprising feature of cell wall synthesis
is that the global peptidoglycan composition remains unaltered under conditions that cause major changes in cell shape (20).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Muropeptide composition of peptidoglycan from
His+ and Hisc strains and from a
His+ strain treated with aztreonam
|
|
D-Cycloserine causes phenotypic suppression of the cell
division defect of Hisc strains.
To explore the
possibility that the septation defect of Hisc strains might
be caused by lowered levels of PBP3 substrate, we investigated whether
D-cycloserine was able to cause phenotypic suppression of
filamentation in a Hisc mutant. D-Cycloserine
reduces the number of pentapeptide side chains in peptidoglycan and
increases the number of tripeptides (21), an effect caused
by inhibition of D-alanine-D-alanine ligase and
alanine racemase (32). The resulting imbalance increases septation at the expense of elongation, giving rise to rounded cells in
the wild type and restoring cell division in FtsI
mutants
(3).
His
c strains formed wrinkled colonies on both green plates
and E plates containing 2% glucose. In the presence of
D-cycloserine
(20 mg/liter), the colonies formed by the
HisO
c strain TR6753 and those formed by the wild type
were identical:
smooth and small. For microscopic observation of
cultures, strains
LT2 and TR6753 were grown in E medium containing 2%
glucose and
15 mg of
D-cycloserine per liter. Exponential
cultures were transferred
to agar-coated slides and photographed under
phase-contrast optics
(7). Strain LT2 formed rounded cells (data not
shown). Strain
TR6753 underwent nearly complete suppression of
filamentation
and formed rod-shaped cells (Fig.
4). Because these results strongly
resemble those obtained with
ftsI mutants of
E. coli (
3), we
hypothesize that
D-cycloserine
may suppress the septation defect
of His
c strains by
increasing PBP3 substrate. If this view is correct,
the cell division
defect of His
c strains may be tentatively reformulated as a
shortage of tripeptide
side chains, the proposed substrate of the
septal machinery (
21).
View larger version (116K):
[in this window]
[in a new window]
|
FIG. 4.
D-Cycloserine causes phenotypic suppression
of the cell division defect of Hisc strains. (A) Strain
TR6753 grown in E medium contains only filaments. (B) The presence of
D-cycloserine causes suppression of filamentation; strain
TR6753 forms rod-shaped cells. Bar, 10 µm.
|
|
| |
ACKNOWLEDGMENTS |
This study was supported by grant PB93-649 from the Dirección
General de Investigación Científica y Técnica of
the Government of Spain and grant 97-109-96 from the Comunidad de
Madrid.
We thank Miguel Vicente for the gift of pMFV26 and Boris Magasanik,
Dick D'Ari, and Molly Schmid for helpful discussions. The assistance
of Gloria Chacón, Ana Moreno, José Córdoba, and Luis
Romanco is also appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Genética, Facultad de Biología, Universidad de Sevilla,
Apartado 1095, Seville 41080, Spain. Phone: 34 95 455 7105. Fax: 34 95 455 7104. E-mail: genbac{at}cica.es.
| |
REFERENCES |
| 1.
|
Alifano, P.,
R. Fani,
P. Liò,
A. Lazcano,
M. Bazzicalupo,
M. S. Carlomagno, and C. B. Bruni.
1996.
Histidine biosynthetic pathway and genes: structure, regulation, and evolution.
Microbiol. Rev.
60:44-69[Free Full Text].
|
| 2.
|
Ayala, J. A.,
C. Goffin,
M. Nguyen-Distèche, and J. M. Ghuysen.
1994.
Site-directed mutagenesis of penicillin-binding protein 3 of Escherichia coli: role of Val545.
FEMS Microbiol. Lett.
121:251-256[Medline].
|
| 3.
|
Begg, K. J.,
A. Takasuga,
D. H. Edwards,
S. J. Dewar,
B. G. Spratt,
H. Adachi,
T. Ohta,
H. Matsuzawa, and W. D. Donachie.
1990.
The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide.
J. Bacteriol.
172:6697-6703[Abstract/Free Full Text].
|
| 4.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 5.
|
Casadesús, J., and J. R. Roth.
1989.
Absence of insertions among spontaneous mutants of Salmonella typhimurium.
Mol. Gen. Genet.
216:210-216[Medline].
|
| 6.
|
Dai, K., and J. Lutkenhaus.
1992.
The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli.
J. Bacteriol.
174:6145-6151[Abstract/Free Full Text].
|
| 7.
|
Donachie, W. D.,
K. J. Begg, and M. Vicente.
1976.
Cell length, cell growth and cell division.
Nature (London)
264:328-333[Medline].
|
| 8.
|
Donachie, W. D.,
K. J. Begg, and N. F. Sullivan.
1984.
Morphogenes of Escherichia coli, p. 27-62.
In
R. Losick, and L. Shapiro (ed.), Microbial development. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 9.
|
Flores, A., and J. Casadesús.
1995.
Suppression of the pleiotropic effects of HisH and HisF overproduction identifies four novel loci on the Salmonella typhimurium chromosome: osmH, sfiW, sfiX, and sfiY.
J. Bacteriol.
177:4841-4850[Abstract/Free Full Text].
|
| 10.
|
Flores, A.,
M. Fox, and J. Casadesús.
1993.
The pleiotropic effects of Salmonella typhimurium his overexpression do not involve AICAR-induced mutagenesis.
Mol. Gen. Genet.
240:360-364[Medline].
|
| 11.
|
Frandsen, N., and R. D'Ari.
1993.
Excess histidine enzymes cause AICAR-independent filamentation in Escherichia coli.
Mol. Gen. Genet.
240:348-354[Medline].
|
| 12.
|
Gibert, I., and J. Casadesús.
1990.
sulA-independent division inhibition in His-constitutive strains of Salmonella typhimurium.
FEMS Microbiol. Lett.
69:205-210.
|
| 13.
|
Glauner, B., and U. Schwarz.
1983.
The analysis of murein composition with high-pressure-liquid-chromatography, p. 29-34.
In
R. Hackenbeck, J. V. Höltje, and H. Labisnchinski (ed.), The target of penicillin. Walter de Gruyter, Berlin, Germany.
|
| 14.
|
Glauner, B.,
J. V. Höltje, and U. Schwarz.
1988.
The composition of the murein of Escherichia coli.
J. Biol. Chem.
263:10088-10095[Abstract/Free Full Text].
|
| 15.
|
Hiraga, S.,
H. Niki,
T. Ogura,
C. Ichinose,
H. Mori,
B. Ezaki, and A. Jaffé.
1989.
Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells.
J. Bacteriol.
171:1496-1505[Abstract/Free Full Text].
|
| 16.
|
Johnston, H. M., and J. R. Roth.
1981.
Genetic analysis of the histidine operon control region of Salmonella typhimurium.
J. Mol. Biol.
145:715-734.
|
| 17.
|
Klem, T. J., and V. J. Davisson.
1993.
Imidazole glycerol phosphate synthase: the glutamine amidotransferase in histidine biosynthesis.
Biochemistry
32:5177-5186[Medline].
|
| 18.
|
Lutkenhaus, J.
1993.
Escherichia coli cell division.
Curr. Opin. Genet. Dev.
3:783-788[Medline].
|
| 19.
|
Murray, M. L., and P. E. Hartman.
1971.
Overproduction of hisH and hisF gene products leads to inhibition of cell division in Salmonella.
Can. J. Microbiol.
18:671-681.
|
| 20.
|
Nanninga, N.
1991.
Cell division and peptidoglycan assembly in Escherichia coli.
Mol. Microbiol.
5:791-795[Medline].
|
| 21.
|
Pisabarro, A. G.,
R. Prats,
D. Vázquez, and A. Rodríguez-Tébar.
1986.
Activity of penicillin-binding protein 3 from Escherichia coli.
J. Bacteriol.
168:199-206[Abstract/Free Full Text].
|
| 22.
|
Pla, J.,
A. Dopazo, and M. Vicente.
1990.
The native form of FstA, a septal protein of Escherichia coli, is located in the cytoplasmic membrane.
J. Bacteriol.
173:5097-5102.
|
| 23.
|
Quintela, J. C.,
M. Caparrós, and M. A. de Pedro.
1995.
Variability of peptidoglycan structural parameters in Gram-negative bacteria.
FEMS Microbiol. Lett.
125:95-100[Medline].
|
| 24.
|
Quintela, J. C.,
M. A. de Pedro,
P. Zöllner,
G. Allmaier, and F. García-del Portillo.
1997.
Peptidoglycan structure of Salmonella typhimurium growing within cultured mammalian cells.
Mol. Microbiol.
23:693-704[Medline].
|
| 25.
|
Roth, J. R.,
D. N. Antón, and P. E. Hartman.
1966.
Histidine regulatory mutants of Salmonella typhimurium. I. Isolation and general properties.
J. Mol. Biol.
22:305-323[Medline].
|
| 26.
|
Smith, D. W. E., and B. N. Ames.
1964.
Intermediates in early steps of histidine biosynthesis.
J. Biol. Chem.
239:1848-1855[Free Full Text].
|
| 27.
|
Spratt, B. G.
1975.
Distinct penicillin-binding proteins involved in the division, elongation and shape of Escherichia coli K12.
Proc. Natl. Acad. Sci. USA
72:2999-3003[Abstract/Free Full Text].
|
| 28.
|
Spratt, B. G., and A. B. Pardee.
1975.
Penicillin-binding proteins and cell shape in E. coli.
Nature (London)
254:516-517[Medline].
|
| 29.
|
Sykes, R. B.,
D. P. Bonner,
K. Bush, and N. H. Georgopapadakou.
1982.
Azthreonam (SQ 26,776), a synthetic monobactam specifically active against aerobic gram-negative bacteria.
Antimicrob. Agents Chemother.
21:85-92[Abstract/Free Full Text].
|
| 30.
|
Tamaki, S.,
H. Matsuzawa, and M. Matsuhashi.
1977.
Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli.
J. Bacteriol.
141:53-57.
|
| 31.
|
Tormo, A.,
J. A. Ayala,
M. A. de Pedro,
M. Aldea, and M. Vicente.
1986.
Interaction of FtsA and PBP3 proteins in the Escherichia coli septum.
J. Bacteriol.
166:985-992[Abstract/Free Full Text].
|
| 32.
|
van Heijenoort, J.
1996.
Murein synthesis, p. 1025-1034.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
|
| 33.
|
Vogel, H., and D. Bonner.
1956.
Acetylornithase of Escherichia coli: partial purification and some properties.
J. Biol. Chem.
218:97-106[Free Full Text].
|
| 34.
|
Winkler, M. E.
1996.
Biosynthesis of histidine, p. 485-505.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
|
Journal of Bacteriology, October 1998, p. 5231-5234, Vol. 180, No. 19
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Rosenberger, C. M., Finlay, B. B.
(2002). Macrophages Inhibit Salmonella Typhimurium Replication through MEK/ERK Kinase and Phagocyte NADPH Oxidase Activities. J. Biol. Chem.
277: 18753-18762
[Abstract]
[Full Text]
-
Mattick, K. L., Jørgensen, F., Legan, J. D., Cole, M. B., Porter, J., Lappin-Scott, H. M., Humphrey, T. J.
(2000). Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity. Appl. Environ. Microbiol.
66: 1274-1279
[Abstract]
[Full Text]
Top
Abstract
Text
