Download Effects of Antibiotics that Inhibit the Bacterial Peptidoglycan

Plant Cell Physiol. 44(7): 776–781 (2003)
JSPP © 2003
Short Communication
Effects of Antibiotics that Inhibit the Bacterial Peptidoglycan Synthesis
Pathway on Moss Chloroplast Division
Nami Katayama 1, Hiroyoshi Takano 1, 7, Motoji Sugiyama 1, Susumu Takio 2, Atsushi Sakai 3, Kan Tanaka 4,
Haruko Kuroiwa 5 and Kanji Ono 6
1
Graduate School of Science and Technology, Kumamoto University, Kumamoto, 860-8555 Japan
Center for Marine Environment Studies, Kumamoto University, Kumamoto, 860-8555 Japan
3
Department of Biological Science, Faculty of Science, Nara Women’s University, Nara, 630-8506 Japan
4
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, 113-0032 Japan
5
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, 113-0033 Japan
6
Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto, 860-8555 Japan
2
;
type. The function of bacterial peptidoglycans is to preserve
cell integrity by withstanding the internal osmotic pressure.
Since bacteria usually divide by building a central septum
across the middle of the cell, the peptidoglycan synthesis pathway is intimately involved in bacterial cell division (for
reviews, see van Heijenoort 2001, Bramhill 1997). Consequently, its loss required the invention of a new mode of morphogenesis and division involving the coevolution of the endocytobiont and host. Chloroplast division is fundamental to plant
cells, because plastids multiply by the binary division of preexisting plastids, as do bacteria. In addition, the loss of the
endocytobiont cell wall was probably accompanied by the loss
of certain antigenic properties of the endocytobiont and by
changes in the flow of metabolites between the symbiotic partners, i.e. the cytoplasm and chloroplasts (Kies 1988). Regardless of the importance of peptidoglycans for bacteria, their loss
in organelles has not received as much attention.
Peptidoglycans are continuous covalent macromolecular
structures found on the outside of the cytoplasmic membrane
of almost all eubacteria. In bacteria, the sacculus, a bag-shaped
structure formed from peptidoglycans, is generated in several
steps (van Heijenoort 2001, Bramhill 1997, Bugg and Walsh
1992) (Fig. 1). The first step in peptidoglycan synthesis is the
formation of UDP-N-acetylmuramic acid (MurNAc) from Nacetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP).
The second step is the formation of UDP-MurNAc pentapeptides by the sequential addition of L-alanine, D-glutamic
acid, diaminopimelic acid (A2pm), and D-alanyl-D-alanine
in Escherichia coli. Next, GlcNAc-MurNAc (pentapeptide)pyrophosphoryl-undecaprenol is formed and used in the formation of linear glycan chains. Finally, this is cross-linked to preexisting peptidoglycan to form the sacculus. Since there is no
peptidoglycan layer in animal cells, the peptidoglycan synthesis pathway is a major target for antibiotics (Fig. 1). b-lactam
antibiotics, including penicillin and ampicillin, form covalent
complexes with the penicillin-binding proteins (PBPs) of bac-
Moss chloroplasts should prove useful for studying the
cyanobacteria-derived system in chloroplasts. To determine the effects of antibiotics that inhibit bacterial peptidoglycan synthesis, the numbers of chloroplasts in treated
Physcomitrella patens cells were counted. Ampicillin and
D-cycloserine caused a rapid decrease in the number of
chloroplasts per cell. Fosfomycin affected half of the cells,
while vancomycin affected a few cells. Conversely, bacitracin had no effect. With the decrease in chloroplast
number, macrochloroplasts appeared in antibiotic-treated
cells. Removal of the antibiotics resulted in the recovery of
chloroplast number, suggesting that the decrease in number
was directly dependent on the antibiotic treatment. Microscopic observations showed that the decrease in the number
of chloroplasts resulted from cell division without chloroplast division. These results suggest that enzymes derived
from the bacterial peptidoglycan synthesis pathway are
related to moss chloroplast division.
Keywords: D-Cycloserine — b-Lactam antibiotics —Moss —
Peptidoglycan — Physcomitrella — Plastid division.
Abbreviations: PBP, penicillin-binding protein; GlcNAc, Nacetylglucosamine; PEP, phosphoenolpyruvate; MurNAc, N-acetylmuramic acid; A2pm, meso-diaminopimelic acid; undecaprenyl-P,
undecaprenyl phosphate; undecaprenyl-PP, undecaprenyl pyrophosphate; PEPC, phosphoenolpyruvate carboxylase.
The endosymbiotic theory states that all chloroplasts are
derived from a single cyanobacterial ancestor (for reviews, see
Cavalier-Smith 2000, Gray 1992). It is widely agreed that the
chloroplasts of red algae and higher plants have no peptidoglycan layer. Therefore, the evolution from endocytobiont into a
wall-less, photosynthetic organelle involved a reduction in and
loss of the cyanobacterial cell wall, which is of Gram-negative
7
Corresponding author: E-mail, [email protected]; Fax, +81-96-342-3432.
776
Effects of antibiotics on chloroplast division
Fig. 1 The bacterial peptidoglycan synthesis pathway and antibiotics
(bold) that interfere with polymerization.
teria, including cyanobacteria, and kill them by interfering with
their ability to synthesize a cell wall.
b-lactam antibiotics block the division of cyanelles, which
fulfill the functions of chloroplasts in the Glaucocystophyceae
Cyanophora, Gloeochaete, and Glaucocystis (Kies 1988,
Berenguer et al. 1987). Some studies have shown that the cyanelles are surrounded by a peptidoglycan wall, in which the
structure of the cyanelle peptidoglycans resembles that of
cyanobacteria, and cyanelle envelope membrane preparations
have been observed to catalyze the lipid-linked steps of peptidoglycan synthesis (Aitken and Stanier 1979, Plaimauer et al.
1991, Pfanzagl and Löffelhardt 1999). Moreover, the cyanelle
envelope contains seven PBPs ranging in size from 110 to
35 kDa (Berenguer et al. 1987). However, no genes for the peptidoglycan synthesis pathway have been identified in glaucocystophytes, except for ftsW, which is predicted to activate
PBP3, in the Cyanophora cyanelle genome (Löffelhardt et al.
1997). Of the bacterial PBPs, PBP3, which is encoded by the
ftsI gene, is essential for bacterial cell division (Wang et al.
1998, Weiss et al. 1999). Recently, ftsI genes were found in the
chloroplast genomes of the two most basal green algae,
Nephroselmis olivacea and Mesostigma viride (Turmel et al.
1999, Lemieux et al. 2000), while other sequenced chloroplast
genomes lacked PBP genes. In 1997, it was reported that treatment with three different b-lactam antibiotics resulted in the
appearance of macrochloroplasts in the moss Physcomitrella
777
patens (Kasten and Reski 1997). We showed that the b-lactam
antibiotic ampicillin also causes the appearance of macrochloroplasts in the liverwort Marchantia polymorpha and the pteridophyte Selaginella nipponica (Tounou et al. 2002, Izumi et al.
2003). These results suggest that enzymes derived from the
bacterial peptidoglycan synthesis pathway have been retained
in chloroplast biogenesis in lower plants. Although b-lactams
do not appear to affect chloroplast division in the higher plant
Lycopersicon esculentum (Kasten and Reski 1997), we think
that the effects of antibiotics on the cyanobacteria-derived division system of chloroplasts should prove to be of considerable
interest.
No quantitative data on changes in the numbers of chloroplasts with treatment of b-lactam antibiotics have been
reported. Therefore, we counted the number of chloroplasts in
each apical and subapical cell because protonemata undergo
apical growth (Fig. 2, 3). Without ampicillin, each P. patens
protonema contained 40–50 chloroplasts. In this experiment,
the average chloroplast number before ampicillin treatment
was 47.0±7.9 (SD) (n = 80). After a 2-d treatment, the average
chloroplast number decreased to 15.4±6.1. After 4 d, it
declined to 8.4±4.8 and no cells with more than 30 chloroplasts were observed. The chloroplast number decreased to
3.8±2.4 after 10 d of treatment. Ampicillin had similar effects
on chloroplast numbers in Funaria hygrometrica and Polytrichum commune cells (data not shown). These results suggest
that the effects of b-lactam antibiotics are general in moss cells.
To confirm the effect of ampicillin, we removed the antibiotic after a 2-d treatment (Fig. 3). Initially, the average chloroplast number in apical and subapical P. patens cells was
52.9±10.5. After a 2-d treatment with 100 mM ampicillin, it
decreased to 21.6±8.4 and no cells had more than 40 chloroplasts. Subsequently, the sample was transferred to liquid
medium without ampicillin. Two d later (i.e. on day 4), the
average number of chloroplasts increased to 27.3±14.9, and the
average recovered to the level of non-treated cells 6 d after
transfer (i.e. day 8). These results demonstrate that the decrease
in chloroplast number was directly dependent on the antibiotic
treatment. Continuous observation of one apical P. patens cell
during ampicillin treatment indicated that the huge chloroplasts were not derived from the fusion of small chloroplasts,
but resulted from cell division without chloroplast division
(Fig. 4). Since many nucleoids appeared as yellow spots with
SYBR Green I DNA staining in the huge chloroplasts (data not
shown), b-lactam treatment might not inhibit chloroplast DNA
multiplication.
There is no report on the effects of antibiotics other than
b-lactam antibiotics, which inhibit bacterial peptidoglycan
synthesis on chloroplast morphology or division. Therefore,
P. patens cells were treated with four antibiotics (Fig. 2, 3).
Severe inhibition of cell growth by these antibiotic treatments
was not observed. Treatment with 100 mM D-cycloserine, an
inhibitor of D-alanine:D-alanine ligase, resulted in a few macrochloroplasts in all cells, as occurred with ampicillin treatment
778
Effects of antibiotics on chloroplast division
Fig. 2 Effects of antibiotics that inhibit bacterial
peptidoglycan synthesis on moss protonemata.
Untreated protonema is shown in (A). Protonemata were treated with 100 mM ampicillin (B),
100 mM D-cycloserine (C), 100 mM fosfomycin
(D), 500 mM vancomycin (E), and 100 mM bacitracin (F). Bar = 20 mm.
(Fig. 2, 3). Before treatment, the average chloroplast number
was 47.0±7.9. After a 2-d treatment, the chloroplast number
decreased to half the normal number, and it decreased to
4.8±3.5 after a 10-d treatment. Although the decrease in chloroplast number in D-cycloserine-treated cells was similar to that
which occurred in ampicillin-treated cells, it was found that
cells were occasionally round and often smaller than normal
cells. By contrast, treatment with 100 mM fosfomycin, a phosphoenolpyruvate analog, led to the appearance of macrochloroplasts in half of the cells (Fig. 2, 3). The quantitative data suggest that there were both affected and unaffected cells; the
chloroplast number in the treated cells ranged from a few to 70
after a 10-d treatment. The same result was seen with 500 mM
fosfomycin (data not shown). Removal of these antibiotics
caused recovery of chloroplast number (data not shown). Continuous observations of single cells during D-cycloserine and
fosfomycin treatments showed cell division without chloroplast division, as seen in ampicillin-treated cells (data not
shown). Treatment with 100 mM vancomycin led to the appearance of macrochloroplasts in a few cells (data not shown), but
most cells were unaffected; similar results occurred with
500 mM vancomycin (Fig. 2) and approximately 4.5% of cells
had fewer than 20 chloroplasts. Bacitracin did not have a clear
effect on chloroplast numbers or cell morphology at either 100
or 500 mM (Fig. 2).
Since protonemata undergo apical growth, the number of
chloroplasts increased mainly in apical cells. In untreated cells,
apical cells, in which the number of chloroplasts reached 75–
110, divided to create subapical and new apical cells. In the
treated cells, the decrease in chloroplast number and increase in
volume of each chloroplast are thought to be related to cell
division (Fig. 4). These relationships were confirmed because
the chloroplast number in non-dividing basal cells was unaffected by antibiotic treatment, and because the decrease in
chloroplast number was slow in poorly conditioned protonemata (data not shown). If cultured cells of P. patens were synchronized, the relationship between cell cycles, antibiotic treatments and chloroplast biogenesis may be clarified.
We did not observe cells without chloroplasts. With the
decrease in chloroplast number, each chloroplast was huge
(Fig. 2, 4). These huge chloroplasts filled most of the cell interior. Since cell division occurs at the midpoint of a cell, the
Effects of antibiotics on chloroplast division
779
Fig. 3 Effects of ampicillin (A, B), D-cycloserine (C), and fosfomycin (D) on the number of chloroplasts per cell. The number of chloroplasts in
apical and subapical cells was counted at 2-d intervals. The number of cells containing each number of chloroplasts is shown (n = 80). The bar at
the right indicates the number of cells containing more than 70 chloroplasts. (B) Histogram showing the results when ampicillin was removed
after 2 d (arrow).
production of cells without chloroplasts may be rare. Possibly,
any cells without chloroplasts died, although dead protonemata were rarely observed.
All the antibiotics tested, with the exception of bacitracin,
affected chloroplast division in mosses, although the effects
depended on the antibiotic. D-cycloserine inhibits the ligation of
two D-alanines in Gram-negative and -positive bacteria (Bugg
and Walsh 1992). Fosfomycin is widely used to inhibit the
enzyme UDP-N-acetylglucosamine-1-carboxy-vinyltransferase,
which catalyzes the first committed step in peptidoglycan biosynthesis, in both Gram-negative and -positive bacteria (Wanke
and Amrhein 1993). Vancomycin forms a complex with the
peptidyl-D-Ala-D-Ala termini to prevent transglycosylation and
transpeptidation, while bacitracin inhibits dephosphorylation of
undecaprenyl-PP to block translocation of the peptidoglycan
precursor across the bacterial plasma membrane. Since vancomycin and bacitracin mainly inhibit peptidoglycan synthesis in
Gram-positive bacteria (Bugg and Walsh 1992), their minimal
effects on chloroplast division may depend on differences in
peptidoglycan synthesis between the ancestral cyanobacteria
and Gram-positive bacteria. Alternatively, the ineffectiveness
of bacitracin might be due to differences in the membrane
topology of chloroplasts and bacteria. Perhaps translocation of
the precursor that is inhibited by bacitracin is not needed in
chloroplasts. If the entire synthesis pathway remains intact, the
ineffectiveness of fosfomycin, vancomycin, and bacitracin may
depend on how permeable they are. The permeabilities of these
antibiotics may have been altered in the affected cells for
unknown reasons. Conversely, the inhibition of chloroplast
division may be due to a different effect of these antibiotics.
There are two reports on the relationship between these antibiotics and eukaryotic plant enzymes. The antibiotic D-cycloserine inhibits the activity of serine hydroxymethyltransferase isolated from mung bean seedlings (Rao and Rao 1982). MújicaJiménez et al. (1998) reported that fosfomycin acts as a potent,
reversible non-essential activator of phosphoenolpyruvate carboxylase from C4 maize leaves by binding to the same allosteric site as glucose-6-phosphate. Although there is no report
780
Effects of antibiotics on chloroplast division
Fig. 4 Cells were continuously observed under an inverted microscope (a, 36 h; b, 48 h; c, 60 h; d, 72 h). The chloroplast numbers are shown
near each cell. Bar = 20 mm.
on the inhibition of plant cysteine synthase by D-cycloserine,
the activity of Salmonella cysteine synthase is decreased
(Nakamura et al. 1984). Although these enzymes are involved
in metabolic pathways in chloroplasts, the relationship between
changes in their enzymatic activities and chloroplast division is
unknown. As stated above, the effects of these antibiotics on
chloroplast morphology in higher plants are now being tested.
The findings in this study suggest that enzymes derived
from the bacterial peptidoglycan synthesis pathway are related
to moss chloroplast division. Two structures are found at the
site of chloroplast division. One is the plastid-dividing ring,
which was first detected by transmission electron microscopy
(Mita et al. 1986, Kuroiwa et al. 1998, Miyagishima et al.
2001a). The other is the FtsZ ring, which plays a central role
in bacterial division and is involved in plastid division
(Osteryoung and Vierling 1995, Osteryoung et al. 1998, Strepp
et al. 1998, Araki et al. 2003). Recently, a biochemical and
immunocytochemical study of the synchronized chloroplast
division system in the red alga Cyanidioschyzon merolae, by
Miyagishima et al. (2001b), showed that the plastid FtsZ ring is
distinct and separable from the plastid-dividing ring. Their
results suggest that the FtsZ ring-based system, which originated
from cyanobacteria, the plastid ancestor, and the plastid-dividing ring-based system, which probably originated, in turn, from
host eukaryotic cells, form a complex and are involved in distinct aspects of plastid division. Our results suggest that enzymes
derived from the bacterial peptidoglycan synthesis pathway are
also involved in chloroplast division, but only in lower plants.
The role of these enzymes in land plants remains to be resolved.
We searched the Arabidopsis genome (Arabidopsis
Genome Initiative 2000) for genes related to the peptidoglycan
synthesis pathway and found putative murE [diaminopimelic
acid (A2pm)-adding enzyme] and murG [GlcNAc transferase to
MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol] genes
with putative chloroplast transit peptides, but no PBPs, including no ftsI homologs. Since ESTs of the murE and murG genes
were present in the database, these results suggest that at least
two peptidoglycan pathway enzymes are active in the chloro-
plasts of higher plants. The relationship between these products and chloroplast biogenesis is unclear at present. The genes
may have other functions in chloroplasts. We are now in the
process of isolating genes for the peptidoglycan synthesis pathway, including PBPs, from P. patens. If any genes are isolated,
we will be able to use the gene-targeting technique developed
for P. patens cells (Schaefer 2001) to resolve the relationship
between the eukaryotic peptidoglycan synthesis pathway and
chloroplast biogenesis in mosses.
The Funariaceae P. patens (Hedw.) B. S. G. was grown
axenically on medium solidified with 0.8% agar in a regulated
chamber at 25°C under continuous light (approx. 35 mmol
m–2 s–1) (Nishiyama et al. 2000). For antibiotic treatment, protonemata were transferred to liquid media. The antibiotics were
dissolved in water and added to the medium under sterile
conditions. The antibiotics used were ampicillin, penicillin,
D-cycloserine, fosfomycin, vancomycin, and bacitracin (Wako).
The number of chloroplasts in a cell was counted under
light microscopy (BX60, Olympus, Tokyo, Japan, or Axioskop
2 plus, Zeiss, Germany). Protonemata were harvested after 2, 4,
6, 8, and 10 d of incubation with antibiotics. Light-field images
of cells were recorded with a CCD camera (DXM1200, Nikon,
Tokyo, Japan, or Zeiss Axiocam). For continuous observation
of one protonema, P. patens was grown on agar medium
including 100 mM ampicillin in a glass-bottom Petri dish and
observed under an inverted microscope (Nikon Diaphot) with a
CCD camera.
Acknowledgments
This study was supported by Grants-in-Aid for the Encouragement of Young Scientists to H.T. from the Japan Society for the Promotion of Science and by the Program for the Promotion of Basic
Research Activities for Innovative Biosciences (PROBRAIN to H.T.).
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(Received January 20, 2003; Accepted May 8, 2003)