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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.). References Aitken, A. and Stanier, R.Y. (1979) Characterization of peptidoglycan from the cyanelles of Cyanophora paradoxa. J. Gen. Microbiol. 112: 219–223. Effects of antibiotics on chloroplast division Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815. Araki, Y., Takio, S., Ono, K. and Takano, H. (2003) Two types of plastid ftsZ genes in the liverwort Marchantia polymorpha. Protoplasma 221: 163–173. Berenguer, J., Rojo, F., De Pedro, M.A., Pfanzagl, B. and Löffelhardt, W. (1987) Penicillin-binding proteins in the cyanelles of Cyanophora paradoxa, a eukaryotic photoautotroph sensitive to b-lactam antibiotics. FEBS Lett. 224: 401–405. 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