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American Society of Plant Biologists Detection and Localization of a Chloroplast-Encoded HU-Like Protein That Organizes Chloroplast Nucleoids
a Department of Biological Science, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan 1 To whom correspondence should be addressed. E-mail tamaki{at}biol.s.u-tokyo.ac.jp; fax 81-3-3814-1408
Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.
It is believed that chloroplasts and mitochondria arose from prokaryotic endosymbionts during eukaryotic evolution. According to this concept, chloroplasts originated from cyanobacteria (Margulis, 1970  ; Gray, 1992) and therefore have their own DNA. The chloroplast genome (cp-genome) often is divided into highly condensed, discrete structures called chloroplast nucleoids (cp-nucleoids) (Hewson and Wolstenholme, 1969; Kuroiwa et al., 1981; Kuroiwa, 1982; Yurina et al., 1995).
The cp-nucleoids are mainly classified into three types according to differences in their shape, size, and distribution (Kuroiwa et al., 1981
Accordingly, it is essential to investigate nucleoid proteins to understand the function of cp-nucleoids. The organization of cpDNA may offer a key to understanding the mechanism of gene expression in the cp-genome. However, little is known about the structures or mechanisms of action of proteins that bind to and organize cpDNA topology (Nemoto et al., 1991
The fundamental unit of chromatin in the cell nucleus is the nucleosome. Within the nucleosome, DNA is wrapped approximately twice around the histone core (Wolffe, 1998
The 70-kD pea protein was dissociated selectively from nucleoids by salt washing and was identified as sulfite reductase (Sato et al., 2001 However, at present, there is no evidence to suggest that these proteins localize in vivo at cp-nucleoids or that they are involved in the organization or maintenance of the chloroplast nucleoid structure. Nucleoids are dispersed throughout the chloroplasts in higher plants such as pea and tobacco. As a result, the cp-nucleoid fraction is easily contaminated with fragments from cell nuclei during nucleoid isolation. Moreover, it is possible for basic proteins to contaminate isolated cp-nucleoids because DNA is negatively charged. Uncontaminated nucleoid fractions may be obtained from organisms with a simple structure that have cp-nucleoids concentrated at the center of chloroplasts.
Here, we report a DNA binding protein purified from the chloroplasts of the primitive red alga Cyanidioschyzon merolae. C. merolae is ideal for both cp-nucleoid protein isolation and the study of cpDNA interactions for the following reasons: (1) cp-nucleoids are restricted to the center of chloroplasts and resemble bacterial nucleoids; (2) mitotic and organelle division cycles can be highly synchronized by light/dark cycles (Suzuki et al., 1994 We isolated an abundant cpDNA binding protein from C. merolae. The protein was very similar to the bacterial histone-like protein HU and was named HC (histone-like protein of chloroplast). We demonstrate that the HC localized within cp-nucleoids is a cpDNA binding protein. HC has DNA-compacting activity and complements the deletion of HU in Escherichia coli.
Identification of DNA Binding Proteins in cp-Nucleoids To analyze the cpDNA binding protein, we isolated cp-nucleoids from C. merolae. Phase-contrast and 4',6-diamidino-2-phenylindole (DAPI)–stained images of whole cells, isolated chloroplasts, and cp-nucleoids from C. merolae are shown in Figure 1 . Whole cells contained a single chloroplast, mitochondrion, and nucleus per cell, and cp-nucleoids were concentrated at the center of chloroplasts (Figures 1A and 1B). Nonsynchronous cells were disrupted in a French pressure cell after hypotonic treatment and layered on Percoll gradients. The intact chloroplast fraction was collected by centrifugation.
Observation of the isolated chloroplasts (Figures 1C and 1D) showed that cp-nucleoids maintained the same compact profiles seen in whole cells (Figure 1B) and were not contaminated by mitochondria or nuclei. The cp-nucleoids were isolated from this fraction by solubilizing the membrane with the nonionic detergent Nonidet P-40 and removing the solubilized components by centrifugation. Morphologically, the isolated cp-nucleoids (Figures 1E and 1F) remained intact and identical to those observed in whole cells (Figure 1B) and in isolated chloroplasts (Figure 1D). The combination of phase-contrast and DAPI-fluorescence microscopy showed that most of the chloroplast membrane had been removed (cf. Figures 1C and 1D with 1E and 1F). Next, to isolate DNA binding proteins involved in packaging cpDNA into nucleoid structures, we treated isolated cp-nucleoids with DNaseI. The proteins that dissociated from the cp-nucleoids were subjected to DNA-cellulose column chromatography and then to SDS-PAGE analysis. Figure 2 shows the protein patterns in the different fractions. After elution with start buffer containing 300 mM NaCl, several proteins were detected (lane 4). Proteins that bound DNA more strongly, with molecular masses of 35 kD (arrowhead) and 17 kD (double arrowhead), were eluted with 1 M NaCl (lane 5). The 17-kD protein was more abundant than the 35-kD protein, and two-dimensional gel electrophoresis of the cp-nucleoids revealed one spot with a molecular mass of 17 kD and a basic pI of 10.0 (data not shown). These results suggest that the 17-kD DNA binding protein is very basic, is abundant in cp-nucleoids, and binds DNA strongly.
Identification of the 17-kD Protein Gene To identify the gene encoding the 17-kD protein, its N-terminal sequence was determined. The first 12 N-terminal residues were MDKTELISAVAE. The program FASTA (Pearson and Lipman, 1988 ) showed that this sequence was similar to part of HU, a bacterial histone-like protein (Rouviere-Yaniv and Gros, 1975). The 17-kD protein was similar to HU in many ways, including molecular mass, basic pI, and N-terminal sequence. This sequence also is similar to that of HlpA, the gene of which (hlpA) was found in the G. theta cp-genome (Wang and Liu, 1991).
Because G. theta is a cryptomonad alga with chloroplasts that are thought to have originated from red algae through secondary endosymbiosis, we expected to find the gene encoding the 17-kD protein in the cp-genome of C. merolae. Therefore, we examined the C. merolae cp-genome (Ohta et al., 1999
The amino acid sequence of HC is shown in Figure 3
. It encodes a protein with 113 amino acid residues, a molecular mass of 12.6 kD, and a calculated pI of 9.8. The molecular mass of HC was estimated to be 17 kD by SDS-PAGE analysis, and not 12.6 kD as deduced from the amino acid sequence (see above). Because the protein is very basic (estimated pI of 9.8), the band likely was shifted upward on SDS-PAGE, as is that of histone (Weber et al., 1972
There is no CmhupA homolog in the mitochondrial genome (Ohta et al., 1998 ) or in the nuclear genome, which was sequenced recently (our unpublished data). These results show that CmhupA is a single-copy gene that is encoded only by the cp-genome.
Complementation of HU-Lacking E. coli Mutants The double mutants were transformed by an HC-expressing vector, and HC was expressed directly in the E. coli double mutants. Morphologically, they exhibited the wild-type phenotype, and the cold-sensitive phenotype was rescued (Figure 4) . Furthermore, they are viable. In all cases, the double mutants carrying the parent vector retained the double mutant phenotypes. These results indicate that HC has not only sequence similarity to HU but also is functionally similar to HU.
Localization and Expression of HC To analyze HC localization, we prepared an antibody against 6xHis-tagged HC expressed in E. coli. Immunoblot analysis showed that this antibody reacted with a single band in a whole cell lysate, which had the same mobility as HC. The antibody also reacted with the same band in isolated chloroplasts and cp-nucleoids (Figure 5) . This result indicates that HC is present in cp-nucleoids.
Immunofluorescence with anti-HC antibody was used to localize HC in vivo (Figures 6A and 6B) . HC was located uniformly throughout the cp-nucleoid but was not seen in cell nuclei or in mitochondrial nucleoids (Figures 1B, 6A, and 6B). Fluorescein isothiocyanate fluorescence was seen clearly at exactly the same position in the chloroplast where the nucleoid was stained with DAPI fluorescence (Figures 6A and 6B). The use of anti-HC antibodies and immunoelectron microscopy allowed more precise localization of HC in cp-nucleoids at the ultrastructural level (Figures 6C and 6D).
Gold particles were observed in the chloroplast but not in the nucleus or the mitochondrion (Figure 6C), supporting the results of the immunofluorescence study (Figures 6A and 6B). Under higher magnification, gold particles were observed in the central part of the chloroplast where the cp-nucleoid was located (Figure 6D). Together, these results clearly demonstrate that HC is localized in cp-nucleoids (Figures 5 and 6).
Next, we studied the relationship between the HC expression pattern and the chloroplast division cycle. Cell division and chloroplast division were synchronized by subjecting them to a 12-h-light/12-h-dark cycle. cpDNA replicates during the second light period, and chloroplasts divide during the second dark period (Itoh et al., 1997
Bands were detected at all time points, and signal intensities were constant. This result indicates that CmhupA was transcribed regardless of cpDNA replication. Immunoblot analysis showed that HC was expressed constitutively in the cell (Figure 7). When dividing chloroplasts were observed by immunofluorescence, HC was localized in the cp-nucleoids, and its fluorescence was approximately the same intensity as that of nondividing chloroplasts (Figures 6A and 6B, inset).
DNA Condensation Assay in Vitro and in E. coli
First, we confirmed that the purified HC was not contaminated with other proteins (Figure 8A). The purified HC was mixed with C. merolae cpDNA and dialyzed against isolation buffer. A number of small particles similar to nucleoids formed, although they were not the same size as cp-nucleoids in vivo (Figure 8D). In control experiments, neither cpDNA alone nor cpDNA plus BSA generated nucleoid-like particles (Figures 8B and 8C). This result suggests that HC can condense DNA into small particles without the help of other proteins. To clarify the effect of HC on E. coli DNA, we expressed HC in E. coli (Figure 9). In the absence of the inducer IPTG, entire cells were stained uniformly. In HC-expressing cells, the nucleoid was condensed extensively, whereas no such change was observed in control cells. When we observed E. coli cells that were stained with the vital nucleic acid stain SYTO 11 to identify possible artifacts associated with fixation, only E. coli nucleoids expressing HC were condensed (data not shown). This result indicates that excess HC condenses nucleoids more compactly in E. coli.
cpDNA is arranged into protein/DNA complexes that are similar to bacteria and are called cp-nucleoids or cp-nuclei (Kuroiwa, 1991 ). Proteins bound to DNA may determine the structure of the nucleoid and influence DNA function as a template for either transcription or replication (Kuroiwa, 1991). In this study, we used a newly developed method to purify DNA binding protein from cp-nucleoids that were isolated from C. merolae (Figures 1 and 2). Seventeen- and 35-kD proteins were eluted with the high-salt fraction, and the 17-kD protein was more abundant than the 35-kD protein. These results suggest that the 17-kD protein is the more abundant DNA binding protein in cp-nucleoids and that it binds DNA strongly.
We determined the N-terminal sequence of the 17-kD protein and performed a homology search. The sequence was completely different from those of other DNA binding proteins that have been isolated from cp-nucleoids (CND41 from tobacco [Nakano et al., 1997
Immunogold staining with anti-HU antibodies revealed the distribution of HU in E. coli cells, and the antibodies were observed primarily at the nucleoid periphery (Dürrenberger et al., 1988 In our study, both immunofluorescence and immunoelectron microscopy showed that HC was distributed uniformly throughout the entire cp-nucleoid (Figure 6). This observation is consistent with the localization of HU within the nucleoids, indicating that the distribution also is conserved between HC and bacterial HU. Furthermore, HC was expressed constitutively within the cells and located uniformly throughout the cp-nucleoids during chloroplast division (Figures 6 and 7). These results suggest that HC localizes within cp-nucleoids, that the DNA binding ability of HC is not restricted to any specific region of the nucleoid, and that HC plays an essential role in the maintenance of cp-nucleoids.
It is known that bacterial HU contributes to the compaction of DNA into nucleoid structures (Rouviere-Yaniv et al., 1979 These results suggest that HC is an important protein in the organization of cpDNA into nucleoids. However, HC organized DNA into a smaller size than the cp-nucleoids observed in vivo (Figure 8). This finding suggests that other proteins, such as the 35-kD protein eluted with 1 M NaCl (Figure 2), are necessary to organize DNA into natural cp-nucleoids. More detailed study is needed to clarify this point.
E. coli mutants lacking HU exhibited phenotypes that were different from that of the wild-type strain, such as filamentous and cold-sensitive phenotypes (Figure 4) (Wada et al., 1988
The primary structure of HU is highly conserved among bacterial species, including cyanobacteria, which are thought to be the ancestors of chloroplasts. In this study, the HU homolog HC also was found in the cp-genome of the primitive red algae C. merolae, and HC organized the cpDNA into nucleoids. A HU-like protein gene (hlpA) has been found in the G. theta cp-genome (Wang and Liu, 1991
The HU homolog also has been found in several sequencing projects of apicomplexan parasites, including Toxoplasma gondii, Plasmodium yoelii, and Plasmodium berghei. The amino acid sequence of the HU homolog from T. gondii is aligned in Figure 3. These parasites have been shown to contain a vestigial nonphotosynthetic plastid, the apicoplast, which may have been derived from a red algal ancestor by secondary endosymbiosis (Fast et al., 2001
No HU homolog has been detected in any of the chloroplast genomes of green algae or land plants. The immunological detection of an HU homolog in spinach chloroplast has been reported (Briat et al., 1984
Culture Conditions Cyanidioschyzon merolae was synchronized according to the method described by Suzuki et al. (1994) . Cells were cultured in Allen's medium (Allen, 1959) at pH 2.5, and flasks were shaken under continuous light (40 W/m2) at 40°C. The cells were subcultured to <107 cells/mL and then synchronized by subjecting them to a 12-h-light/12-h-dark cycle at 45°C in aerated medium. After the second cycle, cells multiplied synchronously. During the light period, cells were in interphase, whereas during the dark period, the chloroplast, mitochondrion, and nucleus divided (in that order) before cytokinesis. Most chloroplasts divided in the middle of the second dark period.
Isolation of Chloroplast Nucleoids Lysates were centrifuged for 50 min at 28,000g, and a band of intact chloroplasts was harvested from the 60 to 80% Percoll interface. Chloroplasts were washed with isolation buffer containing 300 mM Suc and suspended at a concentration of 1 mg protein/mL in isolation buffer containing 2% (v/v) Nonidet P-40. The lysed chloroplasts were incubated at room temperature for 15 min and then centrifuged at 18,500g for 15 min. The resulting pellet was resuspended in isolation buffer and 2% Nonidet P-40, incubated at room temperature for 15 min, and centrifuged at 18,500g for 15 min. The final nucleoid pellet was suspended in isolation buffer and stored at 4°C. Cells, chloroplasts, and chloroplast nucleoids were fixed in 0.5% glutaraldehyde (distilled grade; TAAB, Aldermaston, UK), stained with 1.0 µg/mL 4',6-diamidino-2-phenylindole (DAPI), and pressed between a microscope slide and a cover slip. The samples were observed with a fluorescence microscope (BHS-RFC; Olympus, Tokyo, Japan) using a UV excitation beam at 334 and 365 nm.
Identification of the DNA Binding Protein in Chloroplast Nucleoids Proteins were eluted with start buffer containing 300 and 1000 mM NaCl. Proteins eluted with start buffer containing 1000 mM NaCl were mixed with one-third volume of 4 x Laemmli sample buffer (50 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.01% bromphenol blue) and separated by SDS-PAGE on 15% polyacrylamide gels at 150 V for 1 h. Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) for N-terminal sequencing or visualized by silver staining. The N-terminal sequence of the 17-kD protein was determined using a HP G1005A protein sequencing system (Hewlett Packard, Palo Alto, CA).
Plasmids
RNA Gel Blot Analysis
Complementation Analysis of HU-Lacking Escherichia coli Mutants Using HC
The E. coli hupA hupB double mutant (JR1672) was transformed with plasmid pQE50-CmhupA. Transformed strains were grown in LB medium supplemented with 2% Glc, 50 µg/mL kanamycin, 12.5 µg/mL chloramphenicol, and 50 µg/mL ampicillin until they reached an optical density of 0.4 at 600 nm, at which time isopropylthio- To detect the effect of cold shock, strains JR1669 (wild type), JR1672 (double mutant), JR1672/pQE50, and JR1672/pQE50-CmhupA were grown to log phase in LB medium containing 2 mM IPTG and the appropriate antibiotics, transferred to 0°C for 0, 1, 2, and 3 h, and plated on LB agar. JR1672/pQE50 and JR1672/pQE50-CmhupA were grown in LB medium in the presence of the inducer IPTG. The plates were incubated at 37°C overnight, and the number of colonies was scored. Colony number immediately before the transfer was set at 1.0.
Antibody Preparation and Immunoblot Analysis Proteins were separated by SDS-PAGE using 15% gel as described above and then transferred to a PVDF membrane (Millipore) in buffer (25 mM Tris, 190 mM Gly, 20% methanol, and 0.03% SDS) for 2 h at 100 V. The PVDF membrane was blocked for 1 h at room temperature in TBS (0.2 M Tris-HCl, pH 7.5, and 5 M NaCl) containing 3% gelatin. Blocked membranes were probed with rabbit polyclonal anti-HC antibody (1:10,000 in TBS containing 0.05% Tween 20) for 1 h at room temperature. Membranes then were washed with 1 x TBS containing 0.05% Tween 20 and exposed to an anti-rabbit IgG alkaline phosphatase conjugate (1:1000 dilution). Bands were resolved by incubation with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
Immunofluorescence and Immunoelectron Microscopy Using Anti-HC Antibody
Immunogold labeling was performed as described previously (Miyagishima et al., 2001
DNA Condensation Assay in Vitro
DNA Condensation Assay in E. coli
Accession Numbers
We thank Josette Rouviere-Yaniv (Institut de Biolgie Physico-Chimique, Paris, France) for providing the E. coli strains (JR1669, JR1670, and JR1671) and Tatsushi Mogi (University of Tokyo, Tokyo, Japan) for the kind gift of P1 phage. This work was supported by Grants 12440222 and 13206011 to T.K. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a grant from the Program for the Promotion of Basic Research Activities for Innovative Bioscience.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002717. Received March 4, 2002; accepted March 27, 2002.
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Abstract
INTRODUCTION
-galactoside (IPTG). Cells were fixed in 0.5% glutaraldehyde, stained with 1.0 µg/mL DAPI, and visualized by phase-contrast and fluorescence microscopy. Bar = 3 µm for all panels.
-32P-dCTP, and hybridization, washing, and detection were performed as described above except that the hybridization buffer contained 50% formamide and washing was performed at 65°C (high-stringency conditions). 
