Genetic and Biochemical Characterization of EshA, a Protein That Forms Large Multimers and Affects Developmental Processes in Streptomyces griseus*

Natsumi Saito, Keiko MatsubaraDagger, Masakatsu Watanabe§, Fumio Kato, and Kozo Ochi||

From the National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan

Received for publication, August 21, 2002, and in revised form, December 16, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 52-kDa protein, EshA, whose expression is controlled developmentally, is produced during the late growth phase of Streptomyces spp. We found that disruption of the eshA gene, which encodes the EshA protein, abolishes the aerial mycelium formation and streptomycin production in Streptomyces griseus when grown on an agar plate. The eshA disruptant KO-390 demonstrated a reduced amount of expression of the transcriptional activator strR, thus accounting for the failure to produce streptomycin. KO-390 was found to accumulate deoxynucleoside triphosphates at high levels, including dGTP, at late growth phase. The accumulation of dGTP was a cause for the impaired ability of KO-390 to produce aerial mycelium, because the ability to form aerial mycelium was completely repaired by addition of decoyinine, an inhibitor of GMP synthetase. The accumulation of dNTP in KO-390 coincided with a reduced rate of DNA synthesis. The developmental time frame of these phenomena in KO-390 matched a burst of EshA expression in the wild-type strain. In contrast to S. griseus, the eshA disruption did not affect the ability for Streptomyces coelicolor to form aerial mycelium and did not result in the aberrant accumulation of dNTP accompanied by arrest of DNA synthesis, implying qualitative differences in addition to quantitative differences between the two EshA proteins. We propose that the S. griseus EshA protein somehow positively affects (or regulates) the replication of DNA in wild-type cells at late growth phase but leads to aberrant phenotypes in mutant cells due to the disturbed DNA replication. The EshA protein was found to exist as a multimer (~20-mers) creating a cubic-like structure with a diameter of 27 nm and located predominantly in cytoplasm.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The Gram-positive, filamentous, soil bacterial streptomycetes are a unique group of prokaryotic organisms that display complex morphological differentiation and the ability to produce a wide variety of secondary metabolites (called physiological differentiation), including antibiotics and other useful medicinal compounds (1-3). In some bacteria, especially the genus Streptomyces, morphological and physiological differentiation often commence at the same time in response to environmental signals such as nutrient limitation. Only a limited number of Streptomyces species, for example Streptomyces griseus, can produce spores when cultured in liquid medium (4, 5). Although a molecular based mechanism that connects antibiotic production with morphological differentiation has not yet been fully elucidated, it is apparent that the stringent response plays an essential role in detecting and responding to adverse environmental conditions, eventually leading to the activation (or suppression) of various biological functions. The stringent response, a general and ubiquitous response to nutritional environmental stress in prokaryotic microorganisms, is mediated by a unique nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp) (reviewed by Cashel et al. (6)). By analyzing mutants with an impaired ability to elicit the stringent response, Ochi (7-10) has proposed that ppGpp plays a role in triggering the onset of antibiotic production in Bacillus subtilis and Streptomyces spp., whereas morphological differentiation is triggered by detecting a reduced level of GTP.

Several genera of bacteria produce spores when they encounter adverse environmental conditions such as nutrient deficiency (reviewed by Freese and Heinze (11)). A distinguishing characteristic of Streptomyces spp. is the ability to form an aerial mycelium from a substrate mycelium when cultured on solid media, eventually leading to the formation of spores by creating synchronous and regularly spaced septations in the aerial mycelium (for reviews see Refs. 1, 2, and 12). Insight into the mechanisms by which GTP functions came from studying the development of bacteria that provided several clues for understanding the onset mechanism for bacterial differentiation on a molecular level (13, 14). We (9, 10, 15, 16) and Freese and co-workers (11, 17-19) have demonstrated that a decrease in the GTP pool size correlates with the initiation of morphological differentiation in B. subtilis, Streptomyces spp., and Saccharomyces cerevisiae. Thus these organisms can be induced to differentiate in nutritionally rich media, in which cells normally do not sporulate, when the GTP pool size is reduced artificially. Although the role of GTP pool size variations in Streptomyces is unclear, it seems logical that a GTP-binding protein(s) could be involved in detecting a decreased GTP pool level as a signal for differentiation (13).

Recently, Kendrick and co-workers (20) and our laboratory (21) independently found a novel 52-kDa protein, which has been demonstrated to be required for initiating several developmental processes in Streptomyces. Specifically, in the course of studying ribosomes isolated from Streptomyces coelicolor A3(2) and S. griseus, we found a protein that is produced during the late growth phase (21). The disruption of the gene (eshA), which codes for this 52-kDa protein, was shown to abolish the antibiotic production in S. coelicolor A3(2). At the same time, Kwak et al. (20) reported that EshA is required for the extension of sporogenic hyphal branches (thus the origin of the gene designation, eshA) in S. griseus. Although EshA may be a good tool for uncovering and analyzing still unknown biological events that take place in stationary phase cells, the function of the EshA remains obscure. In this paper, we report the results from genetic and biochemical analysis of the EshA protein working with S. griseus, because this organism produces 10-fold more EshA protein than S. coelicolor A3(2). Eventually, we found that the EshA exists as a large multimer with a characteristic shape. This is also discussed in terms of protein chemistry.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Bacterial Strains and Culture Conditions-- S. griseus IFO13189, a prototrophic streptomycin-producing wild-type strain, was used as the parental strain. Escherichia coli DH5alpha was used routinely for plasmid construction. E. coli GM2163 (New England Biolabs), a non-methylating strain, was used to prepare DNA for transformation into S. griseus. Strains of S. griseus were grown at 30 °C using GYM medium (22) or TSB medium. TSB medium contained (per liter): 10.3% (w/v) sucrose; 3% (w/v) tryptic soy broth (Difco); 5 mg of CuSO4·5H2O; 7.5 mg of FeSO4·7H2O; 3.6 mg of MnSO4·5H2O; 15 mg of CaCl2·2H2O; 9 mg of ZnSO4·7H2O; and 5 mM MgCl2. The regeneration medium, SpRR (23), was used for transformation of S. griseus. SPY medium, synthetic medium, and sporulation medium were described previously (10).

Plasmid Construction-- General DNA manipulations such as plasmid isolation and transformation in E. coli were employed as described by Sambrook et al. (24). Characteristics and construction of plasmids used in this study are summarized in Table I. Plasmid pV52SG, which contains a 3.5-kb S. griseus eshA region, was constructed as follows. A 3.2-kb MluI fragment and a 1.3-kb NcoI fragment, which contains the upstream and the downstream region of eshA gene respectively, were excised from pSGRp52 which carries a 8.0-kb fragment containing the eshA gene on pBluescript SK(+) (21). Then the remaining 3.5-kb BamHI fragment was ligated to pV1, resulting in pV52SG.

                              
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Table I
Bacterial strains and plasmids used in this study

In order to construct a vector to prepare a probe for S1 mapping, a 304-bp fragment containing the 5' part of the S. griseus strR gene was amplified by PCR with genomic DNA isolated from strain 13189 as the template using the primers strR-FS1 (5'-TCGGCAATCAAACTGCGGTTTATTTTG-3') and strR-RS1 (5'-CTCGGGGCTGTTCCCTGAAATATGC-3'). The fragment generated was cloned into the plasmid pGEM-T, resulting in pGEMstrRSG. A 351-bp fragment containing the 5' part of the S. griseus hrdB gene was amplified by PCR with genomic DNA isolated from strain 13189 as the template using the primers SghrdB-F02 (5'-AAGGATCCGCGCCGCGCGAGCACTGAC-3'; the underline indicates the cleavage site for BamHI) and SghrdBR1 (5'-GGAACGATGGAAACGGCTTC-3'). The fragment generated was cloned into the plasmid pGEM-T, resulting in pGEMhrdBSG.

Gene Disruption of eshA-- The plasmid pSGRp52 was digested at the unique BstPI site (456th base position) within the eshA-coding region. The ends of the BstPI-digested fragment were flush-ended with the Klenow fragment of DNA polymerase I and then ligated resulting in the plasmid pSGRDelta p52, which contains a frameshift mutation in the eshA gene. This frameshift mutation makes cells to produce a protein containing about one-third (152 amino acid residues) of the native EshA protein (470 amino acids). A 1.4-kb EcoRI fragment from pKK1000 containing the apramycin acetyltransferase gene (aac(3)IV) was introduced into the plasmid pSGRDelta p52 to generate pSGRDelta p52Ap. pSGRDelta p52Ap was passed through E. coli GM2163 (New England Biolabs) to remove methyl groups and subsequently was methylated with methyltransferases, mAluI and mHapII. The methylated DNA was introduced into S. griseus 13189 protoplasts. Transformants that contain pSGRDelta p52Ap in the chromosome as a result of single crossover were selected with apramycin (50 µg/ml). The apramycin-resistant strains were cultured at 30 °C for 72 h in TSB liquid medium without apramycin to allow double crossover. The cells recovered were then spread on GYM agar without apramycin to screen for apramycin-sensitive clones, which represent eshA disruptant. The frameshift mutation in the eshA gene was confirmed by DNA sequencing.

Measurement of the Intracellular Nucleotide Pools-- For plate culture assays, spores (~2 × 107) were spread on TSB agar medium covered with a cellophane sheet. Methods for extraction of intracellular nucleotides from mycelia, which had been grown on a cellophane sheet or in liquid medium, were described previously (26, 27). Nucleotide pool sizes were analyzed using a high performance liquid chromatography system (Hitachi D-7000 HPLC1 series with L-7100 pump and L-7400 UV detector) with an ion-exchange column (Partisil-10 SAX, 4.6 mm × 25 cm; GL Science). Elution was performed at a flow rate of 1 ml/min by a gradient made up of either a low ionic strength buffer (7 mM KH2PO4, adjusted to pH 4.0 by H3PO4) or a high ionic strength buffer (0.5 M KH2PO4 + 0.5 M Na2SO4, adjusted to pH 5.4 by KOH). UV detection was done at 260 nm. Gradient conditions used in this study to separate NTP and dNTP were as follows. The percentage of high ionic strength buffer was increased during the initial 5 min from 0 to 15%, increasing again for the next 60 min from 15 to 60%, and again for 3 min from 60 to 100%, remaining at 100% for 20 min and then reduced from 100 to 0% in 3 min, then remaining at 0% for 10 min. Intracellular concentrations of nucleotides are expressed as pmol per mg wet cell weight (in agar plate culture) or pmol per AM500 (in liquid culture).

Assay for Streptomycin-- For streptomycin production, strains were inoculated on TSB agar medium. After incubation for the indicated time, agar pieces (diameter 8 mm) were cut from agar plates and were used to assay for streptomycin as described previously (10). B. subtilis ATCC6633 was used as a test organism.

RNA Isolation and S1 Nuclease Protection Assay-- Methods for RNA preparation and S1 mapping were carried out as described by Kieser et al. (28). Mycelia grown on the surface of agar medium covered with cellophane were harvested and immediately dispersed in 5 ml of modified Kirby mix solution and mixed thoroughly by vigorous vortexing with glass beads. Total RNA (100 µg, as estimated spectrophotometrically) was used in S1 nuclease protection assay. The hybridization probes were prepared by PCR using linearized plasmid DNA as a template and 32P-labeled primer, resulting in an unique 5'-end-labeled antisense single strand DNA fragment. For constructing the 329 nucleotides strR probe, SphI-digested pGEMstrRSG (as a template) and the primer 5'-CTCGGGGCTGTTCCCTGAAATATGC-3' (nucleotides +85 to +61 from the transcriptional start point (29)), which was 32P-labeled at the 5' end, were used. For constructing the 349 nucleotides hrdB probe, BamHI-digested pGEMhrdBSG (as a template) and the primer 5'-GGAACGATGGAAACGGCTTC-3' (nucleotides +110 to +91 from the transcriptional start point (30)), which was 32P-labeled at the 5' end, were used.

Expression and Purification of the EshA Protein-- Plasmid pAH0 used for the overexpression of the EshA protein in E. coli was constructed as follows. An insert sequence encoding the entire eshA gene was produced by PCR using the S. griseus 13189 genomic DNA as the template and the synthetic oligonucleotides (5'-AGAGCCTGCCATATGACTGTTGAC-3') and (5'-CCGGATCCCTACCTGGGCCAGTT-3') as primers. These primers contain sequences designed to create an NdeI site immediately prior to the initiation codon in the EshA open reading frame and a BamHI site just after the termination codon as designated by underlines. The 1.8-kb PCR product was digested with NdeI and BamHI and then inserted into the expression vector pPROEX-1 previously digested with NdeI and BamHI. The recombinant EshA protein that possesses an N-terminal His tag sequence was purified on nickel-nitrilotriacetic acid matrices (Qiagen). The EshA protein was further purified using an 8% SDS-PAGE and extracted with phosphate-buffered saline containing 0.1% SDS. Purified EshA protein (0.2 mg) was used to raise antibodies in rabbit.

Antiserum and Western Blotting-- Polyclonal antiserum against the EshA protein from S. griseus was prepared in rabbit using the recombinant protein prepared as described above. Crude extracts for Western blotting were prepared as follows. Cells collected from the cellophane sheet were suspended in buffer A (containing 20 mM Tris-HCl (pH 8.0), 10 mM Mg(CH3COO)2, 20 mM NH4Cl, 5 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 3 µg/ml of antipain, chymostatin, and leupeptin) and then disrupted by sonication. After centrifugation at 15,000 × g for 20 min at 4 °C, the resultant supernatant was used as a crude extract. For Western blotting, the crude extracts were subjected to SDS-PAGE (containing 8-10% acrylamide). Western blotting was performed as described previously (13).

Determination of DNA Synthesis-- Strains were grown in 10 ml of TSB medium in a 100-ml flask. When A500 reached 0.5, 1.5, 2.5, or 5.0, [2-14C]thymidine (PerkinElmer Life Sciences) was added to a final concentration of 20 µM (0.025 µCi/ml), and incubation was continued with shaking. Samples (0.5 ml) were withdrawn at the indicated time, and DNA synthesis was determined by measuring the incorporation of [2-14C]thymidine into the acid-precipitable portion as described previously (26).

Determination of Total DNA-- Strains were grown in TSB medium for the indicated time, and an aliquot of cultured broth (0.5 to 4 ml) was acidified with HClO4 to give a final concentration of 0.25 M. After standing in an ice bath for 30 min, samples were centrifuged, and the resulting precipitant (containing total DNA) was hydrolyzed two times by heating at 70 °C for 15 min in 0.5 M HClO4. The amount of total DNA was determined according to the method described by Burton (31) using diphenylamine reaction.

Isolation and Purification of Intact EshA-- The S. griseus wild-type strain 13189 was grown in GYM medium supplemented with 15% (w/v) sucrose for 30-40 h (late growth phase), and then cells were collected by filtration with filter paper (ADVANTEC, number 2). The resultant cells were suspended in buffer A (see above) without 2-mercaptoethanol, but containing lysozyme (2 mg/ml), and was incubated at 30 °C for 20 min, followed by a gentle sonication for 40 s. After cell debris was removed by centrifugation (8,000 × g for 10 min), EshA was precipitated by ultracentrifugation at 100,000 × g for 2.5 h. The precipitant was then suspended in buffer A and subjected to density gradient centrifugation using 10-30% linear sucrose gradient in buffer A. Finally, EshA protein was isolated by ion-exchange chromatography using a Mono-Q HR 5/5 column (0.5 × 5 cm, Amersham Biosciences).

Gel Filtration with EshA-- Gel filtration was carried out using the AKTA chromatography system equipped with a Superose 6 HR 10/30 column (1.0 × 30 cm, Amersham Biosciences). A molecular weight curve was obtained using the elution profiles of thyroglobulin, ferritin, catalase, aldolase (Gel Filtration HMW calibration Kit, Amersham Biosciences), and bovine serum (Wako) as standard proteins.

Electron Microscopy-- A drop of native EshA sample was placed on a carbon film and stained with uranyl acetate. Electron microscopy was performed with TEM (2010, JEOL) at an acceleration voltage of 80 kV and a magnification of 50,000.

Immunoelectron Microscopy-- Cells, which were grown on GYM agar, were fixed with 0.2% glutaraldehyde and 4% paraformaldehyde, dehydrated, and embedded in LR White resin (Nisshin EM) at 60 °C for 20 min. The resin was cooled and cut into thin sections using an ultramicrotome. Immunolabeling was performed using anti-EshA antiserum as the first antibody and anti-rabbit IgG conjugated to colloidal gold (diameter 10 nm) using the method described by Roth (32). Labeled cells were stained with 2% uranyl acetate and visualized by electron microscopy.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Disruption of eshA Gene-- Because Kwak et al. (20) reported previously that EshA is required for the extension of sporogenic hyphae during submerged spore formation in liquid culture, we assumed that disruption of the eshA gene may cause an impairment of aerial mycelium formation. Thus, we attempted to clarify the function of EshA in S. griseus by disrupting the eshA gene. Gene disruption by frameshift mutation was carried out using the wild-type strain 13189 as briefly illustrated in Fig. 1A. The resultant eshA disruptants grew normally, suggesting that EshA is not essential for viability. Disruption of the eshA gene was confirmed by sequence analysis and also by the lack of EshA protein production as determined by Western blotting (Fig. 1B). Furthermore, we detected the reappearance of EshA protein upon transforming disrupted cells with the plasmid pV52SG, which contains the wild-type eshA gene (Fig. 1B). Strain KO-390 was used for further study as a representative eshA disruptant.


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Fig. 1.   Gene disruption of eshA and its consequence. A, schematic of the strategy used for the disruption of eshA. Plasmid pSGRDelta p52Ap contains the eshA gene with a frameshift mutation at the unique BstPI site and the apramycin resistance gene (aac (3)IV) in the pBluescript SK(+) vector. The designation B represents BamHI. B, Western blot analysis of the EshA protein. Crude extracts were prepared from cells grown in SPY medium for 24 h. C, effect of eshA disruption on aerial mycelium formation. Spores were spread on TSB agar medium or TSB agar medium containing 0.2 mM decoyinine. The plates were incubated at 30 °C for 3 days. White color represents the development of aerial mycelium. D, effect of eshA disruption on streptomycin production. Agar pieces (diameter 8 mm) cut from each of the TSB agar plates after incubation for the indicated time were put on the assay plate, followed by 12 h of incubation.

First, the ability of KO-390 to form aerial mycelium was studied. Wild-type strain 13189 produced abundant aerial mycelium 2 days after incubation on TSB agar medium. In contrast, disruptant KO-390 almost completely lacked the ability to form aerial mycelium (Fig. 1C). The defects in aerial mycelium formation was, however, completely repaired (or even enhanced compared with the wild-type strain) when plasmid pV52SG was introduced into KO-390 (data not shown). KO-390 cells also failed to form aerial mycelium when cells were grown on sporulation agar medium; however, cells could produce aerial mycelium as abundantly as the parental strain when grown on GYM or SPY agar medium (data not shown). Thus, the ability of KO-390 to form aerial mycelium is conditional based on culture conditions. Previous studies (10) have demonstrated that a reduction of intracellular GTP pool can induce aerial mycelium and spore formation in S. griseus. To study whether a forced reduction of the intracellular GTP pool size could lead to restoration of aerial mycelium formation, decoyinine (a GMP synthetase inhibitor) was used. Notably, the ability for KO-390 to form aerial mycelium was completely restored when 0.2 mM decoyinine was added to the medium (Fig. 1C). Decoyinine was effective only when it was added at the early (until 16 h) growth phase. The supplementation of guanosine or deoxyguanosine together with decoyinine (each 0.2 mM) abrogated the ability of decoyinine to restore aerial mycelium formation (data not shown).

Second, the ability to produce antibiotic was studied (Fig. 1D). The wild-type strain produced streptomycin after a 36-48-h incubation on TSB agar medium, but the eshA disruptant KO-390 was unable to produce the antibiotic. This indicates that the EshA protein somehow plays a role in initiating the secondary metabolism in addition to morphological differentiation. The deficiency in producing streptomycin by the eshA disruptant was confirmed in liquid culture using TSB, SPY, and synthetic medium (data not shown). Interestingly, introduction of the plasmid pV52SG into KO-390 restored or even enhanced streptomycin productivity compared with the wild-type strain (Fig. 1D). Addition of decoyinine did not restore streptomycin production. The impaired ability to produce streptomycin may be the result from a failure of inducing the strR gene, which is a transcriptional activator of genes for streptomycin biosynthesis (33, 34). Therefore, we compared the strR transcription level between the wild-type and eshA disruptant strains. Transcription of hrdB encoding one of the major sigma  factors (30) was monitored as a positive internal control. S1 nuclease protection assay revealed that strR mRNA was transcribed growth phase-dependently in both strains, but the level of strR mRNA was 2-fold less in KO-390 than the wild-type strain (Fig. 2), accounting for the impaired ability to produce streptomycin in the eshA disruptant. There was no substantial difference in the level of hrdB mRNA between the wild-type and KO-390 strains (data not shown). The reduced expression of strR in the mutant is possibly due to the less accentuated accumulation of ppGpp at late growth phase (0.3-0.5 pmol/mg wet weight for the wild-type strain and 0.2-0.3 pmol/mg wet weight for KO-390) as determined using HPLC.


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Fig. 2.   S1 nuclease protection assay for the strR transcript. RNA was isolated from wild-type (WT) and mutant cells grown for the indicated time on TSB agar medium. 32P-Labeled, MspI-digested pBR322 size marker is shown at right. nt, nucleotide.

The eshA Disruptant Accumulates dNTP-- Morphological differentiation of B. subtilis and Streptomyces spp. is known to result from a decrease in intracellular GTP levels (18, 19, 26). Therefore, we speculated that the impaired ability of KO-390 to form aerial mycelium might result from an insufficient reduction of GTP (or dGTP) level. To examine this possibility, we monitored the levels of intracellular GTP and dGTP, together with the level of EshA protein, during growth on TSB agar medium. Expression of EshA (in wild-type cells) began at mid-growth phase (24 h) and then decreased at the end of growth (74 h) (Fig. 3). The GTP levels in the wild-type strain and the mutant strain both decreased sharply during the first 30 h of incubation, where the mutant strain displayed the GTP levels only slightly higher than did the wild-type strain. In contrast, it was striking that the eshA disruptant KO-390 accumulated deoxynucleoside triphosphate (dNTP) within the cell. An HPLC profile of extracted nucleotides from wild-type and mutant cells grown to late growth phase on TSB agar was shown in Fig. 4 as a representative example. It is evident from Fig. 4 that dGTP, dCTP, and dTTP all increased significantly in the mutant cells. The amount of dATP was not determined because ATP and dATP have the same retention time. The changes in the level of dNTP were monitored by cultivating the wild-type and mutant cells on TSB agar, and the results for dGTP are shown in Fig. 3 together with the results for GTP. It is apparent that the increase of dGTP (and also dCTP and dTTP (not shown)) typical for mutant KO-390 is temporary and that this accumulation occurs at the mid-growth phase (30 h). Thus, we concluded that the cell's excessive accumulation of dGTP can be a cause at least in part (if not entirely) for the impaired ability of the eshA disruptant to undergo morphogenesis.


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Fig. 3.   Changes in the intracellular concentrations of GTP and dGTP and levels of the EshA protein during growth on agar medium. Strains were grown on TSB agar medium covered with a cellophane sheet. Cells were harvested at the indicated time to determine biomass, nucleotide concentrations, and the amount of EshA protein produced. Nucleotides were separated by HPLC, whereas EshA protein was determined by Western blot analysis (see "Experimental Procedures"). , wild-type strain 13189; open circle , eshA disruptant KO-390.


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Fig. 4.   Elution profile from HPLC of nucleotides extracted from cells. The strains were grown on TSB agar covered with a cellophane sheet for 28 h. The samples injected were equivalent to 3 mg wet weight of cells. Elution from HPLC was done as described under "Experimental Procedures." The numbered arrows indicate the retention times for: 1, dGDP; 2, GDP; 3, dTTP; 4, UTP; 5, dCTP; 6, CTP; 7, ATP and dATP; 8, dGTP; 9, GTP.

To confirm the above conclusion, we investigated the effect of decoyinine and guanosine on the levels of intracellular GTP and dGTP. For this purpose, we used liquid culture instead of agar plate culture, because nucleotide levels can be studied in more detail in liquid culture. Like the agar plate cultures, wild-type cells grown in TSB liquid medium displayed a more accentuated decrease of GTP compared with the eshA disruptant KO-390 at the middle to late growth phase (Fig. 5). However, when KO-390 cells were treated with 0.5 mM decoyinine at the middle growth phase, there was a significant reduction of GTP and dGTP, accounting for the observed restoration of morphogenesis in this mutant (see Fig. 1C). The addition of guanosine together with decoyinine resulted in an increase of GTP to a level higher than non-treated cells, although dGTP level did not increase (Fig. 5). Addition of 0.2 mM deoxyguanosine, however, was accompanied by an elevated amount of dGTP as determined by HPLC analysis (data not shown). Thus, these results are all in agreement with the observed abolishment of decoyinine-induced aerial mycelium formation by addition of either guanosine or deoxyguanosine.


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Fig. 5.   Changes in the concentration of intracellular GTP and dGTP during growth in liquid medium. Wild-type strain 13189 (closed circles) and eshA disruptant KO-390 (open circles) were grown in TSB liquid medium. Decoyinine (0.5 mM; squares) and decoyinine plus guanosine (each 0.5 mM; triangles) were added 19 h after inoculation as indicated by the arrows. Samples were taken at the indicated times to determine culture density (A500) and GTP and dGTP concentrations.

Disruption of eshA Causes Arrest of DNA Synthesis-- dNTP serve as a substrate in DNA synthesis. Therefore, the elevated levels of dNTP in the eshA disruptant imply a disturbance of DNA synthesis at the late growth phase. To assess this possibility, we first investigated the effect of novobiocin, a known inhibitor for DNA synthesis, on intracellular dNTP level. When the wild-type strains grown in TSB liquid medium were treated with a low concentration (20 µg/ml) of novobiocin, dNTP levels were found to increase about 5-fold 1 h after the addition. Growth was inhibited only by 20% (data not shown).

The results of using novobiocin suggest that DNA synthesis in the eshA disruptant is somehow arrested to some extent, eventually leading to elevation of dNTP levels. This was confirmed by measuring directly the rate of DNA synthesis using [2-14C]thymidine. Wild-type (13189) and eshA disruptant (KO-390) strains were grown in TSB liquid medium (Fig. 6A). Both strains grew at the same growth rate until they reached stationary growth phase. Like agar plate cultures (see Fig. 3), the level of intracellular dGTP (and also other dNTP (not shown)) increased sharply at the late (20-24 h) growth phase in KO-390 cells (Fig. 6B). Wild-type cells show an abrupt increase in the amount of EshA protein at the time point b (which corresponds to 20 h in cultivation time) (Fig. 6C). Therefore, we monitored the rate of DNA synthesis at various growth phases using [2-14C]thymidine. Strikingly, 13189 and KO-390 displayed a similar rate of DNA synthesis during the early growth phase (time point a), but later KO-390 exhibited a significantly lower rate compared with 13189 (Fig. 6D). The reduction of the rate of DNA synthesis in KO-390 was most pronounced at the end of the growth phase (time point d). Similar results were obtained when a 5-fold higher concentration of [2-14C]thymidine (100 µM) was used to avoid the effect which pre-existing endogenous dTTP may have had (data not shown). Thus, failure to produce EshA protein in disruptant cells resulted in a reduced DNA synthesis during the middle to late growth phase, accompanied by an increase in dNTP levels. This conclusion was supported by the fact that the disruptant cells had a reduced content of DNA (13.4 versus 15.1 µg/AM500) at late growth phase (time point d), as determined by the method of Burton (31) (Table II).


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Fig. 6.   Growth and DNA synthesis of the wild-type (13189) and eshA disruptant strain (KO-390). Closed circles and open circles represent the wild-type strain and KO-390, respectively. A, growth of wild-type strain and KO-390 in TSB medium at 30 °C. The designation a-d indicate the specified time points in growth. B, changes in intracellular dGTP level during growth. C, Western blot analysis of EshA protein. Cells grown to the time points of a-d were harvested, and crude extracts (each 5 µg of total protein) were subjected to analysis. D, incorporation of [2-14C]thymidine into DNA during growth. [2-14C]Thymidine was added into the culture at the time points of a---d (see "Experimental Procedures"). The incorporation rate of [2-14C]thymidine is expressed as a specific activity (cpm × 102/AM500).

                              
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Table II
Changes in DNA contents during growth of S. griseus
The mean value of six to nine experiments is indicated with the standard deviation.

S. coelicolor eshA Disruptant Does Not Accumulate dNTP-- Our next interest was to know if EshA protein of S. coelicolor can replace in function the S. griseus EshA protein, because these two proteins share an amino acid sequence homology as high as 76% (21).

First, we found that, unlike S. griseus, the S. coelicolor eshA disruptant KO-350 (21) did not accumulate dNTP throughout whole growth cycle. Moreover, there was no substantial difference in the rate of DNA synthesis even at late growth phase when compared between the wild-type strain 1147 and the eshA disruptant KO-350 (data not shown). Thus, arrest of DNA synthesis by disrupting the eshA gene is a property characteristic for S. griseus.

Second, in order to see the effect of high level expression of the S. coelicolor flavor of EshA in an S. griseus eshA mutant, we cloned the S. coelicolor eshA gene into low copy number and high copy number plasmids, and we transformed the S. griseus eshA disruptant KO-390 with the resulting plasmids (pV52SC and pNS52SC; see Table I). However, there was no expression of EshA at all throughout the whole growth cycle as determined by Western analysis, possibly due to inactivity of the S. coelicolor eshA promoter in S. griseus cells.

Isolation and Characterization of the EshA Protein-- We next attempted to isolate the EshA protein in its native form. S. griseus cells (50 g wet wt) grown to the late growth phase (30-40 h) were harvested, disrupted by sonication, and centrifuged to remove cell debris. The supernatant was subjected to ultracentrifugation. The precipitant (containing EshA) was applied to sucrose gradient centrifugation (Fig. 7A), in which the EshA was fractionated with ribosomal 50 S particles as determined by Western blotting using EshA antibody (Fig. 7B). Finally, the EshA fraction was subjected to ion-exchange chromatography to separate EshA from the 50 S particles (Fig. 7C), eventually resulting in 28 mg of purified EshA protein. The purity of EshA protein was confirmed by SDS-PAGE in which EshA was shown to be 52 kDa in size (Fig. 7D) and by native gel electrophoresis in which EshA behaved as a macromolecule as well as ribosomal 30 S, 50 S, or 70 S particles (data not shown). The size of the EshA multimer was estimated to be 1100-1200 kDa as determined on the basis of results from gel filtration. This result indicates that the EshA multimer consists of about 20 EshA molecules.


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Fig. 7.   Purification of EshA by density gradient centrifugation and ion-exchange chromatography. A, ultracentrifuge precipitant containing EshA was fractionated on a 10-30% sucrose gradient. B, fractions containing the EshA protein were detected by Western blot analysis using the EshA antibody. C, the ion-exchange chromatography was performed using a Mono-Q HR 5/5 column. D, purity of EshA was determined by SDS-PAGE. Each lane contained the following: lane 1, low range SDS-PAGE standard (Bio-Rad); lane 2, crude extract; lane 3, fraction 12 after the sucrose gradient; lane 4, EshA protein purified by ion-exchange chromatography.

We performed electron microscopy on the EshA multimers. Surprisingly, the EshA multimer was found to be a cubic-like structure with a diameter of 27 nm (Fig. 8). The shape of each particle is uniform as seen from the photograph. The purified particles were found not to be associated with nucleic acid, as determined by staining with ethidium bromide. The characteristic shape was no longer detected when the EshA sample had been subjected to pH 5.0 or pH 10 before electron microscopy, indicating that this EshA multimer is fragile under physical stress.


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Fig. 8.   Transmission electron microscopy visualization of EshA multimers. EshA protein was stained with uranyl acetate and observed by transmission electron microscopy. B represents a simple expansion of a particle shown in A.

Localization of the EshA Protein-- Finally, we studied the localization of EshA proteins using immunoelectron microscopy. The results clearly indicated that the majority (if not all) of EshA protein is localized in the cytoplasm but not in the membrane (Fig. 9). To confirm this conclusion, we prepared protoplasts, burst the cells, and then subjected the sample to ultracentrifugation at 30,000 × g for 30 min. Majority (80%) of EshA was found in supernatant (cytoplasmic fraction), whereas only 20% was found in precipitate (membrane fraction), as determined by Western blotting (data not shown). Thus, we concluded that the EshA protein exists as a cytoplasmic protein at least in the experimental conditions used here.


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Fig. 9.   Localization of EshA proteins determined by immunostained electron micrograph of vegetative cells. EshA protein localized in a vegetative cell section was immunolabeled using anti-EshA antibodies and anti-rabbit IgG conjugated to 10 nm gold.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

One of the most intriguing challenges in biology is to elucidate mechanisms by which cells detect and respond to extracellular nutritional conditions. Among prokaryotes, B. subtilis and Streptomyces spp. offer a feasible system for studying such mechanisms by displaying a wide range of adaptations to nutrient limitations, including production and secretion of antibiotics and enzymes and formation of aerial mycelium (in Streptomyces spp.) and endospores (in B. subtilis) as an extreme response to nutrient limitation (35). In this regard, nutritional status and sporulation have been successfully linked in B. subtilis (14), especially CodY has been shown to be able to detect and respond to nutrient limitations. Recent works by Inaoka and Ochi (36) support its proposal. In this paper we described a novel function for the S. griseus EshA protein in DNA synthesis. Apparently, EshA is required to sustain the rate of DNA synthesis during the late growth phase, even though the lack of EshA does not affect the growth rate (i.e. increase in biomass) (Fig. 6A). Also, it is possible that the inability to form aerial mycelium (and thus sporulation) in the eshA disruptant mutant is a result of an unusual elevation of intracellular dGTP due to reduced DNA synthesis. Moreover, when the disruptant mutant was treated with decoyinine, a decrease of GTP sufficient to fully induce aerial mycelium formation was demonstrated (Figs. 1C and 5). Therefore, we conclude that disruption of the eshA gene indirectly affects morphogenesis of S. griseus. Rather, the EshA protein may function to regulate directly DNA synthesis. It is worth stressing that the aberrant phenotypes mentioned above for S. griseus are not the case for S. coelicolor; namely, the eshA disruption did not affect the ability for S. coelicolor to form aerial mycelium as examined using a wide variety of media (Ref. 21 and this study) and did not result in the aberrant accumulation of dNTP. Although we could not demonstrate the effect of expression of the S. coelicolor EshA protein within the S. griseus eshA disruptant cells, the differences in the behavior of the two hosts implicate qualitative differences in addition to quantitative (10-fold) differences between the two EshA proteins. Further work is required to elucidate this point.

Although we have demonstrated that disruption of eshA abolishes the aerial mycelium formation (in S. griseus) and antibiotic production (in S. griseus and S. coelicolor) (Ref. 21 and this study), it should be noted that these aberrant phenotypes are conditional. Apparently, this led to a discrepancy between our results and previous results reported by Kwak et al. (20), who stated that eshA disruptant from S. griseus is able to produce aerial mycelium and streptomycin normally. It should be pointed out that the expression of eshA commenced at late growth phase in both S. griseus and S. coelicolor as determined by Western blotting (21; and this study), indicating that EshA is a developmentally regulated protein. These results are in agreement with the previous works reported by Kwak et al. (20). Also important is the fact that propagation of wild-type eshA with low copy number plasmids caused overproduction of antibiotics in both S. griseus (Fig. 1D) and S. coelicolor (21). Therefore, eshA may offer a feasible target for strain improvement, because genes homologous to eshA appear to be widely distributed among streptomycetes (21).

Most interesting is the fact that EshA exists as a multimer in its native state (Fig. 7, A and B), possibly forming an icosahedron as indicated from the results of electron microscopy (Fig. 8), which is an architecture known for coat proteins of certain viruses. Molecular self-assembly is the spontaneous association of molecules into stable, structurally well defined aggregate joined by noncovalent bonds. Understanding self-assembly and the associated noncovalent interactions that connect complementary interacting molecules into aggregates is a central concern in structural biochemistry (37). A recent breakthrough has been reported describing the supramolecular fabrication of large complexes from small molecules by self-assembly, which is called "supramolecular chemistry" (38, 39). As discussed in detail previously by Kwak et al. (20), the EshA protein shows extensive similarity to proteins from mycobacteria and cyanobacterium, namely MMPI in Mycobacterium leprae and SrpI in Synechococcus sp. (40-42). However, there is no EshA homologue in E. coli, B. subtilis, or other prokaryotes whose genomes have been completely sequenced. EshA proteins in S. griseus and S. coelicolor (sharing 76% amino acid identity to one another) are characterized by the presence of a cyclic nucleotide-binding domain, which is absent from the corresponding proteins from mycobacteria and cyanobacterium. Therefore, EshA (termed P3 in the former article) can be classified as a member of the cyclic nucleotide-binding protein superfamily (20). The activity of this family of proteins is modulated by a cyclic nucleotide (43, 44). In prokaryotes, only Crp and DnaA are known to bind cAMP (45-47). The dnaA gene in E. coli has been shown to be an essential element in the initiation of DNA replication at the chromosomal origin, oriC (48). The DnaA protein with a size of 52.5 kDa interacts with cAMP, and this interaction stimulates the DnaA protein to bind to the chromosomal replication origin (49). It has also been shown that the DnaA protein possesses a high affinity for ATP, and ATP activates DnaA protein for the initiation of DNA replication (50). cAMP may also play a role in the reactivation of the DnaA protein by ensuring a rapid release of ADP after each ATP hydrolysis event (45). Although it is unknown whether the EshA protein exerts its influence via the nucleotide-binding domain, recent studies (51, 52) with Streptomyces spp. have demonstrated the existence of cAMP at a high extracellular level during the late growth phase. It is intriguing to investigate whether the nucleotide-binding domain (in the aid of a cyclic nucleotide(s)) plays a role in forming the characteristic multimer as shown in Fig. 8.

Previous studies (20, 42) from several laboratories have emphasized that EshA (and also the MMPI protein of M. leprae) may be a membrane protein. This conclusion is based on the fact that (i) EshA and MMPI were copurified with membrane fractions as determined by ultracentrifugation of crude extract, and (ii) EshA, MMPI, and SrpI all contain putative 14-17-amino acid transmembrane anchor domain at their C termini. However, results presented here indicate that EshA in S. griseus exists predominantly in cytoplasm as demonstrated by both immunoelectron microscopy (Fig. 9) and ultracentrifugation (see "Results"). This apparent discrepancy might be a result of EshA having two different forms, i.e. monomers and multimers. Therefore, localization of EshA within the cell may be directed according to these different conformational forms.

EshA is produced abundantly and accumulates during sporulation induced by phosphate starvation and nutritional downshift in S. griseus (20). Our results demonstrate that EshA in S. griseus is produced even in normal culture conditions when cells reach the middle to late growth phase (Fig. 3). Likewise, the SrpI protein is also induced under sulfur deprivation (53) and MMPI during infection by M. leprae (42). According to these facts, Triccas et al. (53) and Kwak et al. (20) proposed that EshA, SrpI, and MMPI may define a new family of bacterial stress-response proteins. Our present observations may be helpful to provide clues for understanding the mechanism of stress response in relation to protein chemistry and regulation of DNA synthesis.

    ACKNOWLEDGEMENTS

We are grateful to T. Yoshinaga, A. Lezhava, A. Hesketh, and L. Skhirtladze for preliminary work done in several of the experiments.

    FOOTNOTES

* This work was supported in part by a grant from the Organized Research Combination System of the Science and Technology Agency of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Translational Research Department, Mitsubishi Kagaku Institute of Life Sciences, Yokohama Research Center, 1000, Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan.

§ Present address: Dept. of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuda-Cho, Midori-ku, Yokohama 226-8501, Japan.

Present address: Dept. of Microbiology, School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8501, Japan.

|| To whom correspondence should be addressed: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Tel.: 81-29-838-8125; Fax: 81-29-838-7996; E-mail: kochi@affrc.go.jp.

Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M208564200

    ABBREVIATIONS

The abbreviation used is: HPLC, high performance liquid chromatography.

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RESULTS
DISCUSSION
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