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Journal of Bacteriology, June 2008, p. 4061-4068, Vol. 190, No. 11
0021-9193/08/$08.00+0     doi:10.1128/JB.01954-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

CabC, an EF-Hand Calcium-Binding Protein, Is Involved in Ca²⁺-Mediated Regulation of Spore Germination and Aerial Hypha Formation in Streptomyces coelicolor

Sheng-Lan Wang, Ke-Qiang Fan, Xu Yang, Zeng-Xi Lin, Xin-Ping Xu, and Ke-Qian Yang^*

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China

Received 16 December 2007/ Accepted 14 March 2008

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Ca²⁺ was reported to regulate spore germination and aerial hypha formation in streptomycetes; the underlying mechanism of this regulation is not known. cabC, a gene encoding an EF-hand calcium-binding protein, was disrupted or overexpressed in Streptomyces coelicolor M145. On R5– agar, the disruption of cabC resulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, while cabC overexpression significantly delayed development. Manipulation of the Ca²⁺ concentration in R5– agar could reverse the phenotypes of cabC disruption or overexpression mutants and mimic mutant phenotypes with M145, suggesting that the mutant phenotypes were due to changes in the intracellular Ca²⁺ concentration. CabC expression was strongly activated in aerial hyphae, as determined by Western blotting against CabC and confocal laser scanning microscopy detection of CabC::enhanced green fluorescent protein (EGFP). CabC::EGFP fusion proteins were evenly distributed in substrate mycelia, aerial mycelia, and spores. Taken together, these results demonstrate that CabC is involved in Ca²⁺-mediated regulation of spore germination and aerial hypha formation in S. coelicolor. CabC most likely acts as a Ca²⁺ buffer and exerts its regulatory effects by controlling the intracellular Ca²⁺ concentration.

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The importance of Ca²⁺ as a cell function regulator is well established in eukaryotes; however, the regulatory roles of Ca²⁺ in prokaryotes are less clear. In recent years, it has become increasingly evident that Ca²⁺ is involved in the regulation of many cellular behaviors in various prokaryotes, as reviewed previously by Dominguez (3). The intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in prokaryotes is tightly regulated in the 100 to 300 nM range (10, 13). There have been reports that Ca²⁺ is involved in the regulation of cell division (36), chemotaxis (27, 30), and cell differentiation processes such as sporulation, heterocyst formation, and fruiting body development (19, 21, 23, 37).

In eukaryotes, Ca²⁺ signaling is mediated by a large and functionally diverse family of calcium-binding proteins (CaBPs) containing EF-hand motifs. The minimal structural and functional unit of CaBP consists of a pair of EF hands, which form a discrete domain so that most family members have two, four, or six EF hands. An EF hand is defined by its helix-loop-helix secondary structure as well as the ligands in the loop to bind Ca²⁺; a pair of EF hands interacts through two short antiparallel β-sheets strategically located in the C-terminal ends of each Ca²⁺-binding loop (7). Ca²⁺-binding loops of 11 to 14 amino acid residues were reported for CaBPs, but the most common (canonical) EF hand has a 12-residue Ca²⁺-binding loop that starts with an aspartate and ends with a glutamate (7). Functionally, CaBPs can be divided into two groups: calcium sensors translate calcium concentrations into signaling cascades by interactions with target proteins in response to Ca²⁺ binding, whereas calcium buffers are proposed to modify the spatiotemporal calcium concentrations in a cell (9).

Although proteins containing a single EF-hand motif were identified in many prokaryotes (14, 20), authentic EF-hand CaBPs (with at least a pair of interacting EF hands) were found only in complex high-G+C-content actinomycetes (32) and a Rhizobium strain (31). The EF-hand CaBP identified from Saccharopolyspora erythraea (formerly Streptomyces erythreus), later named calerythrin, is the first authentic EF-hand CaBP discovered in prokaryotes (26).

Streptomycetes are known for their complex life cycle and ability to produce secondary metabolites. Eaton and Ensign first reported that Ca²⁺ regulates spore germination in Streptomyces viridochromogenes (4); later, Natsume et al. (16) reported that Ca²⁺ regulates aerial mycelium formation in many streptomycetes and that Ca²⁺-chelating agent, Ca²⁺ channel blocker, and calmodulin inhibitors repressed aerial hypha growth in Streptomyces alboniger (15). With the discovery of calerythrin from Saccharopolyspora erythraea (26) and several other EF-hand CaBPs from streptomycetes (35), one would expect that these CaBPs play important regulatory roles in these bacteria; however, several attempts to detect the in vivo functions of these proteins gave inconclusive results (25, 34, 35).

At least four EF-hand CaBP genes were identified on the Streptomyces coelicolor genome (1, 32): cabB (SCO5464) contains two EF hands, while cabC (SCO7647), cabD (SCO4411), and cabE (SCO0141) contain four EF hands. In this paper, we report evidence that one of these four EF-hand CaBPs, designated cabC, is involved in the regulation of spore germination and aerial hypha formation in S. coelicolor.

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Bacterial strains, plasmids, and growth conditions. Escherichia coli DH5, BL21(DE3), and ET12567(pUZ8002) cells, used for plasmid manipulation (demethylation) or conjugation, were grown and transformed according to standard procedures (12). Plasmids pSET152, pIJ8660, pKC1139 (gifts from Hua-Rong Tan and Keith Chater), and pIMEP (self-constructed) were used for vector construction; pET28a was used for cabC expression in E. coli cells. Total DNA extraction from Streptomyces and conjugation of shuttle plasmids into S. coelicolor M145 were done according to standard procedures (12).

Growth conditions and media. S. coelicolor strains were grown at 30°C on MS (2% mannitol and 2% soya flour), minimal medium (MM), R5 medium (rich medium), and R5– agar (12). R5– agar was modified from R5 medium by leaving out KH₂PO₄, CaCl₂, and L-proline.

Construction of a cabC expression plasmid. cabC was amplified by PCR with primers 5'-AGGATCCATGACGCCCACCAGCG-3' (the BamHI site is underlined) and 5'-GGAATTCTGACCGCACCCGCGAG-3' (the EcoRI site is underlined) using S. coelicolor M145 genomic DNA as a template. The PCR products were digested with BamHI and EcoRI inserted between BamHI/EcoRI-digested pET28a to obtain the expression plasmid pET28a-C. pET28a-C was transformed into E. coli BL21(DE3) cells.

Purification of recombinant CabC. A culture of E. coli BL21(DE3) cells carrying pET28a-C grown overnight was diluted 100-fold in LB broth containing 100 µg/ml kanamycin and grown at 30°C until the optical density at 590 nm reached 0.3 to 0.4. Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to the culture at a final concentration of 1 mM and incubated for 3 h. E. coli cells were harvested by centrifugation and disrupted by ultrasonication for 3 min with a 10-s pulse and 15-s interval. The N-terminal His-tagged CabC (23,006.86 Da) was purified by His-Bind columns (Novagen) according to the manufacturer's protocol. One milligram of purified protein was sent to the Animal Research Center, Institute of Genetics, Chinese Academy of Sciences, to inject rabbits and to generate anti-CabC serum.

Detection of calcium in CabC. Purified His-CabC was concentrated by ultrafiltration in an Amicon Ultra-15 (Millipore) tube at 1,500 x g at 4°C. The concentrated protein was washed three times with ultrapure water to eliminate buffer and other potential contaminants. The prepared protein was further treated according standard procedures and examined by inductively coupled plasma optical emission spectroscopy (ICP-OES) (Iris Intrepid II XSP; Thermo USA); emission at 317.9 nm was measured and compared to the specification curve produced from known concentrations of calcium. A radio frequency power of 1,150 W and a nebulizer flow of 26.0 lb/in² were used to run ICP-OES.

Construction of a disruption plasmid. A 2,249-bp region upstream of cabC was amplified by PCR with primers 5'-GTAAAGCTTGAACCAGCCACGATC-3' (the HindIII site is underlined) and 5'-CTAGATCTCCAGGTAGCCGTTTCCG-3' (the BglII site is underlined); an 1,874-bp region downstream of cabC was amplified with primers 5'-GTAGATCTTCCGAGTTCATCGCACTG-3' (the BglII site is underlined) and 5'-GGAATTCTGCCGACTCGATGG-3' (the EcoRI site is underlined). These fragments were digested by the bordering enzymes and inserted between the EcoRI and HindIII sites of pKC1139 by three-fragment ligation to obtain pWSL2. The plasmid was verified by restriction digestion and sequencing.

Disruption of cabC. pWSL2 was transformed into E. coli ET12567(pUZ8002) cells and conjugated into S. coelicolor M145 cells. Transconjugants were grown for 1 week on MM agar with apramycin at 37°C. Recovered spores were spread onto MM agar without apramycin. Apramycin-sensitive colonies were isolated and screened by PCR using primers 5'-AGGATCCATGACGCCCACCAGCG-3' and 5'-GGAATTCTGACCGCACCCGCGAG-3'. Colonies that gave the expected PCR product size were cabC disruptants; PCR products were sequenced to further confirm the deletion of an internal fragment.

Construction of a cabC complementation plasmid. A 1,249-bp DNA fragment containing cabC and its possible promoter was amplified with two primers: 5'-CAGAACGGGAACCCGAAGG-3' and 5'-GCTGGGCGTCGAGAATCG-3'. The PCR fragments were inserted into the T-easy vector and digested with EcoRI. The digested fragments were inserted into the EcoRI site of pSET152 to obtain plasmid pWSL7. The plasmid was examined by restriction digestion and sequencing. pWSL7 was transformed into S. coelicolor WSL-6 cells to obtain the complementation strain S. coelicolor WSL-7. WSL-7 partially complemented the phenotypes of WSL-6 but did not restore the phenotypes to those of M145 on R5– agar.

cabC overexpression plasmid construction. pIMEP was constructed in our laboratory; it contains an ermE* promoter and ribosomal binding site sequence upstream of the multiple cloning sites of pSET152. A 777-bp fragment containing cabC was amplified by PCR with two primers, 5'-AGGATCCATGACGCCCACCAGCG-3' (the BamHI site is underlined) and 5'-GGAATTCTGACCGCACCCGCGAG-3' (the EcoRI site is underlined). The amplified products were trimmed into a BamHI-EcoRI fragment and then inserted into pIMEP to obtain pWSL3.

Complementation and overexpression of cabC. pWSL3 was transformed into E. coli ET12567(pUZ8002) cells, conjugated into the cabC mutant (WSL-6) to obtain the complementation strain S. coelicolor WSL-3, and conjugated into S. coelicolor M145 to obtain the overexpression strain S. coelicolor WSL-4. Transformants were selected by apramycin resistance. S. coelicolor WSL-3 complemented the phenotypes of WSL-6 and restored the phenotypes to those of M145 on R5– agar.

Construction of cabC-EGFP gene fusion plasmid pWSL1. pKC1139 was digested with EcoRI and HindIII and used as a backbone to construct pWSL1. A 1.22-kb DNA fragment containing the entire cabC coding region was amplified by PCR using S. coelicolor M145 genomic DNA as a template with forward primer 5'-GATAAGCTTCGGGCGGTCTCGCAGTGAC-3' (the HindIII site is underlined) and reverse primer 5'-GATAGATCTGCCGGAGAGGCCGGCGG-3' (the BglII site is underlined). The forward primer was designed to contain a HindIII site, and the reverse primer was designed so that the stop codon (TGA) of cabC was removed and changed to cysteine (TGC) with the introduction of a BglII site. The 0.72-kb enhanced green fluorescent protein (EGFP) gene was amplified from pIJ8660 with forward primer 5'-GAAGATCTAAAGCCACCATGGTGAGCAAGGG-3' (the BglII site is underlined) and reverse primer 5'-GCCGATATCCTATCATCATTACTTGTACAGCTCGTC-3'(the EcoRV site is underlined). The forward primer was designed to introduce five additional amino acids (RSKAT) between CabC and EGFP as well as a BglII site, and the reverse primer was designed to contain three stop codons (TGA) and to contain an EcoRV site. Another 1.22-kb fragment carrying the flanking DNA downstream of cabC was PCR amplified from M145 with forward primer 5'-GTAGATATCCGGCCTCTCCGGCTGAC-3' (the EcoRV site is underlined) and reverse primer 5'-ATAGAATTCGGTGCTGGTCCACGGTATCG-3' (the EcoRI site is underlined). All PCRs were carried out using Pfu polymerase with hot start, and the PCR products were digested with the corresponding restriction enzymes and purified by using the Omega PCR purification kit (Omega, Beijing). The purified products were cloned into EcoRI-HindIII-digested pKC1139 by ligation to generate pWSL1.

Isolation of a cabC::EGFP gene fusion expression mutant. pWSL1 was transformed into S. coelicolor M145 by conjugation. Exconjugants were inoculated onto MM plates containing 10 µg/ml apramycin and incubated at 28°C for 7 days. Spore suspensions were prepared and spread onto an MM plate containing 10 µg/ml apramycin and incubated at 37°C. The spores were then restreaked onto an MM plate without apramycin. Spores were prepared again and plated out on an MM plate without apramycin to give single colonies. Total DNAs from apramycin-sensitive colonies were prepared and screened by PCR using primers 5'-AGGATCCATGACGCCCACCAGCG-3' and 5'-GGAATTCTGACCGCACCCGCGAG-3. The possible cabC replacement mutants were verified by PCR and sequencing of the PCR product.

Morphology observation under a light microscope. The wild-type, cabC disruption mutant, and cabC overexpression and complementation strains were inoculated onto R5– agar for observation on a plate. Coverslips were inserted after inoculation and observed after 3 days with a light microscope (CLSM 510; Zeiss, Germany).

Confocal microscopy and image processing. S. coelicolor WSL-1 was inoculated onto R5– agar, and coverslips were inserted after inoculation and observed after 3 days with a confocal laser scanning microscope. The confocal laser scanning microscope equipped with a Zeiss inverted microscope and an argon laser (excitation at 488 nm and 515 nm) was used to examine the fluorescence of EGFP in S. coelicolor WSL-1. A laser at 488 nm was used to excite EGFP; fluorescence images and transmission images (using a separate detector) were obtained simultaneously. All images were stored in a workstation, and further image processing was carried out using Adobe Photoshop after conversion of the images into JPEG format.

Scanning electron microscopy. Spores and hyphae of M145 and mutant strains grown at 30°C for 3 days on R5– agar were examined by scanning electron microscopy. To prepare specimens, agar blocks were cut from the R5– agar. Agar blocks were fixed with 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) and incubated at 4°C overnight. After being rinsed for 15 min with 0.1 M sodium phosphate buffer three times, they were postfixed in 1% (wt/vol) osmium tetroxide for 2 h at 4°C and rinsed for 15 min with the same buffer three times. Samples were then dehydrated in a graded series of ethanol solutions (30%, 50%, 70%, 85%, 95%, and 100% ethanol) for 20 min in each solution. Finally, 100% ethanol treatment was replaced with two 3-methylbutyl acetate treatments. Samples were critical-point dried, sputter coated with platinum, and observed under a scanning electron microscope (model FEI Quanta 200).

Protein sample preparation for Western blotting. Sterilized cellophane was placed onto the surface of R5– agar and inoculated with a spore suspension. Mycelia were collected from the cellophane at intervals during growth, resuspended in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄ [pH 7.2]) buffer, and disrupted by ultrasonication at 200 V for 6 min with a 10-s pulse and 15-s internal. The disrupted cell mixtures were centrifuged at 10,000 x g for 15 min. The supernatants were used as samples for Western blotting.

Western blotting. The protein concentration was measured using the bicinchoninic acid method (24). Samples of 30 µg of total protein were mixed with sodium dodecyl sulfate loading buffer and heated in boiling water for 10 min. Total protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%). The separated proteins on a polyacrylamide gel were transferred onto a polyvinylidene difluoride membrane (Bio-Rad) using a Bio-Rad Mini Trans-Blot system. The membrane was blocked for 1 h with 2% bovine serum albumin (Sigma) in Tris-buffered saline (20 mM Tris, 0.5 M NaCl [pH 7.5]). The primary antibodies, anti-CabC serum, were diluted at a 1:3,000 ratio in Tris-buffered saline with 0.05% Tween 20 containing 1% bovine serum albumin and incubated with the membrane for 2 h. Goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate (Beijing Zhong Shan Jin Qiao Company) was used as a secondary antibody. The membrane was incubated with chromogen and exposed to X-ray films for 1 min.

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Characteristics of CabC. cabC encodes a protein of 179 amino acids with a calculated molecular mass of 19,463 Da and pI of 4.18. Blast searches showed that CabC is most similar to several proteins from actinomycetes and numerous CaBPs from eukaryotes. It is similar to a hypothetical protein (CAE45695) from Streptomyces parvulus (58% identity), a putative calcium binding protein (CAJ89382) from Streptomyces ambofaciens (50% identity), and calerythrin from Saccharopolyspora erythraea (25% identity).

The secondary structure of CabC predicted by PROF (http://cubic.bioc.columbia.edu/predictprotein/) fitted the nuclear magnetic resonance-determined structure of calerythrin (28) very well. Both proteins consist of 4 EF-hand motifs (Fig. 1), although only the first three EF hands in CabC contain the amino acid ligands to bind Ca²⁺, whereas in calerythrin, EF hands I, III, and IV were shown to bind Ca²⁺ (28).

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FIG. 1. Sequence alignment of CabC and calerythrin. The secondary structural elements of nuclear magnetic resonance-determined calerythrin are labeled below the alignment: cylinders mark -helices, blank arrows mark β-sheets, and black lines mark loops. The four EF hands are numbered I to IV; the conserved Ca2+-binding amino acid residues at positions 1, 3, 5, and 12 of the EF-hand loop regions are marked with *.

Overall, the sequences, EF-hand arrangements, and structures of CabC and calerythrin resemble those of sarcoplasmic CaBPs found in invertebrates (8, 28) that normally bind two to three Ca²⁺ ions and are presumed to function as intracellular calcium buffers. To determine whether CabC shows large conformational changes in response to Ca²⁺, far-UV circular dichroism spectra of CabC in the presence or absence of Ca²⁺ were recorded. The circular dichroism spectra of CabC in 10 mM CaCl₂ or in 10 mM EGTA were similar. Both showed characteristics of -helices, with negative peaks at about 208 and 222 nm and a positive peak at about 194 nm. The only difference between the two spectra was that the spectrum in CaCl₂ solution was slightly reduced at 208 nm, indicating that the conformational changes of CabC in response to Ca²⁺, if any, were very small. The small conformational changes of CabC were in sharp contrast to those of CabB, a two-EF-hand Ca²⁺-binding protein of S. coelicolor A3(2) studied previously by Yonekawa et al. (35), which showed large conformational changes in response to Ca²⁺. The sequence and structure similarity between CabC, calerythrin, and sarcoplasmic CaBPs implied that they had similar functions.

Calcium in CabC. The calcium content in purified His-CabC was determined using ICP-OES; the calculated molar ratio of calcium to CabC is 2.63:1. The ratio suggests that CabC binds about three Ca²⁺ ions under the conditions that we used, which is consistent with the three functional EF hands in CabC predicted by structural modeling. The bound Ca²⁺ could not be titrated out with chelating agents.

Phenotypes of cabC disruption mutants. cabC locates upstream of an oppositely oriented gene (SCO7648) that encodes a probable response regulator of the two-component regulatory system. Upstream of cabC and oriented in the same direction lies a gene (SCO7646) that encodes a possible membrane protein; SCO7646 and cabC are separated by 308 bp (Fig. 2). This gene organization pattern suggests that cabC may be a single transcript controlled by an upstream promoter within the 308-bp intergenic region. The disruption of cabC is not expected to influence the function of the downstream gene (SCO7648) since it is oppositely transcribed from cabC.

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FIG. 2. Gene organization around cabC.

The cabC disruption mutants of S. coelicolor M145, designated WSL-6 and WSL-18, in which a 270-bp fragment within the coding region of cabC was deleted were isolated. Both mutants were verified by PCR and sequencing of PCR products. The absence of CabC expression in two mutants grown on R5–agar was confirmed by Western blotting with M145 as a positive control (data not shown).

Spores of WSL-6 and WSL-18 were streaked onto different solid media including MS, R5–, R5, and MM supplemented with different carbon sources. Both mutants produced aerial hyphae earlier on R5– agar than did M145 (Fig. 3A); no obvious phenotypical differences were observed on other media. The phenotypes with R5– agar were observed for 10 days; the most obvious phenotypical differences were observed after 3 to 4 days incubation. This time point (3 to 4 days of incubation on R5– agar) was chosen for further observations under a light microscope and a scanning electron microscope.

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FIG. 3. Phenotypes of the cabC disruption mutants and S. coelicolor M145 on R5– agar. (A) Picture of cabC disruption mutants and M145 on an R5– plate after 3 days. a, S. coelicolor M145; b, WSL-6; c, WSL-18. (B to D) Phenotypes of M145 on R5– agar after 3 days. (B) Aerial hyphae under a light microscope; (C) spore chains under a light microscope; (D) aerial hyphae under a scanning electron microscope. (E to G) Phenotypes of WSL-6 on R5– agar after 3 days. (E) Dense aerial hyphae with many short branches (white arrows) under a light microscope; (F) early-germinated spores (white arrows) on spore chains under a light microscope; (G) swollen aerial hyphal tips (white arrows) under a scanning electron microscope. Bars, 5 µm.

Observations of WSL-6 under a light microscope and a scanning electron microscope showed that it produced aerial hyphae with many short branches, and many branches appeared near the hyphal tips (Fig. 3E), suggesting that the disruption of cabC caused hyperbranching. Under a scanning electron microscope, WSL-6 was also observed to show swollen hyphal tips (Fig. 3G).

Noticeably, the disruption of cabC also caused early spore germination (Fig. 3F). Under light microscopy, some spores germinated on the spore chain of WSL-6, while no early germination was observed for M145 after 3 days of incubation (Fig. 3C). These results indicate that the regulation of spore germination was affected in WSL-6.

Overexpression of cabC inhibited aerial hypha formation and spore formation. To further confirm the effects of cabC on hyphal growth, it was overexpressed in S. coelicolor M145; pWSL3 was transformed into M145 cells by conjugation. The transformant was designated S. coelicolor WSL-4.

The overexpression of cabC delayed aerial hypha growth and spore formation significantly. After being grown for 4 days on R5– agar, aerial hyphae of WSL-4 were sparse compared to those of M145 (Fig. 4A), and no spores could be observed even after 7 days of incubation. Light microscopy and scanning electron microscopy gave similar results. Few aerial hyphae could be observed for WSL-4 grown on R5– agar after 4 days under light microscopy (Fig. 5B). Under scanning electron microscopy, small granules appeared over the aerial hyphae except at the zone near hyphal tips (Fig. 4C).

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FIG. 4. Phenotypes of cabC overexpression strain S. coelicolor WSL-4 on R5– agar. (A) WSL-4 and M145 on R5– agar. a, WSL-4; b, M145. (B) WSL-4, with sparse aerial hyphae under a light microscope. (C) WSL-4, with tiny granules (white arrows) on the hyphal surface under a scanning electron microscope. (D) M145, with aerial hyphae under a light microscope. (E) M145, with aerial hyphae under a scanning electron microscope. Bars, 5 µm.

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FIG. 5. Phenotypes of S. coelicolor M145 on R5– agar with 10 mM CaCl2 or with 10 mM EGTA. (A to D) Phenotypes of M145 on R5– agar with 10 mM CaCl2. (A) Dense aerial hyphae; (B) dense aerial hyphae with many short branches; (C) geminated spore under a light microscope; (D) swollen hyphal tips under a scanning electron microscope. (E to G) Phenotypes of M145 on R5– agar with 10 mM EGTA. (E) Sparse aerial hyphae; (F) sparse aerial hyphae under a light microscope; (G) tiny granules (white arrows) on the hyphal surface under a scanning electron microscope. Bars, 5 µm.

Ca²⁺ concentration in R5– agar affected hyphal growth in S. coelicolor M145. The above-described results suggested that both the disruption and the overexpression of cabC affected aerial hyphal growth in S. coelicolor M145; we deduced that the phenotypes are due to intracellular Ca²⁺ concentration changes caused by a loss or gain of CabC function.

To test the above-mentioned hypothesis, we tried to change the intracellular Ca²⁺ concentration in M145 cells by preparing modified R5– agar containing either 10 mM EGTA or 10 mM CaCl₂. Hyphal growth and spore germination were observed under a light microscope and a scanning electron microscope.

M145 cells exhibited dense aerial hyphae on R5– agar with 10 mM CaCl₂ (i.e., many short branches appeared along the aerial hyphae) (Fig. 5A and B), swollen hyphal tips (Fig. 5D), and early spore germinations on the spore chain (Fig. 5C) after 4 days of incubation. All these phenotypes are similar to those of cabC mutants (WSL-6 and WSL-18), suggesting that these phenotypes maybe caused by increased intracellular Ca²⁺ concentrations when CabC function was abolished (in cabC mutants).

In contrast, M145 cells grown on R5– agar with 10 mM EGTA showed phenotypes similar to those of a cabC overexpression mutant (WSL-4). Aerial hyphae were sparse (Fig. 5E); tiny granules appeared over the hyphal surface (Fig. 5G), as also observed in WSL-4, supporting the hypothesis that the observed phenotypes of WSL-4 are due to low intracellular Ca²⁺ concentrations caused by the chelating effect of EGTA.

The above-described results support the hypothesis that CabC regulates the hypha growth pattern by controlling intracellular Ca²⁺ concentrations in M145 cells. When the Ca²⁺ concentration (10 mM CaCl₂) was high, M145, WSL-6 (cabC disruption mutant), and WSL-4 (cabC overexpression mutant) cells all produced aerial hyphae on R5– agar, although WSL-6 produced the most aerial hyphae. In contrast, on R5– agar with a low Ca²⁺ concentration (10 mM EGTA), M145 and WSL-4 did not produce any aerial hyphae, while WSL-6 still produced sparse aerial hyphae. The facts that WSL-4 regained the ability to produce aerial hyphae when the Ca²⁺ concentration is high in environment and that WSL-6 produced less aerial hyphae when the Ca²⁺ concentration is low in the environment all suggest that these phenotypes are regulated by an internal Ca²⁺ concentration, which is dynamically influenced by CabC's buffering function and environmental Ca²⁺ concentration.

Temporal and spatial expression of CabC in S. coelicolor. To investigate the temporal and spatial expression of CabC in M145, EGFP was used as a reporter. To visually observe CabC expression in M145 cells, cabC was replaced by a cabC C-terminal EGFP fusion gene (cabC::EGFP gene) on the genome; the strain carrying this fused gene was designated S. coelicolor WSL-1. WSL-1 showed phenotypes similar to those of M145 on R5– agar, suggesting that the CabC::EGFP fusion protein did not disturb the function of CabC.

The fluorescence of WSL-1 grown on R5– agar was observed using a confocal laser scanning microscope over 7 days (Fig. 6A to E). Fluorescence was weak and evenly distributed in substrate mycelia at 24 h (Fig. 6A), and it remained weak during substrate mycelium growth at 36 h (Fig. 6B). In young aerial hyphae, the florescence turned strong and was evenly distributed (Fig. 6C); it remained strong and evenly distributed during aerial hypha growth (Fig. 6D). Fluorescence in spores was relatively strong and evenly distributed but was slightly weaker than that in aerial hyphae (Fig. 6E). The fluorescence disappeared in lysed substrate or aerial hyphae (data not shown). These results indicated that cabC expression is turned on before or at the onset of aerial hypha formation and matched well with Western blot results using anti-CabC antibody (Fig. 7). The expression of CabC was weakly detected at early stages of growth, increased to the highest levels after 3 to 4 days, and decreased after 5 to 7 days. Under the growth conditions used, M145 cells grew as substrate mycelia until 36 h, as a mixture of substrate and aerial mycelia from 36 to 84 h, and as a mixture of aerial mycelium and spores after 84 h.

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FIG. 6. Detection of CabC-EGFP's spatial and temporal expression in S. coelicolor WSL-1 on R5– agar by confocal microscopy. Transmission images of samples taken at different incubation times are shown on the left; the corresponding EGFP fluorescence images by confocal microscopy are shown on the right. Sample inoculation times were 24 h (A), 36 h (B), 72 h (C), 96 h (D), and 144 h (E). (A and B) Substrate hyphae; (C and D) aerial hyphae; (E) spores. Bars, 5 µm.

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FIG. 7. Western blot detection of CabC expression in S. coelicolor M145 cells grown on R5– agar using anti-CabC polyclonal antibodies. Lanes: M, marker; 1, WSL-6 protein extract (negative control) after 4 days; 2, His-CabC (positive control) purified from E. coli BL21 cells; 3 to 9, protein extracts of M145 cells prepared at different incubation times, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, and 144 h, respectively. The arrow on the left indicates the position of His-CabC (20 kDa) purified from E. coli BL21 cells; the arrow on the right indicates the position of CabC (19 kDa) expressed in M145 cells.

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EF-hand CaBPs were reported for several complex high-G+C-content actinomycetes (26, 32, 34, 35). When expressed in Streptomyces lividans, calerythrin, the Saccharopolyspora erythraea CaBP, did not show apparent effects on growth or differentiation (25, 26). The disruption or overexpression of cabA, a four-EF-hand CaBP gene, in S. ambofaciens showed no detectable phenotypes on YMA agar (0.4% yeast extract, 1% malt extract, 1% glucose, and 2% agar) (34). cabA was stably expressed at between 30 h (substrate hyphae) and 54 h (a mixture of substrate and aerial hyphae) on YMA agar supplemented with 5 mM CaCl₂. A two-EF-hand CaBP gene, designated cabB, was identified in S. coelicolor, S. ambofaciens, Streptomyces griseus, and Streptomyces avermitilis. In S. coelicolor, cabB was stably expressed at between 36 and 184 h; in S. ambofaciens, the expression of cabB was examined at between 30 and 54 h. A mutant of S. coelicolor cabB grew, produced actinorhodin, and formed aerial mycelia and spores normally on R2YE agar (35) containing 20 to 100 mM CaCl₂, but growth of the mutant on minimal agar containing more than 20 mM CaCl₂ was delayed compared to the growth of the wild-type strain; the overexpression of cabB in S. coelicolor and S. lividans resulted in a decrease in CFU from spores on the medium containing Ca²⁺ at a high concentration, but no other apparent phenotypes were detected with various media (including MM) containing different carbon and nitrogen sources. Therefore, data on the regulatory roles of cabB, if any, are inconclusive (35). In our study, phenotype changes of cabC mutants were observed on R5– agar, a rich medium modified from R5 medium by deleting CaCl₂, KH₂PO₄, and proline. The omission of CaCl₂ lowered the concentration of free Ca²⁺ in R5– agar, while the omission of KH₂PO₄ prevented the formation of CaHPO₄ precipitation. In R5– agar, important nutrients and ions such as phosphate and Ca²⁺ are supplied in yeast extract and agar (solid medium); further manipulation of the Ca²⁺ concentration in R5– agar is much easier. Our results showed that R5– agar worked as a suitable medium for the observation of Ca²⁺-regulated phenotypes. It was much harder to manipulate the Ca²⁺ concentration in MM due to the presence of KH₂PO₄; nevertheless, we could show that high Ca²⁺ concentrations induced aerial hypha formation (data not shown).

CabC is a four-EF-hand calcium-binding protein based on sequence analyses (1, 32) and crystal structure (X.-Y. Zhao et al., unpublished data). If CabC functions as a Ca²⁺ buffer as discussed above, its loss or gain of function is expected to influence the intracellular Ca²⁺ concentration, thus causing a physiological shift. On R5– agar, the disruption of cabC resulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, while the overexpression of cabC led to delayed aerial hyphae and spores. Manipulation of the environmental calcium concentration in M145 cells could mimic the phenotypes of cabC disruption or overexpression mutants, supporting the notion that CabC acts as a Ca²⁺ buffer.

Unlike CabA (S. ambofaciens) and CabB proteins (S. coelicolor and S. ambofaciens) (34, 35), which were stably expressed in substrate hyphae and aerial hyphae under their respective growth conditions, CabC was not expressed or was only weakly expressed in substrate hyphae but strongly expressed in aerial mycelia on R5– agar.

In aerial hyphae and spores, the high level of CabC should keep the free Ca²⁺ concentration very low. We suspect that in spores, a low Ca²⁺ concentration prevents spore germination. However, under certain circumstances, high environmental Ca²⁺ concentrations could trigger spore germination; this is supported by the observation that spores germinated on the spore chains in CabC mutants or in high-Ca²⁺ environments. During substrate hypha growth, due to the dilution of CabC in the rapidly growing cell, the intracellular Ca²⁺ concentration may gradually increase, or at certain point in growth, a Ca²⁺ influx function maybe turned on, which again causes the increase of the Ca²⁺ concentration to a value that triggers the onset of aerial hypha formation. During aerial hypha growth or sporulation, the high level of CabC serves to reduce the Ca²⁺ concentration to a low level again.

Streptomycetes, unlike most other bacteria, grow as branching hyphae in a manner highly analogous to that of filamentous fungi, characterized by polar growth at the hyphal tips, the apices. This uneven growth is due to the de novo incorporation of peptidoglycan precursors at the apices but not at other sites of the hyphae, in sharp contrast to the even growth in simple rod-shaped bacteria such as E. coli. The mechanisms of creating apices in streptomycetes are largely unknown. DivIVA, a protein that is present in all gram-positive bacterial genomes, is the first one implicated in polar growth in S. coelicolor (5, 6). DivIVA was distinctively localized to hyphal tips and lateral branches; its overexpression led to swollen hyphal tips, while its partial deletion led to irregularly shaped hyphae and irregular branches. SsgA was also recognized for its key role in regulating polar growth; SsgA-like proteins are found exclusively in sporulating actinomycetes. SsgA-like proteins play a chaperonin-like role in supporting the function of enzymes involved in peptidoglycan maintenance (penicillin-binding proteins and autolysins) and most likely mark the sites where changes in local cell wall morphogenesis are required, in particular, septum formation and germination (17, 18). The swollen hyphal tips and hyperbranching observed in cabC mutants suggest that the Ca²⁺ concentration (not CabC) regulates polar growth during spore germination and aerial hypha formation.

In eukaryotes, polar growth is regulated by a tip-high cytoplasmic Ca²⁺ gradient (2); the local high Ca²⁺ concentration could directly regulate the functions of many apically located Ca²⁺-dependent enzymes, or it could initiate a regulatory cascade (11, 22, 29, 33). The large body of information on the mechanisms to generate and maintain a tip-high Ca²⁺ gradient and on the major protein complexes recruited to apices and their roles in polar growth are beyond the scope of this paper. However, it is reasonable to speculate that streptomycetes use Ca²⁺ concentrations as a signal to regulate polar growth-related morphogenesis.

 
We thank Hua-Rong Tan and Keith Chater for kindly providing S. coelicolor M145 and plasmids pKC1139, pIJ8660, and pSET152; Gang Liu, Hai-Hua Yang, and Yu-Qing Tian for their technical assistance; Lu-Hua Lai and Ao-Neng Cao for their assistance in operating the circular dichroism spectrometer; Chun-Li Li and Jia-Yi Xie for their assistance in operating the confocal microscope and scanning electron microscope; and Xiao-Yan Zhao.

This work was supported by the National Natural Science Foundation of China (grant 30400259).

 
* Corresponding author. Mailing address: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China. Phone: 86-10-64807457. Fax: 86-10-64807459. E-mail: yangkq{at}im.ac.cn

Published ahead of print on 28 March 2008.

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Journal of Bacteriology, June 2008, p. 4061-4068, Vol. 190, No. 11
0021-9193/08/$08.00+0     doi:10.1128/JB.01954-07
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