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A calmodulin-like protein in the bacterial genus Streptomyces
FEMS Microbiology Letters 244 (2005) 315–321 www.fems-microbiology.org
A calmodulin-like protein in the bacterial genus Streptomyces Tohru Yonekawa, Yasuo Ohnishi, Sueharu Horinouchi
*
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received 5 January 2005; received in revised form 31 January 2005; accepted 2 February 2005 First published online 13 February 2005 Edited by R.P. Gunsalus
Abstract The gene, named cabB, encoding a calmodulin-like protein of 70 amino acids containing two helix–loop–helix EF-hand motifs was cloned from Streptomyces coelicolor A3(2). cabB was transcribed from a single promoter throughout growth. The CabB protein produced in Escherichia coli was a monomer in solution, although it corresponded to one half of a dumbbell shape of the eukaryotic calmodulins. CabB bound calcium and upon binding of calcium its a-helix content was increased, as determined by circular dichroism spectroscopy. The growth of cabB-disruptants (mutant DcabB) on minimal agar medium containing calcium higher than 20 mM was delayed, suggesting that CabB has a role in calcium homeostasis by serving as a calcium buffer or transporter, as suggested for calerythrin in actinomycetes and the invertebrate sarcoplasmic calcium-binding proteins. Wide distribution of cabB almost exclusively in actinomycetes suggests a common role of EF-hand CabB-type proteins in these filamentous, soil-dwelling Gram-positive bacterial genera. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Ca-binding protein; EF-hand motif; Streptomyces ambofaciens; Streptomyces coelicolor A3(2)
1. Introduction In eukaryotes, helix–loop–helix ‘‘EF-hand’’ proteins including calmodulin are ubiquitous and involved in various physiologically important regulatory or buffering functions. Calcium thus serves as a second messenger, controlling various functions. In prokaryotes, several calmodulin-like proteins containing two or more authentic EF-hand motifs have been deposited in the databases. However, the role of calmodulin-like proteins in bacteria is poorly understood, although involvement of calcium in many processes, such as sporulation, virulence, septation, chemotaxis, and phosphorylation, has been observed [1,2]. The first prokaryotic protein shown to
*
Corresponding author. Tel.: +81 3 5841 5123; fax: +81 3 5841 8021. E-mail address: [email protected] (S. Horinouchi).
contain typical EF-hand motifs and to bind calcium ions was calerythrin from the actinomycete Saccharopolyspora erythraea [3,4], which probably functions as a calcium ion buffer rather than having a regulatory role [5]. A motif search of the databases by Michiels et al. [1] revealed the presence of many bacterial proteins containing the EF-hand motif, although most of the identified proteins are hypothetical or of unknown function. We previously isolated a gene (named cabA) encoding calerythrin from Streptomyces ambofaciens to determine its possible role in morphological differentiation [6], because calcium is known to be important for aerial mycelium formation in the filamentous, soil-dwelling, Gram-positive bacterial genus Streptomyces [7,8]. Contrary to our expectation, cabA-disruption gave no detectable phenotypic alterations to the host, probably because CabA serves as a calcium ion buffer [6]. We then aimed at SC3D11.21 in Streptomyces coelicolor A3(2)
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.02.003
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[9], which shows high similarity in amino acid sequence to eukaryotic calmodulins and is totally different in overall structure from calerythrin or CabA. In the present study, we cloned SC3D11.21 and expressed it in Escherichia coli to determine its Ca-binding ability. Because it showed a distinct Ca-binding activity, we named it CabB. We also disrupted cabB on the chromosome to determine its role in morphological development and secondary metabolism.
(SDS–PAGE) and blotted onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated with 0.037 MBq/ml 45Ca (Amersham Pharmacia) in 60 mM KCl, 5 mM MgCl2, and 10 mM imidazole, pH 6.8, for 20 min at room temperature. The final concentration of Ca2+ in the incubation mixture was 64 lM. After removal of unbound 45Ca by vigorous washing, bound radioactivity was visualized by autoradiography. 2.4. Circular dichroism spectroscopy
2. Materials and methods 2.1. Cloning of cabB from S. coelicolor A3(2) and S. ambofaciens On the basis of the S. coelicolor A3(2) genome sequence (on cosmid SC3D11), the cabB gene was cloned from S. coelicolor A3(2) M130 by PCR with a pair of primers, 5 0 -GGACCCACATATGGCGGACATCGAGGAAGCACGCAAG-3 0 (the underline indicates an NdeI site) and 5 0 -GGTGGTTGCTCTTCCGCACTTGTTCAGGTGAGCCCGGAA-3 0 (the underline indicates a SapI site). The amplified DNA fragment was inserted between the NdeI and SapI sites of pTWIN1 (New England BioLabs), resulting in pTWIN1-cabB. No errors during PCR were confirmed by nucleotide sequencing. cabB in S. ambofaciens was cloned and sequenced by standard DNA manipulations including Southern hybridization and colony hybridization with the cabB sequence from S. coelicolor A3(2) as the 32P-labelled hybridization probe. The nucleotide sequence of the 2.0-kb SmaI fragment cloned in this way has been deposited to the EMBL, DDBJ, and GenBank databases under Accession No. AB194697. 2.2. Purification of recombinant CabB An overnight culture of E. coli BL21 (DE3) [pTWIN1-cabB] was diluted 100-fold into Luria-Bertani broth containing ampicillin at 100 lg/ml and grown at 37 °C until OD590 (optical density at 590 nm) reached 0.3–0.4. Isopropyl-b-D-thiogalactopyranoside (IPTG) was then added at a final concentration of 1 mM and the culture was continued for additional 3 h. The E. coli cells were harvested by centrifugation and CabB was purified according to the instructions in the New England BioLabs manual. The CabB protein was purified without any tags. 2.3. Ca binding assay Two micrograms each of recombinant CabB and bovine brain calmodulin (Sigma) as a positive control were run on 15% SDS–polyacrylamide gel electrophoresis
Protein solutions (2 mg/ml each of CabB and bovine brain calmodulin) were prepared with distilled water plus 10 mM CaCl2 or 2 mM EGTA [(ethyleneglycolbis(b-aminoethylether)-N,N,N 0 ,N 0 -tetraacetic acid)] and used for collecting circular dichroism (CD) spectra on the Jasco J-710 spectropolarimeter, equipped with quartz cuvettes of a 0.1-cm path length, at room temperature with a response time of 0.25 s and with a data point resolution of 0.2 nm from 190 to 260 nm. 2.5. S1 nuclease mapping RNA was isolated from mycelium grown at 30 °C for 36 to 156 h on cellophane placed on the surface of R2YE agar. 32P-labelled probes were prepared by PCR with the chromosomal DNA of S. coelicolor A3(2) as a template and the following primers: 5 0 GCTCCTCCCTCTGGTTCA-3 0 (corresponding to nucleotide positions from 328 to 311, taking the first G residue of the GTG start codon of cabB as +1) and 5 0 GTTCGACCGGATCGACACGGA-3 0 (positions from +48 to +28). The latter primer was 32P-labelled with T4 polynucleotide kinase. Protected DNA fragments were analyzed on DNA sequencing gels by the method of Maxam and Gilbert [10]. For S1 mapping of cabB in S. ambofaciens, RNA was prepared as for the S1 mapping of cabB in S. coelicolor A3(2). 32P-labelled probe was prepared with the following primers: 5 0 -GCCGCGGGCGGGCGGTTAAT-3 0 (nucleotide positions from 90 to 71, taking the first G residue of the GTG start codon as +1) and 5 0 TCCGTGTCGATCCGGTCGAA-3 0 (positions from +50 to +31). 2.6. Gene disruption of cabB A 754-bp region upstream of cabB was amplified by PCR with primers 5 0 -GGAATTCCGTACGCCGTGTGCTGGCCGG-3 0 (the italic letters indicate an SplI site present on the chromosome, and the underlined letters indicate a synthetic EcoRI site) and 5 0 -CCACAAGCTTGATATCCGGTCCGCCCCTCGTTCC-3 0 (the italic and underlined letters indicate a synthetic EcoRV and HindIII sites, respectively). A 1007-bp
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region downstream of cabB was amplified with 5 0 GAAGCTTGATATCCTGAACAAGTGACGGCACGGT-3 0 (the underlined letters indicate a HindIII site) and 5 0 -ACCTAAGCTTCGTACGCCGGTGACGGGCTCTGGA-3 0 (the italic letters indicate an SplI site present on the chromosome, and the underlined letters indicate a HindIII site). The regions upstream and downstream of cabB were each cloned between the EcoRI and HindIII sites and into the HindIII site of pUC19, respectively. The upstream and downstream regions were excised by digestion with EcoRI plus EcoRV and with HindIII, respectively, by use of the multi-cloning sites and connected with a 1.3-kb SmaI–HindIII fragment carrying the kanamycin (Km) resistance determinant from Tn5 so that the Km resistance gene was sandwiched by the upstream and downstream regions on pUC19, generating pDiscabB (see Fig. 5). pDiscabB prepared from E. coli JM110 containing dam and dcm mutations was digested with SplI, alkali denatured, and introduced by protoplast transformation into S. coelicolor A3(2) to isolate mutants in which the SplI fragment was integrated in the chromosome by homologous recombination. Km-resistant colonies were selected to obtain mutants containing the complete deletion of cabB. Correct deletion of cabB sequence was checked by Southern hybridization with the 3.1 kb SplI fragment from pDiscabB and the Km resistance gene as probes. 2.7. Overexpression of CabB A DNA fragment containing cabB of S. coelicolor A3(2) and its own promoter sequence was amplified by PCR with primers 5 0 -GGGGAATTCGCTCCTCCCTCTGGTTCA-3 0 (corresponding to nucleotide positions from 328 to 311, taking the first G residue of the GTG start codon of cabB as +1. The underlined letters indicate an EcoRI site) and 5 0 -CCCGGATCCC-
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GCCCCTTCGCCGTCCGGGCA-3 0 (positions from +260 to +240; the underlined letters indicate a BamHI site). The amplified DNA fragment was cloned between the EcoRI and BamHI sites of pUC19. After the nucleotide sequence had been checked, the EcoRI–BamHI fragment was inserted between the EcoRI and BamHI sites of pIJ486 [11], generating pIJ486-cabB. pIJ486cabB was introduced by protoplast transformation into S. coelicolor A3(2).
3. Results and discussion 3.1. Alignment of amino acid sequences of CabB and calmodulin The first EF-hand protein in actinomycetes was calerythrin [4], which shows sequence similarity with the subfamily of sarcoplasmic calcium-binding proteins (SCPs) and is therefore believed to be designed rather as an intracellular calcium buffer than as an enzyme activator [5]. A search in the genome database of S. coelicolor A3(2) [9] predicted the presence of four EF-hand proteins, SCJ33.05c, SC6F11.09, SC10F4.20, and SC3D11.21. These proteins, except for SC3D11.21, show similarity to SCPs. SC3D11.21 shows high sequence similarity to calmodulins of Dictyostelium discoideum (M64089) and Oryza sativa (AB060552). The three-dimensional structures of calmodulins are like a dumbbell, in which the amino-terminal lobe containing two EF-hand motifs is linked with the carboxy-terminal lobe containing two EF-hand motifs via the central helix. The amino- and carboxy-terminal lobes show sequence similarity to each other. SC3D11.21 consisting of 70 amino acids corresponds to one of the terminal lobes and therefore contains two EF-hand motifs (Fig. 1). Because SC3D11.21 showed a distinct calcium-binding
(a)
(b)
Fig. 1. Comparison of CabB proteins. (a) CabB from S. coelicolor A3(2) is aligned with N-terminal (CaM-N) and C-terminal (CaM-C) lobes of calmodulin of Dictyostelium discoideum. Residues that are identical to the CabB sequence are highlighted by reverse shading and residues that are similar are shaded in gray. The helix–loop–helix EF-hand motifs are indicated above the alignment. (b) Comparison of CabB proteins in Streptomyces. Sco, S. coelicolor A3(2); Sam, S. ambofaciens; Sav, S. avermitilis; and Sgr, S. griseus. Amino acid residues identical in alignment to CabB from S. coelicolor A3(2) are highlighted by reverse shading.
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activity, as described below, we named it CabB (calcium-binding protein). We also cloned and sequenced a 2.0-kb SmaI fragment carrying a cabB-like sequence from S. ambofaciens. The 2.0-kb fragment contained two complete open reading frames and two truncated open reading frames. CabB consisting of 70 amino acids in this strain shows 93% amino acid identity to that in S. coelicolor A3(2) and contains two EF-hand motifs (Fig. 1). We therefore assume that CabB-type proteins are distributed in a wide variety of actinomycetes. In fact, the genome databases of S. avermitilis [12] and S. griseus (our unpublished data) reveal that both strains contain a CabB-like protein. The CabB-like protein (SAV2780) of 70 amino acids in S. avermitilis shows 90% amino acid identity to CabB in S. coelicolor A3(2) and that of 71 amino acids in S. griseus shows 66% identity (Fig. 1). 3.2. Transcription of cabB To determine the transcription of cabB in S. coelicolor A3(2), we performed S1 nuclease mapping by using RNAs prepared from mycelium grown for various periods on agar medium. Under the growth conditions, S. coelicolor A3(2) grew as substrate mycelium until 36 h, as a mixture of substrate and aerial mycelium from 36 to 84 h, and as a mixture of aerial mycelium and spores after 84 h. As seen in Fig. 2(a), cabB was apparently transcribed throughout the growth. hrdB encoding a principal r factor of RNA polymerase was used as a constitutively expressed internal control. In front of the transcriptional start points 39 and 40 nucleotides upstream of the GTG start codon, a TAATCT sequence
similar to the 10 sequence, TAGRRT (R: A or G), for housekeeping genes in Streptomyces [13] is present. However, no sequence similar to the 35 sequence, TTGACA, is present at an appropriate position. cabB in S. ambofaciens was also transcribed throughout growth (Fig. 2(b)). The transcriptional start points were identified to be 62 and 63 nucleotides upstream of the GTG start codon. The TAATCT sequence upstream of the transcriptional start points might serve as a 10 sequence. No sequence similar to the 35 consensus sequence was present. 3.3. Calcium-binding activity of CabB The CabB protein in S. coelicolor A3(2) produced in the soluble fraction of E. coli [pTWIN1-cabB] was purified as a native form without any tags. We applied the purified CabB protein to gel filtration column chromatography with a Shodex PROTEIN KW-802.5 to determine its ternary structure, since CabB is a half size of calmodulin. Both CabB and Ca-bound CabB eluted at the position corresponding to about 14 kDa (data not shown), which suggests that CabB is a monomer in the solution. We also performed cross-linking experiments by using 0–10 mM BS3 [bis(sulfosuccinimidyl) suberate; Pierce], followed by SDS–PAGE, in which most population of the CabB protein remained a monomer and, at high concentrations of BS3, only a small amount of CabB migrated at a position of a dimer (data not shown). We therefore concluded that CabB is a monomer in solution. Each of the amino- and carboxy-terminal lobes of bovine brain calmodulin contains two EF-hand motifs and
Fig. 2. Transcriptional analysis of cabB in S. coelicolor A3(2) (a) and S. ambofaciens (b). RNA was prepared from cells grown on agar medium for the indicated number of hours. hrdB served as an internal control. The 32P-labelled probes in the absence of RNA were run in lane p. The open triangles indicate the positions of the S1 protected fragments. The 5 0 termini of the transcripts were assigned the nucleotides indicated by the arrows, because the fragments generated by the chemical sequencing reactions migrate 1.5 nucleotides further than the corresponding fragments generated by S1 nuclease digestion of the DNA–RNA hybrids. Probable 10 sequences, together with the transcriptional start points (tsp), in the promoter sequences are indicated.
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25 20
CaM
[θ ] (mdeg)
15 10 5
EGTA
0 -5 -10 -15
Ca2+
-20 200
220
230
240
250
260
25 20
[θ ] (mdeg)
Fig. 3. Calcium-binding activity of CabB from S. coelicolor A3(2). CabB purified from E. coli [pTWIN1-cabB], together with bovine brain calmodulin (CaM), was separated by SDS–polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) (left). Proteins transferred to a PVDF membrane were incubated with 45 CaCl2 and the membrane was examined by autoradiography (right).
210
CabB
15 10 EGTA
5 0 -5
one molecule of calmodulin therefore binds four calcium ions. Since CabB represents one half of the calmodulin and contains two EF-hand motifs, we expected the ability of CabB to bind calcium ions. CabB and bovine brain calmodulin as a positive control were electrophoresed on a Tricine–SDS–polyacrylamide gel and the proteins were transferred to a PVDF membrane. The membrane was incubated in a buffer containing 45Ca, washed, and analyzed by autoradiography (Fig. 3). CabB showed distinct calcium-binding activity to the same extent as calmodulin, whereas 45Ca did not bind to any of the molecular size markers. Because of the presence of two EF-hand motifs in CabB, we suppose that CabB binds two calcium ions. 3.4. Conformational change of CabB upon calciumbinding Upon binding calcium ions, calmodulins take a rigid conformation, thereby binding to the target enzymes and proteins and modulating their enzyme activities. We therefore examined possible conformational change of CabB upon binding calcium by CD spectroscopy (Fig. 4). In the presence of calcium, the values at 210 and 225 nm of both calmodulin and CabB were smaller than those in the absence of calcium, indicating that the a-helix content of both proteins was increased in the presence of calcium. The similar overall change of the CD spectra between CabB and calmodulin suggests that the conformational changes of these proteins upon binding calcium were similar. However, differences between these proteins in the wavelength giving a minimum absorption around at 210 nm in the absence of calcium and in the degree of decrease of the value at 220 nm in the presence of calcium predict that calcium causes CabB a larger conformational change than for calmodulin. This may result from the linkage of eukaryotic cal-
-10
Ca2+
-15 -20
200
210
220
230
240
250
260
Wavelength (nm) Fig. 4. CD spectra in the far UV of CabB and bovine brain calmodulin (CaM) in the presence and absence of calcium. CD spectra were obtained by scanning from 190 to 260 nm. The concentration of the samples was 2 mg/ml in distilled water containing 10 mM CaCl2 or 2 mM EGTA. h, molar ellipticity.
modulins between the amino- and carboxy-terminal lobes via the central helix. 3.5. Gene disruption and overexpression of cabB In several Streptomyces species, calcium affects morphological differentiation, especially aerial mycelium formation [7,8]. We first deleted cabB on the chromosome of S. coelicolor A3(2) by insertion of the Km resistance determinant into the cabB-coding region (Fig. 5). After Southern hybridization with two different probes, four cabB-disruptants (mutant DcabB) having correct replacement were obtained. Mutants DcabB grew normally and formed aerial hyphae and spores as the parental strain on R2YE medium containing various concentrations (20–100 mM) of CaCl2. No change in actinorhodin production, which was detected by blue pigmentation, was also observed. However, the growth rate of mutant DcabB was delayed on minimal medium containing CaCl2 at concentrations higher than 20 mM, when compared to that of the parental strain (data not shown). The delay of growth of mutant DcabB was restored by introduction of pIJ486-cabB, indicating that this delay was caused solely by the cabB mutation. If the growth delay is associated with a decrease in calcium
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Colony forming frequency (%)
M130 [pIJ486]
resistance of mutant DcabB, we speculate that CabB has a role in buffering or transport of Ca2+, thereby serving as an influx and efflux system that tightly controls the low cytoplasmic Ca2+ levels. We next examined the effects of cabB overexpression in S. coelicolor A3(2) and S. lividans by placing the promoter and coding regions of cabB on a high-copy-number plasmid pIJ486 with its copy number of 40–100 [11]. However, no phenotypic alterations were observed; S. coelicolor A3(2) [pIJ486-cabB] and S. lividans [pIJ486cabB] grew and formed aerial mycelium and spores at the same time course as the parental strain on various media containing different carbon and nitrogen sources and on minimal medium. During these experiments, we noticed that the colony forming frequency from spores on R2YE medium was lower than on other media. R2YE medium has been used for protoplast regeneration and contains a high concentration (20 mM) of calcium than other routine media. We then examined colony formation on R2YE containing various concentrations of CaCl2 (Fig. 6). Although the colony forming frequency of the parental strain and mutant DcabB both harbouring the vector pIJ486 was almost the same on
M130 [pIJ486-cabB]
100 80 60 40 20 0
Fig. 5. Isolation of cabB-disruptants from S. coelicolor A3(2) M130. (a) The strategy used for complete deletion of the chromosomal cabB gene by replacement by the Km resistance (Kmr) gene as a result of double cross-over is schematically shown. (b) Southern hybridization with probe 1 of 3.1-kb from pDisCabB against the SplI-digested chromosomal DNA revealed the presence of a signal of the expected size of 3.1-kb in mutant DcabB. Similar Southern hybridization with probe 2 (the Kmr gene) as 32P-labelled probe revealed the presence of the SplI fragment at the expected position at 3.1-kb.
∆cabB [pIJ486]
10 30 50 100 10 30 50 100 10 30 50 100 CaCl 2 (mM)
Fig. 6. Colony forming frequency of S. coelicolor A3(2) [pIJ486] or [pIJ486-cabB]. As a control, spores (about 2000/ml) were prepared from the parental strain M130, spread on R2YE medium containing 10 mM CaCl2, and incubated at 30 °C for 5 days, and the colonies formed were counted. Taking the colony forming unit as 100%, the colony forming frequency of strain M130 [pIJ486] or [pIJ486-cabB] on R2YE medium containing various concentrations of CaCl2 was calculated. The colony forming frequency of mutant DcabB was also calculated. The values are the average obtained from three independent experiments. The colony forming frequency of strain M130 [pIJ486-cabB] decreased as the concentration of CaCl2 increased, whereas those of M130 [pIJ486] and mutant DcabB [pIJ486] did not change in response to Ca2+.
R2YE medium containing Ca2+ up to 100 mM, the parental strain M130 [pIJ486-cabB] did not efficiently form colonies on medium containing high concentrations of Ca2+; only 50% of spores formed colonies on R2YE medium containing 30 mM CaCl2. The low frequency of colony formation of the parental strain M130 [pIJ486-cabB] might be explained in terms of a disturbance of the possible CabB function as a calcium buffer or transporter as a result of overexpression.
4. Conclusions CabB in S. coelicolor A3(2) is able to bind calcium ions and upon binding calcium it takes a conformational change so that the a-helix content increases, as determined by CD spectroscopy. Conceivably, the CabB homologues in other Streptomyces species also show the same properties because of the high homology to the S. coelicolor A3(2) CabB. In prokaryotes, calcium affects the growth rates of some bacteria, such as Staphylococcus aureus, Clostridium perfringens, and Streptococcus mutans [2]. On the other hand, calcium appears not to significantly affect the growth rate or morphological development of S. coelicolor A3(2), because this strain grows and develops aerial hyphae and spores on routinely used rich media containing various concentra-
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tions (0–100 mM) of CaCl2. However, disruption of cabB made it apparent that calcium at concentrations higher than 20 mM in minimal medium delays the growth rate. These findings suggest that CabB has a role in calcium buffering or transport of Ca2+, as suggested for calerythrin of S. erythraea [5], and not in modulating enzyme activity. The low frequency of colony formation of the parental strain M130 [pIJ486-cabB] may also be explained in terms of the calcium buffering or transport activity of CabB. Our preliminary experiments to see the effect of CabB on the cAMP-phosphodiesterase activity, one of the targets of eukaryotic calmodulins, denied its regulatory role (data not shown).
Acknowledgements We thank Tomoyuki Fujii for the CD analysis of CabB. This work was supported by a Grant-in-Aid for Exploratory Research of JSPS and by the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
References [1] Michiels, J., Xi, C., Verhaert, J. and Vanderleyden, J. (2002) The functions of Ca2+ in bacteria: a role for EF-hand proteins?. Trends Microbiol. 10, 87–93. [2] Norris, V., Chen, M., Goldberg, M., Voskuil, J., McGurk, G. and Holland, I.B. (1991) Calcium in bacteria: a solution to which problem?. Mol. Microbiol. 5, 775–778. [3] Leadlay, P.F., Roberts, G. and Walker, J.E. (1984) Isolation of a novel calcium-binding protein from Streptomyces erythreus. FEBS Lett. 178, 157–160.
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[4] Swan, D.G., Hale, R.S., Dhillon, N. and Leadlay, P.F. (1987) A bacterial calcium-binding protein homologous to calmodulin. Nature 329, 84–85. [5] Cox, J.A. and Bairoch, A. (1988) Sequence similarities in calciumbinding proteins. Nature 331, 491. [6] Yonekawa, T., Ohnishi, Y. and Horinouchi, S. (2001) A calciumbinding protein with four EF-hand motifs in Streptomyces ambofaciens. Biosci. Biotechnol. Biochem. 65, 156–160. [7] Natsume, M., Yasui, K. and Marumo, S. (1989) Calcium ion regulates aerial mycelium formation in actinomycetes. J. Antibiot. 42, 440–447. [8] Natsume, M. and Marumo, S. (1992) Calcium signal modulators inhibit aerial mycelium formation in Streptomyces alboniger. J. Antibiot. 45, 1026–1028. [9] Bentley, S.D., Chater, K.F., Cerdeno-Ta´rraga, A.-M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.-H., Kieser, T., Larke, L., Murphy, L., Oliver, K., OÕNeil, S., Rabbinowitsch, E., Rajandream, M.-A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J. and Hopwood, D.A. (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147. [10] Maxam, A.M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific cleavages. Methods Enzymol. 65, 499–560. [11] Ward, J.M., Janssen, G.R., Kieser, T., Bibb, M.J., Buttner, M.J. and Bibb, M.J. (1986) Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol. Gen. Genet. 203, 468–478. [12] Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakasi, Y., Hattori, M. and Omura, S. (2003) Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nature Biotechnol. 21, 526–531. [13] Strohl, W.R. (1992) Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 20, 961–974.
A calmodulin-like protein in the bacterial genus Streptomyces Tohru Yonekawa, Yasuo Ohnishi, Sueharu Horinouchi
*
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received 5 January 2005; received in revised form 31 January 2005; accepted 2 February 2005 First published online 13 February 2005 Edited by R.P. Gunsalus
Abstract The gene, named cabB, encoding a calmodulin-like protein of 70 amino acids containing two helix–loop–helix EF-hand motifs was cloned from Streptomyces coelicolor A3(2). cabB was transcribed from a single promoter throughout growth. The CabB protein produced in Escherichia coli was a monomer in solution, although it corresponded to one half of a dumbbell shape of the eukaryotic calmodulins. CabB bound calcium and upon binding of calcium its a-helix content was increased, as determined by circular dichroism spectroscopy. The growth of cabB-disruptants (mutant DcabB) on minimal agar medium containing calcium higher than 20 mM was delayed, suggesting that CabB has a role in calcium homeostasis by serving as a calcium buffer or transporter, as suggested for calerythrin in actinomycetes and the invertebrate sarcoplasmic calcium-binding proteins. Wide distribution of cabB almost exclusively in actinomycetes suggests a common role of EF-hand CabB-type proteins in these filamentous, soil-dwelling Gram-positive bacterial genera. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Ca-binding protein; EF-hand motif; Streptomyces ambofaciens; Streptomyces coelicolor A3(2)
1. Introduction In eukaryotes, helix–loop–helix ‘‘EF-hand’’ proteins including calmodulin are ubiquitous and involved in various physiologically important regulatory or buffering functions. Calcium thus serves as a second messenger, controlling various functions. In prokaryotes, several calmodulin-like proteins containing two or more authentic EF-hand motifs have been deposited in the databases. However, the role of calmodulin-like proteins in bacteria is poorly understood, although involvement of calcium in many processes, such as sporulation, virulence, septation, chemotaxis, and phosphorylation, has been observed [1,2]. The first prokaryotic protein shown to
*
Corresponding author. Tel.: +81 3 5841 5123; fax: +81 3 5841 8021. E-mail address: [email protected] (S. Horinouchi).
contain typical EF-hand motifs and to bind calcium ions was calerythrin from the actinomycete Saccharopolyspora erythraea [3,4], which probably functions as a calcium ion buffer rather than having a regulatory role [5]. A motif search of the databases by Michiels et al. [1] revealed the presence of many bacterial proteins containing the EF-hand motif, although most of the identified proteins are hypothetical or of unknown function. We previously isolated a gene (named cabA) encoding calerythrin from Streptomyces ambofaciens to determine its possible role in morphological differentiation [6], because calcium is known to be important for aerial mycelium formation in the filamentous, soil-dwelling, Gram-positive bacterial genus Streptomyces [7,8]. Contrary to our expectation, cabA-disruption gave no detectable phenotypic alterations to the host, probably because CabA serves as a calcium ion buffer [6]. We then aimed at SC3D11.21 in Streptomyces coelicolor A3(2)
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.02.003
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[9], which shows high similarity in amino acid sequence to eukaryotic calmodulins and is totally different in overall structure from calerythrin or CabA. In the present study, we cloned SC3D11.21 and expressed it in Escherichia coli to determine its Ca-binding ability. Because it showed a distinct Ca-binding activity, we named it CabB. We also disrupted cabB on the chromosome to determine its role in morphological development and secondary metabolism.
(SDS–PAGE) and blotted onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated with 0.037 MBq/ml 45Ca (Amersham Pharmacia) in 60 mM KCl, 5 mM MgCl2, and 10 mM imidazole, pH 6.8, for 20 min at room temperature. The final concentration of Ca2+ in the incubation mixture was 64 lM. After removal of unbound 45Ca by vigorous washing, bound radioactivity was visualized by autoradiography. 2.4. Circular dichroism spectroscopy
2. Materials and methods 2.1. Cloning of cabB from S. coelicolor A3(2) and S. ambofaciens On the basis of the S. coelicolor A3(2) genome sequence (on cosmid SC3D11), the cabB gene was cloned from S. coelicolor A3(2) M130 by PCR with a pair of primers, 5 0 -GGACCCACATATGGCGGACATCGAGGAAGCACGCAAG-3 0 (the underline indicates an NdeI site) and 5 0 -GGTGGTTGCTCTTCCGCACTTGTTCAGGTGAGCCCGGAA-3 0 (the underline indicates a SapI site). The amplified DNA fragment was inserted between the NdeI and SapI sites of pTWIN1 (New England BioLabs), resulting in pTWIN1-cabB. No errors during PCR were confirmed by nucleotide sequencing. cabB in S. ambofaciens was cloned and sequenced by standard DNA manipulations including Southern hybridization and colony hybridization with the cabB sequence from S. coelicolor A3(2) as the 32P-labelled hybridization probe. The nucleotide sequence of the 2.0-kb SmaI fragment cloned in this way has been deposited to the EMBL, DDBJ, and GenBank databases under Accession No. AB194697. 2.2. Purification of recombinant CabB An overnight culture of E. coli BL21 (DE3) [pTWIN1-cabB] was diluted 100-fold into Luria-Bertani broth containing ampicillin at 100 lg/ml and grown at 37 °C until OD590 (optical density at 590 nm) reached 0.3–0.4. Isopropyl-b-D-thiogalactopyranoside (IPTG) was then added at a final concentration of 1 mM and the culture was continued for additional 3 h. The E. coli cells were harvested by centrifugation and CabB was purified according to the instructions in the New England BioLabs manual. The CabB protein was purified without any tags. 2.3. Ca binding assay Two micrograms each of recombinant CabB and bovine brain calmodulin (Sigma) as a positive control were run on 15% SDS–polyacrylamide gel electrophoresis
Protein solutions (2 mg/ml each of CabB and bovine brain calmodulin) were prepared with distilled water plus 10 mM CaCl2 or 2 mM EGTA [(ethyleneglycolbis(b-aminoethylether)-N,N,N 0 ,N 0 -tetraacetic acid)] and used for collecting circular dichroism (CD) spectra on the Jasco J-710 spectropolarimeter, equipped with quartz cuvettes of a 0.1-cm path length, at room temperature with a response time of 0.25 s and with a data point resolution of 0.2 nm from 190 to 260 nm. 2.5. S1 nuclease mapping RNA was isolated from mycelium grown at 30 °C for 36 to 156 h on cellophane placed on the surface of R2YE agar. 32P-labelled probes were prepared by PCR with the chromosomal DNA of S. coelicolor A3(2) as a template and the following primers: 5 0 GCTCCTCCCTCTGGTTCA-3 0 (corresponding to nucleotide positions from 328 to 311, taking the first G residue of the GTG start codon of cabB as +1) and 5 0 GTTCGACCGGATCGACACGGA-3 0 (positions from +48 to +28). The latter primer was 32P-labelled with T4 polynucleotide kinase. Protected DNA fragments were analyzed on DNA sequencing gels by the method of Maxam and Gilbert [10]. For S1 mapping of cabB in S. ambofaciens, RNA was prepared as for the S1 mapping of cabB in S. coelicolor A3(2). 32P-labelled probe was prepared with the following primers: 5 0 -GCCGCGGGCGGGCGGTTAAT-3 0 (nucleotide positions from 90 to 71, taking the first G residue of the GTG start codon as +1) and 5 0 TCCGTGTCGATCCGGTCGAA-3 0 (positions from +50 to +31). 2.6. Gene disruption of cabB A 754-bp region upstream of cabB was amplified by PCR with primers 5 0 -GGAATTCCGTACGCCGTGTGCTGGCCGG-3 0 (the italic letters indicate an SplI site present on the chromosome, and the underlined letters indicate a synthetic EcoRI site) and 5 0 -CCACAAGCTTGATATCCGGTCCGCCCCTCGTTCC-3 0 (the italic and underlined letters indicate a synthetic EcoRV and HindIII sites, respectively). A 1007-bp
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region downstream of cabB was amplified with 5 0 GAAGCTTGATATCCTGAACAAGTGACGGCACGGT-3 0 (the underlined letters indicate a HindIII site) and 5 0 -ACCTAAGCTTCGTACGCCGGTGACGGGCTCTGGA-3 0 (the italic letters indicate an SplI site present on the chromosome, and the underlined letters indicate a HindIII site). The regions upstream and downstream of cabB were each cloned between the EcoRI and HindIII sites and into the HindIII site of pUC19, respectively. The upstream and downstream regions were excised by digestion with EcoRI plus EcoRV and with HindIII, respectively, by use of the multi-cloning sites and connected with a 1.3-kb SmaI–HindIII fragment carrying the kanamycin (Km) resistance determinant from Tn5 so that the Km resistance gene was sandwiched by the upstream and downstream regions on pUC19, generating pDiscabB (see Fig. 5). pDiscabB prepared from E. coli JM110 containing dam and dcm mutations was digested with SplI, alkali denatured, and introduced by protoplast transformation into S. coelicolor A3(2) to isolate mutants in which the SplI fragment was integrated in the chromosome by homologous recombination. Km-resistant colonies were selected to obtain mutants containing the complete deletion of cabB. Correct deletion of cabB sequence was checked by Southern hybridization with the 3.1 kb SplI fragment from pDiscabB and the Km resistance gene as probes. 2.7. Overexpression of CabB A DNA fragment containing cabB of S. coelicolor A3(2) and its own promoter sequence was amplified by PCR with primers 5 0 -GGGGAATTCGCTCCTCCCTCTGGTTCA-3 0 (corresponding to nucleotide positions from 328 to 311, taking the first G residue of the GTG start codon of cabB as +1. The underlined letters indicate an EcoRI site) and 5 0 -CCCGGATCCC-
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GCCCCTTCGCCGTCCGGGCA-3 0 (positions from +260 to +240; the underlined letters indicate a BamHI site). The amplified DNA fragment was cloned between the EcoRI and BamHI sites of pUC19. After the nucleotide sequence had been checked, the EcoRI–BamHI fragment was inserted between the EcoRI and BamHI sites of pIJ486 [11], generating pIJ486-cabB. pIJ486cabB was introduced by protoplast transformation into S. coelicolor A3(2).
3. Results and discussion 3.1. Alignment of amino acid sequences of CabB and calmodulin The first EF-hand protein in actinomycetes was calerythrin [4], which shows sequence similarity with the subfamily of sarcoplasmic calcium-binding proteins (SCPs) and is therefore believed to be designed rather as an intracellular calcium buffer than as an enzyme activator [5]. A search in the genome database of S. coelicolor A3(2) [9] predicted the presence of four EF-hand proteins, SCJ33.05c, SC6F11.09, SC10F4.20, and SC3D11.21. These proteins, except for SC3D11.21, show similarity to SCPs. SC3D11.21 shows high sequence similarity to calmodulins of Dictyostelium discoideum (M64089) and Oryza sativa (AB060552). The three-dimensional structures of calmodulins are like a dumbbell, in which the amino-terminal lobe containing two EF-hand motifs is linked with the carboxy-terminal lobe containing two EF-hand motifs via the central helix. The amino- and carboxy-terminal lobes show sequence similarity to each other. SC3D11.21 consisting of 70 amino acids corresponds to one of the terminal lobes and therefore contains two EF-hand motifs (Fig. 1). Because SC3D11.21 showed a distinct calcium-binding
(a)
(b)
Fig. 1. Comparison of CabB proteins. (a) CabB from S. coelicolor A3(2) is aligned with N-terminal (CaM-N) and C-terminal (CaM-C) lobes of calmodulin of Dictyostelium discoideum. Residues that are identical to the CabB sequence are highlighted by reverse shading and residues that are similar are shaded in gray. The helix–loop–helix EF-hand motifs are indicated above the alignment. (b) Comparison of CabB proteins in Streptomyces. Sco, S. coelicolor A3(2); Sam, S. ambofaciens; Sav, S. avermitilis; and Sgr, S. griseus. Amino acid residues identical in alignment to CabB from S. coelicolor A3(2) are highlighted by reverse shading.
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activity, as described below, we named it CabB (calcium-binding protein). We also cloned and sequenced a 2.0-kb SmaI fragment carrying a cabB-like sequence from S. ambofaciens. The 2.0-kb fragment contained two complete open reading frames and two truncated open reading frames. CabB consisting of 70 amino acids in this strain shows 93% amino acid identity to that in S. coelicolor A3(2) and contains two EF-hand motifs (Fig. 1). We therefore assume that CabB-type proteins are distributed in a wide variety of actinomycetes. In fact, the genome databases of S. avermitilis [12] and S. griseus (our unpublished data) reveal that both strains contain a CabB-like protein. The CabB-like protein (SAV2780) of 70 amino acids in S. avermitilis shows 90% amino acid identity to CabB in S. coelicolor A3(2) and that of 71 amino acids in S. griseus shows 66% identity (Fig. 1). 3.2. Transcription of cabB To determine the transcription of cabB in S. coelicolor A3(2), we performed S1 nuclease mapping by using RNAs prepared from mycelium grown for various periods on agar medium. Under the growth conditions, S. coelicolor A3(2) grew as substrate mycelium until 36 h, as a mixture of substrate and aerial mycelium from 36 to 84 h, and as a mixture of aerial mycelium and spores after 84 h. As seen in Fig. 2(a), cabB was apparently transcribed throughout the growth. hrdB encoding a principal r factor of RNA polymerase was used as a constitutively expressed internal control. In front of the transcriptional start points 39 and 40 nucleotides upstream of the GTG start codon, a TAATCT sequence
similar to the 10 sequence, TAGRRT (R: A or G), for housekeeping genes in Streptomyces [13] is present. However, no sequence similar to the 35 sequence, TTGACA, is present at an appropriate position. cabB in S. ambofaciens was also transcribed throughout growth (Fig. 2(b)). The transcriptional start points were identified to be 62 and 63 nucleotides upstream of the GTG start codon. The TAATCT sequence upstream of the transcriptional start points might serve as a 10 sequence. No sequence similar to the 35 consensus sequence was present. 3.3. Calcium-binding activity of CabB The CabB protein in S. coelicolor A3(2) produced in the soluble fraction of E. coli [pTWIN1-cabB] was purified as a native form without any tags. We applied the purified CabB protein to gel filtration column chromatography with a Shodex PROTEIN KW-802.5 to determine its ternary structure, since CabB is a half size of calmodulin. Both CabB and Ca-bound CabB eluted at the position corresponding to about 14 kDa (data not shown), which suggests that CabB is a monomer in the solution. We also performed cross-linking experiments by using 0–10 mM BS3 [bis(sulfosuccinimidyl) suberate; Pierce], followed by SDS–PAGE, in which most population of the CabB protein remained a monomer and, at high concentrations of BS3, only a small amount of CabB migrated at a position of a dimer (data not shown). We therefore concluded that CabB is a monomer in solution. Each of the amino- and carboxy-terminal lobes of bovine brain calmodulin contains two EF-hand motifs and
Fig. 2. Transcriptional analysis of cabB in S. coelicolor A3(2) (a) and S. ambofaciens (b). RNA was prepared from cells grown on agar medium for the indicated number of hours. hrdB served as an internal control. The 32P-labelled probes in the absence of RNA were run in lane p. The open triangles indicate the positions of the S1 protected fragments. The 5 0 termini of the transcripts were assigned the nucleotides indicated by the arrows, because the fragments generated by the chemical sequencing reactions migrate 1.5 nucleotides further than the corresponding fragments generated by S1 nuclease digestion of the DNA–RNA hybrids. Probable 10 sequences, together with the transcriptional start points (tsp), in the promoter sequences are indicated.
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25 20
CaM
[θ ] (mdeg)
15 10 5
EGTA
0 -5 -10 -15
Ca2+
-20 200
220
230
240
250
260
25 20
[θ ] (mdeg)
Fig. 3. Calcium-binding activity of CabB from S. coelicolor A3(2). CabB purified from E. coli [pTWIN1-cabB], together with bovine brain calmodulin (CaM), was separated by SDS–polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) (left). Proteins transferred to a PVDF membrane were incubated with 45 CaCl2 and the membrane was examined by autoradiography (right).
210
CabB
15 10 EGTA
5 0 -5
one molecule of calmodulin therefore binds four calcium ions. Since CabB represents one half of the calmodulin and contains two EF-hand motifs, we expected the ability of CabB to bind calcium ions. CabB and bovine brain calmodulin as a positive control were electrophoresed on a Tricine–SDS–polyacrylamide gel and the proteins were transferred to a PVDF membrane. The membrane was incubated in a buffer containing 45Ca, washed, and analyzed by autoradiography (Fig. 3). CabB showed distinct calcium-binding activity to the same extent as calmodulin, whereas 45Ca did not bind to any of the molecular size markers. Because of the presence of two EF-hand motifs in CabB, we suppose that CabB binds two calcium ions. 3.4. Conformational change of CabB upon calciumbinding Upon binding calcium ions, calmodulins take a rigid conformation, thereby binding to the target enzymes and proteins and modulating their enzyme activities. We therefore examined possible conformational change of CabB upon binding calcium by CD spectroscopy (Fig. 4). In the presence of calcium, the values at 210 and 225 nm of both calmodulin and CabB were smaller than those in the absence of calcium, indicating that the a-helix content of both proteins was increased in the presence of calcium. The similar overall change of the CD spectra between CabB and calmodulin suggests that the conformational changes of these proteins upon binding calcium were similar. However, differences between these proteins in the wavelength giving a minimum absorption around at 210 nm in the absence of calcium and in the degree of decrease of the value at 220 nm in the presence of calcium predict that calcium causes CabB a larger conformational change than for calmodulin. This may result from the linkage of eukaryotic cal-
-10
Ca2+
-15 -20
200
210
220
230
240
250
260
Wavelength (nm) Fig. 4. CD spectra in the far UV of CabB and bovine brain calmodulin (CaM) in the presence and absence of calcium. CD spectra were obtained by scanning from 190 to 260 nm. The concentration of the samples was 2 mg/ml in distilled water containing 10 mM CaCl2 or 2 mM EGTA. h, molar ellipticity.
modulins between the amino- and carboxy-terminal lobes via the central helix. 3.5. Gene disruption and overexpression of cabB In several Streptomyces species, calcium affects morphological differentiation, especially aerial mycelium formation [7,8]. We first deleted cabB on the chromosome of S. coelicolor A3(2) by insertion of the Km resistance determinant into the cabB-coding region (Fig. 5). After Southern hybridization with two different probes, four cabB-disruptants (mutant DcabB) having correct replacement were obtained. Mutants DcabB grew normally and formed aerial hyphae and spores as the parental strain on R2YE medium containing various concentrations (20–100 mM) of CaCl2. No change in actinorhodin production, which was detected by blue pigmentation, was also observed. However, the growth rate of mutant DcabB was delayed on minimal medium containing CaCl2 at concentrations higher than 20 mM, when compared to that of the parental strain (data not shown). The delay of growth of mutant DcabB was restored by introduction of pIJ486-cabB, indicating that this delay was caused solely by the cabB mutation. If the growth delay is associated with a decrease in calcium
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Colony forming frequency (%)
M130 [pIJ486]
resistance of mutant DcabB, we speculate that CabB has a role in buffering or transport of Ca2+, thereby serving as an influx and efflux system that tightly controls the low cytoplasmic Ca2+ levels. We next examined the effects of cabB overexpression in S. coelicolor A3(2) and S. lividans by placing the promoter and coding regions of cabB on a high-copy-number plasmid pIJ486 with its copy number of 40–100 [11]. However, no phenotypic alterations were observed; S. coelicolor A3(2) [pIJ486-cabB] and S. lividans [pIJ486cabB] grew and formed aerial mycelium and spores at the same time course as the parental strain on various media containing different carbon and nitrogen sources and on minimal medium. During these experiments, we noticed that the colony forming frequency from spores on R2YE medium was lower than on other media. R2YE medium has been used for protoplast regeneration and contains a high concentration (20 mM) of calcium than other routine media. We then examined colony formation on R2YE containing various concentrations of CaCl2 (Fig. 6). Although the colony forming frequency of the parental strain and mutant DcabB both harbouring the vector pIJ486 was almost the same on
M130 [pIJ486-cabB]
100 80 60 40 20 0
Fig. 5. Isolation of cabB-disruptants from S. coelicolor A3(2) M130. (a) The strategy used for complete deletion of the chromosomal cabB gene by replacement by the Km resistance (Kmr) gene as a result of double cross-over is schematically shown. (b) Southern hybridization with probe 1 of 3.1-kb from pDisCabB against the SplI-digested chromosomal DNA revealed the presence of a signal of the expected size of 3.1-kb in mutant DcabB. Similar Southern hybridization with probe 2 (the Kmr gene) as 32P-labelled probe revealed the presence of the SplI fragment at the expected position at 3.1-kb.
∆cabB [pIJ486]
10 30 50 100 10 30 50 100 10 30 50 100 CaCl 2 (mM)
Fig. 6. Colony forming frequency of S. coelicolor A3(2) [pIJ486] or [pIJ486-cabB]. As a control, spores (about 2000/ml) were prepared from the parental strain M130, spread on R2YE medium containing 10 mM CaCl2, and incubated at 30 °C for 5 days, and the colonies formed were counted. Taking the colony forming unit as 100%, the colony forming frequency of strain M130 [pIJ486] or [pIJ486-cabB] on R2YE medium containing various concentrations of CaCl2 was calculated. The colony forming frequency of mutant DcabB was also calculated. The values are the average obtained from three independent experiments. The colony forming frequency of strain M130 [pIJ486-cabB] decreased as the concentration of CaCl2 increased, whereas those of M130 [pIJ486] and mutant DcabB [pIJ486] did not change in response to Ca2+.
R2YE medium containing Ca2+ up to 100 mM, the parental strain M130 [pIJ486-cabB] did not efficiently form colonies on medium containing high concentrations of Ca2+; only 50% of spores formed colonies on R2YE medium containing 30 mM CaCl2. The low frequency of colony formation of the parental strain M130 [pIJ486-cabB] might be explained in terms of a disturbance of the possible CabB function as a calcium buffer or transporter as a result of overexpression.
4. Conclusions CabB in S. coelicolor A3(2) is able to bind calcium ions and upon binding calcium it takes a conformational change so that the a-helix content increases, as determined by CD spectroscopy. Conceivably, the CabB homologues in other Streptomyces species also show the same properties because of the high homology to the S. coelicolor A3(2) CabB. In prokaryotes, calcium affects the growth rates of some bacteria, such as Staphylococcus aureus, Clostridium perfringens, and Streptococcus mutans [2]. On the other hand, calcium appears not to significantly affect the growth rate or morphological development of S. coelicolor A3(2), because this strain grows and develops aerial hyphae and spores on routinely used rich media containing various concentra-
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tions (0–100 mM) of CaCl2. However, disruption of cabB made it apparent that calcium at concentrations higher than 20 mM in minimal medium delays the growth rate. These findings suggest that CabB has a role in calcium buffering or transport of Ca2+, as suggested for calerythrin of S. erythraea [5], and not in modulating enzyme activity. The low frequency of colony formation of the parental strain M130 [pIJ486-cabB] may also be explained in terms of the calcium buffering or transport activity of CabB. Our preliminary experiments to see the effect of CabB on the cAMP-phosphodiesterase activity, one of the targets of eukaryotic calmodulins, denied its regulatory role (data not shown).
Acknowledgements We thank Tomoyuki Fujii for the CD analysis of CabB. This work was supported by a Grant-in-Aid for Exploratory Research of JSPS and by the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
References [1] Michiels, J., Xi, C., Verhaert, J. and Vanderleyden, J. (2002) The functions of Ca2+ in bacteria: a role for EF-hand proteins?. Trends Microbiol. 10, 87–93. [2] Norris, V., Chen, M., Goldberg, M., Voskuil, J., McGurk, G. and Holland, I.B. (1991) Calcium in bacteria: a solution to which problem?. Mol. Microbiol. 5, 775–778. [3] Leadlay, P.F., Roberts, G. and Walker, J.E. (1984) Isolation of a novel calcium-binding protein from Streptomyces erythreus. FEBS Lett. 178, 157–160.
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[4] Swan, D.G., Hale, R.S., Dhillon, N. and Leadlay, P.F. (1987) A bacterial calcium-binding protein homologous to calmodulin. Nature 329, 84–85. [5] Cox, J.A. and Bairoch, A. (1988) Sequence similarities in calciumbinding proteins. Nature 331, 491. [6] Yonekawa, T., Ohnishi, Y. and Horinouchi, S. (2001) A calciumbinding protein with four EF-hand motifs in Streptomyces ambofaciens. Biosci. Biotechnol. Biochem. 65, 156–160. [7] Natsume, M., Yasui, K. and Marumo, S. (1989) Calcium ion regulates aerial mycelium formation in actinomycetes. J. Antibiot. 42, 440–447. [8] Natsume, M. and Marumo, S. (1992) Calcium signal modulators inhibit aerial mycelium formation in Streptomyces alboniger. J. Antibiot. 45, 1026–1028. [9] Bentley, S.D., Chater, K.F., Cerdeno-Ta´rraga, A.-M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.-H., Kieser, T., Larke, L., Murphy, L., Oliver, K., OÕNeil, S., Rabbinowitsch, E., Rajandream, M.-A., Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J. and Hopwood, D.A. (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147. [10] Maxam, A.M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific cleavages. Methods Enzymol. 65, 499–560. [11] Ward, J.M., Janssen, G.R., Kieser, T., Bibb, M.J., Buttner, M.J. and Bibb, M.J. (1986) Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol. Gen. Genet. 203, 468–478. [12] Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakasi, Y., Hattori, M. and Omura, S. (2003) Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nature Biotechnol. 21, 526–531. [13] Strohl, W.R. (1992) Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 20, 961–974.