cmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptomyces coelicolorA3(2)
© Xie et al; licensee BioMed Central Ltd. 2009
Received: 12 May 2009
Accepted: 4 August 2009
Published: 4 August 2009
Streptomyces coelicolor is the most studied Streptomyces species and an excellent model for studying differentiation and antibiotic production. To date, many genes have been identified to be required for its differentiation (e.g. bld genes for aerial growth and whi genes for sporulation) and antibiotics production (including actII-orf4, redD, cdaR as pathway-specific regulatory genes and afsR, absA1/A2 as pleiotropic regulatory genes).
A gene cluster containing six genes (SCO4126-4131) was proved to be co-transcribed in S. coelicolor. Deletions of cmdABCDEF (SCO4126-4131) displayed defective sporulation including formation of aberrant branches, and abnormalities in chromosome segregation and spore septation. Disruption mutants of apparently orthologous genes of S. lividans and S. avermitilis also showed defective sporulation, implying that the role of these genes is similar among Streptomyces. Transcription of cmdB, and therefore presumably of the whole operon, was regulated developmentally. Five of the encoded proteins (CmdA, C, D, E, F) were predicted membrane proteins. The other, CmdB, a predicted ATP/GTP-binding protein with an ABC-transporter-ATPase domain shown here to be essential for its function, was also located on the cell membrane. These results indicate that CmdABCDEF proteins mainly affect Streptomyces differentiation at an early stage of aerial hyphae formation, and suggest that these proteins may form a complex on cell membrane for proper segregation of chromosomes. In addition, deletions of cmdABCDEF also revealed over-production of blue-pigmented actinorhodin (Act) via activation of transcription of the pathway-specific regulatory gene actII-orf4 of actinorhodin biosynthesis.
In this study, six co-transcribed genes cmdABCDEF were identified by their effects on differentiation and antibiotic production in Streptomyces coelicolor A3(2). These six membrane-located proteins are possibly assembled into a complex to function.
Streptomyces are Gram-positive eubacteria that are the major natural source of antibiotics, producing about half of all known microbial antibiotics . This genus also has a complex life cycle, in which spores germinate to form a substrate mycelium of branching hyphae on solid medium, from which branches grow into the air, such multi-nucleoid aerial hyphae ultimately becoming septated to form chains of unigenomic spores [2, 3].
Streptomyces coelicolor is the most studied Streptomyces species and an excellent model for studying antibiotic production and differentiation . It produces several chemically different antibiotics, including the blue-pigmented actinorhodin (Act), red-pigmented undecylprodigiosin (Red), calcium-dependent antibiotic (CDA) and plasmid SCP1-encoded methylenomycin (Mmy). Pathway-specific regulatory genes, e.g. actII-orf4, redD, cdaR and mmyB, are required for initiating transcription of the corresponding antibiotics biosynthetic gene clusters; while pleiotropic regulators, e.g. AfsR, often affect multiple secondary metabolism [5, 6]. By using S. coelicolor as a model system, two dozen genes (bld and whi), most of them encoding regulatory proteins, important for initiation of aerial mycelium formation and sporulation have been identified . More than 20 other genes from primary metabolism (e.g. citA encoding citrate synthase; ) and stress-response (rsrA for oxidation-sensing anti-sigma protein; ) etc also affect Streptomyces differentiation, indicating that the regulatory signaling cascades for aerial growth and sporulation extensively interact with metabolic, morphological, homeostatic and stress-related checkpoints . Recently, several key genes affecting apical growth, chromosome segregation and cell division (e.g. divIVA, sffA, ftsZ, ftsQ, ftsK and parA/B; [11–17]) have been identified.
Here we describe identification of a cluster of six co-transcribed genes cmdABCDEF (encoding five membrane proteins and one membrane-located ATP/GTP-binding protein) in S. coelicolor that affect sporulation and antibiotic production.
Co-transcription of six genes SCO4126-4131 of S. coelicolor
To investigate if SCO4126-4131 were involved in plasmid transfer, null mutants of the whole gene cluster were constructed by PCR-targeted mutagenesis . However, no significant difference in transfer frequencies of the SLP2-derived linear plasmid pQC542 which contained genes for DNA replication in linear mode and plasmid conjugal transfer [18, 19] between the mutant and the wild-type was found (data not shown), suggesting that these chromosomal genes could not substitute for the SLP2 genes for plasmid transfer.
Null mutants of SCO4126-4131display defective sporulation
Aberrant branches, defective spore septation and abnormal chromosome segregation in null mutants
CmdB, an ATP/GTP-binding protein with an ABC-transporter ATPase domain, is located on the cell membrane
CmdB contained an ABC-transporter-ATPase domain (from positions 44 to 427) according to Superfamily 1.69 analysis http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/hmm.html. This superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores . To investigate whether this domain was required for the function of CmdB, lysines at conserved positions 90 or 404 were mutated to arginines by site-directed mutagenesis (K90A or K404A). The mutated cmdB genes were cloned into pFX101, and then introduced by conjugation into the cmdB null mutant. In contrast to the functional cmdB gene, the site-mutated cmdB genes could not complement the cmdB null mutant to reverse its phenotype of over-production of blue pigment (Figure 4B) and also to produce dark grey colony to the wild type level (data not shown). These results indicated that the mutated residues were essential for function. It was however also possible that the mutations had destabilised the protein, causing it to degrade much more rapidly than the wild-type form.
Transcription of cmdBduring differentiation
To see if transcription of cmdB was regulated during differentiation, strain M145 grown on MS medium was harvested at different times for RT-PCR and analysed using primers specific for cmdB. As seen in Figure 4C, a small amount of cmdB transcript could be detected from mainly vegetative mycelium (16 h), and a larger amount (at least five-fold) was produced at the stage of aerial mycelium formation (26 h) and continued to increase during sporulation (40–74 h). These results suggested that transcription of cmdB was regulated temporally, possibly developmentally.
The cmdA-F orthologues in S. lividans and S. avermilitisalso affect differentiation
The complete nucleotide sequence of S. avermitilis genome reveals a highly homologous gene cluster (i.e. SAV4098 to SAV4103) to cmdA-F . A null mutant of SAV4098-4103 was constructed in S. avermilitis NRRL8165. Its defective sporulation was displayed on MS medium, and blocking in development of coiled aerial hyphae was observed under microscopy compared with that of the wild type (Figure 5B). No over-production of antibiotic avermectin was detected in the null mutant (data not shown).
Several null mutants of cmdABCDEFreveal over-production of blue pigment
Initiating transcription of the pathway-specific regulatory gene actII-orf4 of actinorhodin biosynthesis at an earlier growth stage in the cmdA-Fnull mutant
In S. coelicolor, pathway-specific regulatory gene actII-orf4 is essential for initiating transcription of the whole biosynthetic gene cluster of blue-pigment actinorhodin . To study transcription of actII-orf4 in the cmdA-F null mutant, we harvested spores/mycelium from MS plates after different growth periods and isolated RNA for RT-PCR. As seen in Figure 6B, transcription of actII-orf4 in the null mutant started as early as 16 h and then reached a maximum at 40 h, ~24 and 34 h earlier than was observed in M145.
Here, we report that an operon of six genes cmdABCDEF (SCO4126-4131) of S. coelicolor, encoding five membrane proteins and one membrane-located ATP/GTP-binding protein, affects differentiation and causes increased production of an antibiotic, actinorhodin. The ΔcmdABCDEF strains reveal aberrant branches and short aerial hyphae. Expression of cmdB, and therefore presumably of the whole operon, was detectable during vegetative growth, but increased substantially as soon as aerial growth was detectable. Similar conserved gene clusters are also found in other Streptomyces species, e.g. S. avermitilis (SAV4098-4103; ), S. griseus (SGR3915-3920; ) and S. lividans (Our unpublished data). Serious block in forming aerial hyphae in S. lividans and in the development of coiled aerial hyphae in S. avermitilis were observed when their cmd operons were disrupted. Together, these results indicate that CmdABCDEF proteins mainly affect Streptomyces differentiation early in aerial hyphae formation.
The ΔcmdABCDEF strains of S. coelicolor also showed defective chromosome segregation during sporulation. In prokaryotes, motor proteins such as FtsK and SpoIIIE containing a conserved RecA domain are often associated with DNA translocation during processes of cell division, conjugation and sporulation . In S. coelicolor, FtsK and ParA/ParB are required for proper chromosome segregation during sporulation [15, 16]. However, despite detectable levels of errors in chromosome segregation in FtsK or ParAB mutants, the majority of chromosomes still appear to segregate properly, suggesting that other proteins are also involved in chromosome partition or segregation. According to analysis using the Protein Homology/analogY Recognition Engine PHYRE http://www.sbg.bio.ic.ac.uk/phyre/html/index.html, CmdB protein was predicted containing a RecA domain (from positions 77 to 407, expectation value 1.7 × 10-21) or E. coli-FtsK motor domain (3.3 × 10-12), suggesting that it might be an ATP/GTP-dependent motor protein. CmdB displays homology with VirB4-like proteins from Frankia, Brevibacterium, Geobacillus and Thermoanaerobacter (expectation values 3 × 10-42, 1 × 10-39, 7 × 10-9 and 2 × 10-9, respectively) etc. The VirB4, an essential component of the bacterial type IV system, interacts with other membrane proteins in the vir operon to assemble a pore for transfer of a DNA-protein complex [26, 27]. Since CmdB is also located on the cell membrane, it is likely that CmdB along with other five membrane proteins from the same gene cluster might form a complex on the cell membrane. Further study will be needed to explore the existence of such a complex and to investigate whether it could form a type IV-like channel on cell membrane for chromosome and/or plasmid translocation in Streptomyces.
About 836 and 69 genes of S. coelicolor genome are predicted to encode membrane and ATP/GTP-binding proteins, respectively (; http://www.sanger.ac.uk/Projects/S_coelicolor/classwise.html#class4.1.0). Among these, SCO6878, SCO6880 and SCO6881, located in a cluster of 14 probably co-transcribed genes SCO6871-6884, highly resemble cmdB, cmdC and cmdD, respectively. However, null mutants of SCO6878 or SCO6881 did not display defective sporulation or over-production of blue pigment on MS medium (our unpublished data). Thus, either these genes are not involved in sporulation and antibiotic production, or their role may be masked by functional overlap with other genes, or the phenotype might be manifested only under particular conditions.
This study describes the identification of six co-transcribed genes cmdABCDEF, deletions of which displayed over-expression of blue-pigmented Act, defective sporulation and especially abnormalities in chromosome segregation, indicating that cmdABCDEF are new genes involved in antibiotic production and differentiation of S. coelicolor.
Bacterial strains, plasmids and general Methods
S. coelicolor M145 , S. lividans ZX7  and S. avermitilis NRRL8165  were hosts for studying functions of cmdABCDEF genes. Streptomyces were cultivated on Mannitol Soya flour medium (MS; 30). A cellophane sheet was placed over the agar medium when it was necessary to collect mycelium/spores or when cultures were to be examined by scanning electron microscopy . Manipulation of Streptomyces DNA and RNA followed Kieser et al. . E. coli strain DH5α (Life Technologies Inc) was used as cloning host. Plasmid isolation, transformation and PCR amplification followed Sambrook et al. . DNA fragments were purified from agarose gels with the Gel Extraction Master kit (Watson).
Construction and complementation of Streptomycesnull mutants
Cosmid SCD72A of S. coelicolor containing cmdABCDEF genes was kindly provided by Professor David Hopwood. Cosmid SAV3-17 of S. avermitilis containing the SAV4098-4103 genes was constructed in our laboratory. PCR-targeted mutagenesis was used to replace precisely the cmdABCDEF or SAV4098-4103 genes with an antibiotic resistant gene and then remove the marker but leaving an 81-bp "scar" sequence when necessary . Derivatives of the Streptomyces chromosomal-integrating plasmid pSET152  or pFX101 containing the functional cmdABCDEF genes were employed for complementing the mutated genes. PCR primers for construction and complementation of Streptomyces null mutants are listed in Additional file 1.
Scanning electron microscopy (SEM)
Streptomyces cultures were grown on MS medium covered with cellophane disks. After 7 days incubation at 30°C, the cells were fixed with fresh 2% glutaraldehyde (pH7.2) and 1% osmium tetroxide. After dehydration, ethanol was replaced by amyl acetate. The samples were then dried with the supercritical drying method in HCP-2 (Hitachi), coated with gold by Fine Coater JFC-1600 (Jeol), and examined with a JSM-6360LV scanning electron microscopy (Jeol).
Streptomyces spores were evenly spread onto MS medium, into which cover-slips were then inserted at an angle of approximate 60°C. After 4 days incubation at 30°C, cells attached to cover-slips were fixed with methanol followed by washing with phosphate-buffered saline. Samples were then stained with 4',6-diamidino-2-phenylindole (DAPI, 25 μg/ml) at room temperature for 30 minutes. After that, samples were observed by laser scanning confocal microscope Fluoview FV1000 (Olympus). Images were processed with Image-Pro Plus 6.0.
Reverse-transcription (RT) PCR assay
S. coelicolor were cultured on MS medium covered with cellophane disks, and RNA was isolated from cultures at a series of incubation times. The RNA samples were treated with DNase (RNase-free, Takara) to remove possible contaminating DNA and, after quantification, reverse-transcribed into cDNA by using "Revert Acid First Strand cDNA Synthesis" kit (MBI Fermentas). Then equal 25-ng products were subjected to PCR amplification (25 cycles). Five paired primers (p67, p78, p89, p90 and p01; see Additional file 2) were used for validating co-transcription of the cmdABCDEF genes. Three paired primers, Pact, PcmdB and P16S (Additional file 2), were used to detect transcription levels of actII-orf4, cmdB and genes for 16S rRNA, respectively. PCR conditions were: template DNA denatured at 94°C for 5 min, then 94°C 30 s, 60°C 30 s, 72°C 50 s, for 25 cycles.
Site-directed mutagenesis of cmdB
The site-directed mutagenesis of cmdB was performed by using the QuikChange kit (Stratagene). Plasmid pFX103 containing the intact cmdB and promoter of cmdABCDEF was used as PCR template. Two paired primers, PcmdBK90A (5'-tcggtgatcaggtgtctgaccacctggacgt-3', 5'-acgtccaggtggtcagacacctgatcaccga-3') and PcmdBK404A (5'-Tctcgagggccgacctgccgttccccgactc-3', 5'-Gagtcggggaacggcgagtcggccctcgaga-3'), were used to change lysines of CmdB at positions 90 and 404 into arginines.
CmdB protein and Western blotting
The PCR-amplified cmdB gene was cloned between the EcoRI and BamHI sites of E. coli plasmid pET-28a (Novagen), and the resulting plasmid was introduced by transformation into E. coli strain BL21 (DE3). Over-expression of CmdB was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 20°C for 12 hours. The six-histidine-tagged CmdB was purified by Ni2+ column chromatography (Qiagen) and used to raise rabbit polyclonal antibodies (the Antibody Center of the Shanghai Institutes for Biological Sciences).
S. coelicolor M145 was cultivated in Typtone-Soya-Broth medium  for 24 hours. Cells were sonicated and debris was removed by centrifugation (12,000 × g, 10 min). Then the lysate was incubated with 0.5 M KCl or 5 mM EDTA at 4°C for 30 min, prior to separation into cytosolic (supernatant) and membrane (precipitate) fractions by ultracentrifugation at 180,000 × g for 2 h . Each fraction together with the cell lysate was electrophoresed in a 12% SDS-polyacrylamide gel, and then transferred onto a PVDF membrane (Immobilon-P, Millipore) by electrophoresis. The PVDF film was incubated with the polyclonal antibody and horse-radish peroxidase-conjugated anti-rabbit IgG (Amersham). After 3 times washing, the signal on the film was directly detected by HRP Substrate Reagent (Shenergy).
We are very grateful to Keith Chater for critical reading of and useful suggestions on the manuscript. These investigations were supported by grants from National Nature Science Foundation of China (30325003, 30770045, 30870067), National "863" project (2007AA021503) and the Chinese Academy of Sciences project (KSCX2-YW-G-014) to Z. Qin.
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