By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Wiley Online Library will be unavailable on Saturday 7th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 08.00 EDT / 13.00 BST / 17:30 IST / 20.00 SGT and Sunday 8th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 06.00 EDT / 11.00 BST / 15:30 IST / 18.00 SGT for essential maintenance. Apologies for the inconvenience.
Correspondence: Kenji Ueda, Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-0880, Japan. Tel.: +81 466 84 3937; fax: +81 466 84 3935; e-mail: email@example.com
Stress-response sigma factor σH is negatively regulated by its cognate anti-sigma factor RshA in Streptomyces griseus. As the overexpression of RshA in the wild-type strain confers a distinctive bald phenotype (deficiency in aerial mycelium formation and streptomycin production), RshA is supposed to associate with not only σH but also another regulatory element that plays a crucial role in the developmental control of S. griseus. Here, we show that an anti-sigma factor antagonist BldG associates with RshA and negatively regulates its activity. The bald phenotype conferred by the overexpression of rshA was restored to the wild-type phenotype by the coexpression with bldG. The in vivo and in vitro protein interaction analyses demonstrated the specific association between RshA and BldG. A bldG mutant exhibited a distinctive bald phenotype and was defective in the σH-dependent transcription activities. The positive regulatory role of BldG regarding the σH activity was verified by an in vitro transcriptional analysis, in which the inhibition of σH-dependent transcription by RshA was abolished by the addition of BldG in a dose-responsive manner. Overall, evidence suggests that BldG serves as a master switch for both stress-response and developmental gene expression based on its association with multiple anti-sigma factors in S. griseus.
Streptomyces and related bacteria harbor a large number of RNA polymerase sigma factors. For example, Streptomyces coelicolor A3(2), the model microorganism for genetic manipulation, harbors four major and 60 minor sigma factors (including 50 factors involved in extracytoplasmic function and nine in stress-response) (Bentley et al., 2002; Hahn et al., 2003). Streptomyces griseus, the streptomycin producer used in this study, retains four major and 48 minor sigma factors (Ohnishi et al., 2008). The presence of these varied sigma factors suggests divergences in the gene expression in this microorganism, and these divergences enable the microorganism to adapt to various environmental and physiological conditions. We studied the role of stress-response sigma factors in S. griseus (streptomycin producer) with regard to the link between the stress response and morphological and physiological differentiation.
In our previous study (Takano et al., 2003), we had characterized an rshA-sigH operon encoding a stress-response sigma factor σH and its antagonist (anti-σH factor) RshA. In that study, the insertion of rshA into a high-copy-number plasmid (pIJ702-rshA) caused marked repression of aerial mycelium formation (Fig. 1a, left) and streptomycin production in S. griseus IFO13350 (the wild-type strain). Therefore, we assumed that this marked phenotypic change was caused by the sequestration of σH and alternative sigma factors by the excess RshA. However, a triple knockout mutant for σH and two σH paralogs (σF and σN) showed the wild-type phenotype (Takano et al., 2007). This finding indicated that these sigma factors are not directly involved in the control of morphological development and secondary metabolism and suggested that RshA binds to another protein regulating the expression of developmental genes.
In this study, we identified BldG, an anti-sigma factor antagonist, to be such a protein associating RshA. BldG has been characterized for its essential role in the developmental control in S. coelicolor A3(2) (Bignell et al., 2000, 2003). The evidence suggests that the cross-talk between BldG and RshA controls the activity of σH and related stress-response sigma factors in S. griseus.
Materials and methods
Bacterial strains, plasmids, and growth conditions
Strains, plasmids, and growth conditions used in this study were as described previously (Takano et al., 2007), except that TA cloning of PCR-generated DNA fragments was done with the help of pMD19 (Takara Shuzo). An integration plasmid pKU463, a derivative of pKU493aad (Komatsu et al., 2010) carrying kanamycin resistance, was obtained from H. Ikeda at Kitasato University.
Plasmid construction and gene disruption
The construction of pIJ702-rshA has been described previously (Takano et al., 2003). This plasmid directed the constitutive expression of rshA by localizing the coding sequence downstream from the promoter of mel operon on the vector. pIJ702-rshAbldG was constructed by inserting a bldG cassette prepared by PCR (primers are shown in Supporting Information, Table S1) between the PstI and KpnI sites of pIJ702-rshA. The bldG cassette contained its own promoter region, which directed the expression of the coding sequence. For genetic complementation, the 957-bp DNA fragment containing the bldG coding sequence (cds) and its promoter region was amplified by PCR using the primers GCF/GCR and cloned between the EcoRI and HindIII sites of pKU463 to form pBG. pBG was then introduced into the bldG mutant by transformation to generate a kanamycin-resistant transformant carrying the plasmid integrated at the K38-1 site (nucleotide sequence accession no. AB251919).
Disruption of bldG was carried out by a homologous recombination technique based on REDIRECT technology (Gust et al., 2003). The cosmid clone SGR1G10 used for bldG knockout in S. griseus containing the region corresponding to nucleotides 3874894–3914177 in the genome sequence database was obtained in our study using SuperCos1 (Stratagene, Japan, Tokyo) as a vector. An apramycin resistance gene cassette was prepared by PCR using the primers DisGF/DisGR and used for the replacement for the bldG cds by in vivo recombination using λ RED. The resulting apramycin-resistant cosmids purified from Escherichia coli GM2163 were introduced into the wild-type strain of S. griseus. Apramycin-resistant recombinants were then screened and checked for true recombination by PCR using appropriate primer sets. All mutants obtained exhibited the identical bald phenotype; hence, one of them was designated as the bldG mutant.
S1 nuclease mapping
The transcriptional activities of the promoters preceding the rshA-sigH operon (PH1, the σH-dependent promoter), sigN (PN1), and rpp operon (Prpp) were studied by S1 protection analysis. Methods and conditions for RNA preparation and S1 nuclease mapping were previously described (Kieser et al., 2000; Kelemen et al., 2001; Takano et al., 2007). The probes for PH1, PN1, and Prpp were prepared by PCR using primers HS1-F/HS1-R* for PH1, NS1-F/NS1-R* for PN1, and RS1-F/RS1-R* for Prpp, respectively (primers indicated with an asterisk were labelled at its 5′-end with [γ-32P] ATP using T4-polynucleotide kinase).
Recombinant protein preparation
Preparation of a C-terminally histidine-tagged RshA (RshA-6xHis) was described previously (Takano et al., 2003). For preparation of RshA carrying an N-terminal glutathione-S-transferase (GST-RshA), an rshA cassette was amplified using the PCR primers Rex-F/Rex-R and cloned between the BamHI/EcoRI sites of pGEX-4T-2 (GE Healthcare, Tokyo, Japan). For BldG-6xHis, a bldG cassette was amplified using primers Gex-F/Gex-R and cloned between the NdeI/HindIII sites of pET26-b+ (Takara Shuzo). The E. coli BL21(DE3) cells harboring each expression plasmid were cultured aerobically at 28 °C in 100 mL Luria–Bertani liquid medium. The culture was supplied with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) when OD600 nm reached 0.8. Cells were grown for 4 h after the addition of IPTG, and harvested by centrifugation. The resultant cellular precipitate was suspended in an appropriate volume of phosphate-buffered saline buffer (Maniatis et al., 1982) and disrupted by sonication. The soluble recombinant proteins were purified from the cell extract with appropriate affinity chromatography following the method recommended by the manufacturer (GE Healthcare).
The interaction between RshA and BldG was studied by a two-hybrid analysis using an E. coli host–vector system (BacterioMatch 2-hybrid Kit, Stratagene). The protocols were similar to those described in previous studies (Takano et al., 2003). The target plasmid was constructed by inserting the bldG cassette between the BamHI and EcoRI sites of pTRG (the PCR primers are summarized in Table S1). The rshA-containing bait plasmid was constructed using protocols described in previous studies (Takano et al., 2003).
The protocol for the pull-down assay was essentially the same as that described in previous studies (Komatsu et al., 2006). The bait (GST-RshA) and the target (BldG-6xHis) proteins were mixed, incubated, and bound to glutathione Sepharose resin. After elution, the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Western blotting using anti-GST and anti-6xHis antibodies.
In vitro run-off transcriptional assay
Methods for the preparation of σH-6xHis, RNA synthesis, and detection were described previously (Takano et al., 2007). The template (PH1 region) was prepared by PCR using the primers P123-F/P23H-R. The reaction mixture contained a commercial RNA polymerase core enzyme of E. coli (E) and various amounts of the recombinant proteins (σH-6xHis, RshA-6xHis, and BldG-6xHis). For the estimation of the transcript sizes, a 100-bp ladder marker (Takara shuzo) denatured by heat treatment was used as a standard.
Cells grown at 28 °C for 3 days on R2YE medium were observed using a scanning electron microscopy. To prepare the specimens, agar blocks were fixed with 2% osmium tetroxide for 30 h and then dehydrated by freeze-drying. Each specimen was sputter-coated with palladium/gold using an E-1010 ion sputter (Hitachi, Tokyo, Japan) and scanned on a VE8800 scanning electron microscope (Keyence, Tokyo, Japan).
Results and discussion
To identify the proteins that associate with RshA, the genes that suppressed the aforementioned inhibitory effect of rshA were screened using pIJ702-rshA as the vector and S. griseus wild-type strain as the DNA donor as well as the cloning host. One of the transformants obtained showed abundant aerial mycelium despite the presence of rshA on the same plasmid. Partial nucleotide sequencing of the DNA fragment cloned into this plasmid revealed that the fragment contained a cds corresponding to SGR3307, an ortholog of bldG of S. coelicolor A3 (2) (our unpublished data). On the basis of this observation, we introduced a bldG cassette into the pIJ702-rshA to generate pIJ702-rshAbldG and transformed the wild-type strain with this plasmid. The wild-type strain harboring this plasmid exhibited the wild-type phenotype; it formed aerial mycelium (Fig. 1a) and produced normal levels of streptomycin (data not shown), thereby indicating that bldG suppresses the inhibitory activity of rshA.
Originally, bldG was identified by Leskiw and colleagues to be an essential regulator for the initiation of aerial mycelium formation and antibiotic production in S. coelicolor A3(2) (Bignell et al., 2000, 2003). The amino acid sequence similarity strongly suggests that the BldG product is an anti-sigma factor antagonist. The bldG gene and a downstream cds for a putative anti-sigma factor (SGR3306 in S. griseus) comprise an operon. This operon, located at a locus different from the rshA-sigH operon, does not contain any cds for sigma factor (Fig. 1b). The gene organization at the bldG locus is highly conserved in the genome of Streptomyces and related bacteria.
To observe the interaction between RshA and BldG, we carried out a two-hybrid analysis using an E. coli host–vector system. The measurement of β-galactosidase activity, which enabled the evaluation of interaction activity, showed that the activity of the transformants harboring the rshA-containing bait and bldG-containing target plasmid (63.6 × 10−5; ΔA410 min−1 μg−1) was considerably higher than that of the control strains harboring an empty bait or target plasmid (8.3–15.1 × 10−5; ΔA410 min−1 μg−1). The interaction activity between RshA and BldG was higher than that between RshA and σH-family sigma factors described previously (23.4–47.0 × 10−5ΔA410 min−1 μg−1) (Takano et al., 2003).
To verify the interaction, we performed an in vitro pull-down assay. As shown in Fig. 2, during glutathione column chromatography for the mixture of GST-RshA and BldG-6xHis recombinants, both proteins were collected in the same fraction (lane 5), indicating that the latter protein was bound to the former. The binding complex of the two proteins was also observed in a native PAGE analysis (Fig. S1).
To study the role of bldG in S. griseus, we generated a knockout mutant by the standard homologous recombination technique. The bldG mutant was unable to form aerial mycelium and produce streptomycin (Fig. 1c), indicating that BldG plays an essential role in the developmental control of S. griseus. The bald phenotype of this mutant was restored to the wild type by introducing an integration plasmid carrying an intact bldG cassette (data not shown).
Transcriptional analysis using a low-resolution S1 protection assay revealed that the activities of σH-dependent promoters were downregulated in the bldG mutant (Fig. 3a). Among the three promoters preceding the rshA-sigH operon (PH1, PH2, and PH3), the activity of PH1, the σH-dependent promoter (Takano et al., 2007), was considerably reduced by bldG knockout. The activity of the σH-independent promoters (PH2 and PH3) were also reduced, but still retained a certain transcription level. The activities of the other σH-dependent promoters preceding sigN (PN1) and rpp operon (Prpp), which are also known for their dependence on σH (Takano et al., 2007), were also significantly downregulated in the bldG mutant (Fig. 3a). These observations supported the hypothesis that BldG regulates the activity of σH and alternative sigma factors by binding to RshA.
Further, the σH-dependent transcription was studied by an in vitro transcription assay (Fig. 3b). As described in previous studies, RshA inhibited the σH-dependent transcription at PH1. This RshA-dependent transcriptional repression was abolished in a dose-dependent manner by the addition of BldG at excess molar ratios (Fig. 3b). This finding can be attributed to the dissociation of σH from RshA, which in turn binds to BldG.
The lines of evidence obtained in this study suggest that the role of BldG is highly pleiotropic. BldG regulates the expression of both developmental and stress-responsive genes in S. griseus. Since σH and its paralogs are not essential for the initiation of development (Takano et al., 2007), BldG probably binds to another sigma-factor antagonist involved in the developmental control. Recently, Parashar et al. (2009) reported that BldG binds to the putative anti-sigma factor encoded by SCO3548, the adjacent cds, to control the key developmental processes in S. coelicolor A3(2). The specific sigma factor regulated by this anti-sigma factor is expected to be involved in developmental control, although this sigma factor has not yet been identified. The conserved gene organization suggests that these findings would also be observed in S. griseus.
During the writing of this paper, Sevcikova et al. (2010) reported a similar observation on the interaction between BldG and RshA in S. coelicolor A3(2). The authors demonstrated specific interaction between BldG and UshX (the RshA ortholog) by pull-down and two-hybrid analyses and showed that the activity of the σH-dependent promoter preceding ushX-sigH operon (sigHp2; equivalent of PH1 of S. griseus) is abolished in a bldG mutant of S. coelicolor. Overall, our results are confirmatory except that the activities of the σH-independent promoters preceding rshA (PH2 and PH3) were reduced in the bldG mutant of S. griseus (Fig. 3a). In contrast, the corresponding promoters of S. coelicolor (sigHp3 and sigHp4) were upregulated in the bldG mutant of S. coelicolor A3(2) (Sevcikova et al., 2010). Currently, we do not know why the σH-independent promoters were also affected by the knockout of bldG, but the difference in the two species implies that this is due to some indirect effect. On the other hand, the identical evidence regarding σH-dependent promoters obtained in the two phylogenetically divergent species strongly suggests that the regulation generally occurs in this group of organisms.
Similarly as in S. coelicolor A3(2), the PH1 promoter preceding rshA-sigH operon is osmotically induced in S. griseus (Takano et al., 2003). This makes us speculate that BldG serves as a hub in the complex network and connects various environmental and physiological signal inputs to developmental and stress-responsive output processes. Detailed biochemical characterization with respect to the association of BldG with its binding targets will provide useful information regarding the mechanism of signal switching.
This study was supported by the High-tech Research Center Project of MEXT, Japan.