Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade


Southern blot analysis of S. griseus
Southern blot analysis revealed that the S. griseus genome contains within a BamHI fragment of ∼5.6 kbp a single copy of an atrA homologue (Fig. 1, panel A). This encouraged us to investigate further whether the regulation of streptomycin production may involve a homologue of S. coelicolor AtrA. In the absence of a publicly available genome sequence for S. griseus, we approached this by constitutively expressing AtrAc in S. griseus. This negated the necessity to clone and sequence the homologue in S. griseus, which would have been a prerequisite for the alternative strategy of generating a targeted knockout using homologous recombination. The atrAc gene was inserted into a pSET152-based integrating expression construct (Tilley 2003). The resulting construct, called pL646, was then introduced by conjugation into S. griseus ATCC12475 (Vallins and Baumberg 1985). To provide controls, pSET152 was also introduced as well as a derivative of pL646 called pL644 that encodes an Arg 71 to Ala mutant, which has greatly reduced DNA-binding activity as shown by an electrophoretic mobility shift assay (Uguru et al. 2005), but remains highly soluble (manuscript in preparation). A combination of Southern blot (panel A) and PCR (panel B) analysis confirmed that plasmids pL646 and pL644 integrated exclusively at the phiC31 attachment site as a single copy to produce an atrA-containing BamHI fragment of approximately 8.6 kbp (Fig. 1). The Southern blot analysis also revealed that the S. griseus gene, which we term atrAg, is likely a close homologue as it binds the probe as well as does atrAc under the conditions used (panel A).
Assay of effects on streptomycin production and str transcription
Streptomycin production was assayed using an established protocol that utilises Bacillus subtilis as an indicator (Horinouchi et al. 1984). We observed that the amount of streptomycin produced after 2 days varied between exconjugants that received the same plasmid; however, it was clear that from comparison of mycelia producing wild-type AtrAc (strain BH646) with those containing an ‘empty’ vector (strain BH152) that the expression of wild-type atrAc promotes a significant reduction in streptomycin production (Fig. 2). This effect was dependent on the DNA-binding activity of AtrA, as production of the R71A mutant (strain BH644) appeared to have no significant effect. Similar results were obtained when these S. griseus strain were incubated for 3 days and 4 days before being assayed for streptomycin production (data not shown).
Next, the relative level of the strR transcript in S. griseus with and without the expression of wild-type atrAc was investigated using a quantitative RT-PCR method described previously (Uguru et al. 2005). As shown in Fig. 3, the level of strR mRNA in BH152 was 4–5-fold higher than in BH646 after incubation for 2 days (panel A). Between 2 days and 3 days, the level of the strR transcript decreased considerably in both, but remained higher in BH152 (panel A). This result indicated that the expression of wild-type atrA interferes with the transcription of strR. The level of strR transcript was also lower in BH646 when compared with BH644 (data not shown).
The above results predicted that the expression of genes downstream of strR would also be reduced in BH646 compared with BH152. We therefore analysed the level of transcripts of strB1 and strD (Fig. 3, panel B), which are in separate operons. The transcription of both genes is known to be activated by StrR (Retzlaff and Distler 1995; Tomono et al. 2005). The transcript level of both strB1 and strD were found to be significantly lower in mycelia expressing wild-type atrA (BH646) compared to those that did not (BH152). As part of this analysis, we also analysed the transcript level of adpA (panel B), the transcriptionally regulated relay immediately upstream of strR in the A-factor cascade (Ohnishi et al. 1999). Interestingly, there was no significant difference in the level of this transcript between BH646 and BH152, as judged by both end-point (Fig. 3) and real-time RT-PCR analysis (data not shown).


The medical importance of many different secondary metabolites has resulted in the study of gene expression in numerous species of the evolutionarily deep genus Streptomyces, as for any particular secondary metabolite increased knowledge about its regulation expands the scope for the rationale design of approaches to improve yield and fermentation performance, at least in principle. Consequently, there is interest in determining to what extent regulators of secondary metabolism identified for one species have homologues with a role in the regulation of secondary metabolites for others. Here we have shown that the production in S. griseus of AtrAc, an activator of transcription of the CSR of the actinorhodin biosynthetic genes in S. coelicolor, causes a reduction in the biosynthesis of streptomycin (Fig. 2) and in the transcription of the corresponding CSR strR (Fig. 3). These results indicate that the atrA homologue in S. griseus, revealed here by Southern blot analysis (Fig. 1), merits further investigation as a potential regulator of streptomycin production.

The advantages of using the approach we used here to determine the extent to which a regulator of secondary metabolism has homologues that also have a role in the regulation of secondary metabolites in other species are that it requires no prior knowledge of the genome sequences of the species being screened and it is relatively straightforward to perform. Most Streptomyces species can readily receive plasmids by conjugation and have an integration site for the expression constructs used here (Combes et al. 2002). We are in the process of determining the effects producing AtrAc has on the production of known secondary metabolites by other species. At this stage we can report that while it stimulated the production of actinorhodin in S. lividans, a close relative, no effects were detected on the production of oxytetracycline or rimocidin by strains of S. rimosus (data not shown). The latter is not unexpected as our previous characterisation of atrAc revealed that it has specificity with regard to the biosynthetic genes it influences. Although disruption of the atrA gene in S. coelicolor reduced the production of actinorhodin, it had no detectable effect on the production of undecylprodigiosin or the calcium dependent antibiotic (Uguru et al. 2005).

We do not, however, conclude from our results that the S. griseus homologue of AtrA likely functions as a repressor of strR transcription. During the time S. coelicolor and S. griseus last shared a common ancestor, which has been estimated to be about 200 million years ago (Embley and Stackebrandt 1994), it is likely that the ancestral AtrA and its targets have coevolved (i.e. each have adapted to changes in the other) within both lineages. Therefore, even though a homologue may be able in a heterologous host to bind sites used by the native activator, this may not be sufficient for the former to stimulate transcription as it may be unable to make necessary contacts with RNA polymerase (for review, see Lloyd et al. 2001). Moreover, the co-expression of such a homologue may antagonize activation by the native activator as a result of competition for DNA binding. Thus, the finding that the expression of S. coelicolor AtrA reduces strR transcription in S. griseus by a DNA binding-dependent mechanism does not exclude the possibility that the native AtrAg homologue functions as an activator of strR transcription. Indeed, deletion analysis has revealed that a region containing a site to which AtrAc binds in vitro is required for maximum transcription of strR by an A-factor-independent mechanism (Vujaklija et al. 1991). Although, in comparison to identifying and knocking out the genes of homologues, the approach we used does not by itself provide a clear indication of the precise function of homologues, it nonetheless has value as a simple screen to investigate whether or not they have a role in regulating the production of a particular secondary metabolite.

Interestingly, the effect on the transcription of strR resulting from the expression of atrAc did not appear to be mediated via a change in the transcription of adpA, the only previously characterised regulator of strR (Fig. 3). In support of the latter point, the expression of atrAc had no discernible effect on S. griseus sporulation (data not shown), which is also under adpA control (for review, see Ohnishi et al. 2005; Horinouchi 2002). From the above results we conclude that, as implicit in the data of Vujaklija et al. (1993), the A-factor-ArpA-AdpA-StrR regulatory cascade represents only part of the full complexity of regulation of streptomycin biosynthesis in S. griseus. That a factor(s) in addition to AdpA regulates transcription from the strR promoter is not unusual, at least from the study of E. coli where it is rare for a promoter to be regulated in response to a single environmental or physiological signal (reviewed in Browning and Busby 2004). This conclusion stands irrespective of the precise role that transpires for the S. griseus homologue of AtrA.

Acknowledgements  This work was funded in part by a grant from a BBSRC research grant (24/G18095) to S.B. and K.J.M. B.H was the recipient of a visiting scholarship from the State Scholarship Fund of China. Support is also acknowledged from the National Natural Science Foundation of China (30572274) and Beijing Municipal Science and Technology Commission to B.H. S.P was supported by funding provided by the Royal Thai Government Scholarship under the Teacher Professional Development Project (TPDP) jointly administered by the Ministry of Science and Technology, the Institute for Promotion of Teaching Science and Technology (IPST), and the Ministry of Education, Thailand. We thank Alice Morningstar for the gift of streptomycin-sensitive and -resistant E. coli strains


Bibb M (1996) The regulation of antibiotic production in Streptomyces coelicolor A3 (2). Microbiology 142:1335–1344

Bibb MJ, White J, Ward JM, Janssen GR (1994) The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome-binding site. Mol Microbiol 14:533–545

Bibb MJ (2005) Regulation of secondary metabolism in streptomycetes. Curr Opin Microbiol 8:208–215

Browning DF, Busby SJ (2004) The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57–65

Challis GL, Hopwood DA (2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci USA 2:14555–14561

Chater KF, Bibb MJ (1997) Regulation of bacterial antibiotic production. In: Kleinkauf H, Von Doehren H.v. (eds) Products of secondary metabolism. VCH Press, Germany

Chater KF, Horinouchi S (2003) Signalling early developmental events in two highly diverged Streptomyces species. Mol Microbiol 48:9–15

Combes P, Till R, Bee S, Smith MC (2002) The streptomyces genome contains multiple pseudo-attB sites for the (phi)C31-encoded site-specific recombination system. J Bacteriol 184:5746–5752

Demain AL, Fang A (2000) The natural functions of secondary metabolites. Adv Biochem Eng Biotechnol 69:1–39

Demain AL (1999) Pharmaceutically active secondary metabolites of microorganisms. Appl Microbiol Biotechnol 52:455–463

Embley TM, Stackebrandt E (1994) The molecular phylogeny and systematics of the actinomycetes. Ann Rev Microbiol 48:257–289

Flett F, Mersinias V, Smith CP (1997) High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol Lett 155:223–229

Gramajo HC, Takano E, Bibb MJ (1993) Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Mol Microbiol 7:837–845

Hesketh A, Sun J, Bibb M (2001) Induction of ppGpp synthesis in Streptomyces coelicolor A3(2) grown under conditions of nutritional sufficiency elicits actII-ORF4 transcription and actinorhodin biosynthesis. Mol Microbiol 39:136–144

Jin W, Ryu YG, Kang SG, Kim SK, Saito N, Ochi K, Lee SH, Lee KJ (2004) Two relA/spoT homologous genes are involved in the morphological and physiological differentiation of Streptomyces clavuligerus. Microbiology 150:1485–1493

Hopwood DA, Chater KF, Bibb MJ (1995) Genetics of antibiotic production in Streptomyces coelicolor A3(2), a model streptomycete. Biotechnology 28:65–102

Horinouchi S, Kumada Y, Beppu T (1984) Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization. J Bacteriol 158:481–487

Horinouchi S (2002) A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus. Frontiers Biosci 7:2045–2057

Kato JY, Suzuki A, Yamazaki H, Ohnishi Y, Horinouchi S (2002) Control by A-factor of a metalloendopeptidase gene involved in aerial mycelium formation in Streptomyces griseus. J Bacteriol 184:6016–6025

Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical streptomyces genetics. The John Innes Foundation, Norwich

Lloyd G, Landini P, Busby S (2001) Activation and repression of transcription initiation in bacteria. Essays Biochem 37:17–31

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

Martin JF, Demain AL (1980) Control of antibiotic biosynthesis. Microbiol Rev 44:230–251

Ohnishi Y, Kameyama S, Onaka H, Horinouchi S (1999) The A-factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus: identification of a target gene of the A-factor receptor. Mol Microbiol 34:102–111

Ohnishi Y, Yamazaki H, Kato JY, Tomono A, Horinouchi S (2005) AdpA, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces griseus. Biosci Biotechnol Biochem 69:431–439

Retzlaff L, Distler J (1995) The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites. Mol Microbiol 18:151–162

Schmitt-John T, Engels JW (1992) Promoter constructions for efficient secretion expression in Streptomyces lividans. Appl Microbiol Biotechnol 36:493–498

Takano E, Gramajo HC, Strauch E, Andres N, White J, Bibb MJ (1992) Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Mol Microbiol 6:2797–2804

Tilley E (2005) The metabolic engineering of Streptomyces sp. PhD Thesis. Department of Pharmaceutical Sciences, University of Strathclyde, Gaosgow, UK

Tomono A, Tsai Y, Yamazaki H, Ohnishi Y, Horinouchi S (2005) Transcriptional control by A-factor of strR, the pathway-specific transcriptional activator for streptomycin biosynthesis in Streptomyces griseus. J Bacteriol 187:5595–5604

Uguru GC, Stephens KE, Stead JA, Towle JE, Baumberg S, McDowall KJ (2005) Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor. Mol Microbiol 58:131–150

van Wezel GP, White J, Hoogvliet G, Bibb MJ (2000) Application of redD, the transcriptional activator gene of the undecylprodigiosin biosynthetic pathway, as a reporter for transcriptional activity in Streptomyces coelicolor A3(2) and Streptomyces lividans. J Mol Microbiol Biotech 2:551–556

Vallins WJ, Baumberg S (1985) Cloning of a DNA fragment from Streptomyces griseus which directs streptomycin phosphotransferase activity. J Gen Microbiol 131:1657–1669

Vujaklija D, Horinouchi S, Beppu T (1993) Detection of an A-factor-responsive protein that binds to the upstream activation sequence of strR, a regulatory gene for streptomycin biosynthesis in Streptomyces griseus. J Bacteriol 175:2652–2661

Vujaklija D, Ueda K, Hong SK, Beppu T, Horinouchi S (1991) Identification of an A-factor-dependent promoter in the streptomycin biosynthetic gene cluster of Streptomyces griseus. Mol Gen Genet 229:119–128

Yamazaki H, Ohnishi Y, Horinouchi S (2000) An A-factor-dependent extracytoplasmic function sigma factor (sigma(AdsA)) that is essential for morphological development in Streptomyces griseus. J Bacteriol 182:4596–4605

Yamazaki H, Ohnishi Y, Horinouchi S (2003a) Transcriptional switch on of ssgA by A-factor, which is essential for spore septum formation in Streptomyces griseus. J Bacteriol 185:1273–1283

Yamazaki H, Takano Y, Ohnishi Y, Horinouchi S (2003b) AmfR, an essential gene for aerial mycelium formation, is a member of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus. Mol Microbiol 50:1173–1187



Fig. 1 The integration of plasmids in exconjugants of S. griseus. (A) The integration of a single copy of each plasmid was confirmed by Southern blot analysis of total DNA digested with BamHI and probed with a DIG-labelled segment of S. coelicolor atrA. The numbers at the top of the panel identify individual isolates of each of the strains. Lane M contains DIG-labelled DNA molecular weight marker II (λ/HindIII, Roche). (B) Integrated of plasmids at the attB site was confirmed by PCR using a primer that bound within this attachment site and another that bound within the backbone of pSET152. The desired integration produces a PCR product of 1.6 kbp. Lane M contains DNA size markers (1-kb DNA ladder, New England BioLabs), whereas lane C corresponds to strain ATCC12475, which served as a negative control. Numbers down the left of each panel indicate the sizes of specific DNA markers (kbp)


Fig. 2 The effect on streptomycin production of expressing S. coelicolor atrA in S. griseus. Multiple isolates of each strain were assayed in triplicate. Each point in the scatterplot represents the mean area of the zones of growth inhibition around a particular isolate. For each strain, a cross and bar indicates the mean and standard deviation, respectively, of the values obtained for different isolates. The arrows indicate the values of isolates that were chosen for further analysis (Fig. 3); these were BH646 #1 and BH152 #1. The insets show the actual zones of inhibition observed for these particular isolates; BH646 #1 on the right and BH152 #1 on the left


Fig. 3 The effects on transcription of strR, its regulon and adpA. (A) The relative abundance of the strR transcript in mycelial patches of strains BH646 #1 and BH152 #1 grown on YMPD agar plates for 2 and 3 d was determined using quantitative, real-time PCR analysis of reverse-transcribed RNA samples. The values were normalised using values obtained for hrdB mRNA. The error bars are derived from three estimates of the abundance of strR mRNA in each sample (see Experimental procedures). (B) The levels of the strB1, strD, hrdB and adpA transcripts in 2 days samples of BH646 #1 and BH152 #1 were analysed semi-quantitatively using end-point PCR analysis of the same reverse-transcribed RNA samples used in panel A




Original Research Paper

Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade

Bin Hong2, Somkiat Phornphisutthimas3, Emma Tilley4, Simon Baumberg1and Kenneth J. McDowall1 

(1)  Faculty of Biological Sciences, University of Leeds, Leeds, LS8 2BH, UK
(2)  Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing, 100050, China
(3)  Institute for Innovation and Development of Learning Process, Mahdiol University, Rama VI, Bangkok, 10400, Thailand
(4)  Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G1 1XW, UK


In Streptomyces coelicolor, AtrA is an activator of transcription of the actinorhodin cluster-situated regulator gene actII-ORF4. In previous work, we showed that S. coelicolor AtrA binds in vitro to the promoter of S. griseus strR, the streptomycin cluster-situated regulator. We show here that S. griseus carries a single close homologue of atrA and that expression of S. coelicolor AtrA in S. griseus causes a DNA binding-dependent reduction in streptomycin production and in the mRNA levels of strR and genes of streptomycin biosynthesis. However, there is no effect on the level of the mRNA of adpA, which is the only transcription factor that has so far been characterised for strR. The adpA gene is directly regulated by ArpA, the receptor protein for the γ-butyrolactone signalling molecule A-factor. Therefore, to our knowledge, our results provide the first in vivo evidence that A-factor-ArpA-AdpA-StrR regulatory cascade represents only part of the full complexity of regulation of streptomycin biosynthesis in S. griseus. The potential biotechnological application of our findings is discussed.

Keywords  Actinorhodin - Secondary metabolite production -  Streptomyces gene regulation -  S. coelicolor  -  S. griseus  - Streptomycin

Biotechnology Letters. © Springer Science+Business Media B.V. 2006.10.1007/s10529-006-9216-2.


Mycelial bacteria of the genus Streptomyces and their relatives produce a plethora of secondary metabolites including antibiotics, antihelminthics and anti-tumour agents in clinical use. Their yield and timing of formation are exquisitely sensitive to growth conditions and have been associated with the accumulation of signalling molecules such as γ-butyrolactones, a family of microbial hormones (Horinouchi 2002; Chater and Horinouchi 2003). In virtually all cases that have been studied in detail, a change in the production of a secondary metabolite has been correlated with a change in the transcription of an activator gene situated with the corresponding biosynthetic genes as part of a cluster (for review, see Chater and Bibb 1997; Bibb 2005). To date, the only system for which there is a well authenticated, completely ungapped regulatory pathway from a signal to the production of a secondary metabolite is the regulation of streptomycin production in S. griseus by the γ-butyrolactone A-factor, whose accumulation may serve to communicate or coordinate the behaviour of individual cells (Ohnishi et al. 2005). A-factor produced by S. griseus binds to the receptor protein ArpA, which then has reduced affinity for DNA and thus is a less effective repressor of AdpA, which directly activates strR (Ohnishi et al. 1999), the activator situated in the streptomycin cluster, as well as several genes required for morphological differentiation (Ohnishi et al. 1999; Yamazaki et al. 2000, 2003a, b; Kato et al. 2002). It has been reported (Vujaklija et al. 1993) that as well as AdpA, other unidentified proteins bind upstream of strR; however, the role of the latter factors in mediating the response to A-factor or some other signal remains to be established.

The actinorhodin system in S. coelicolor has been studied in detail and numerous genes have been identified that affect the production of this secondary metabolite (Hopwood et al. 1995; Bibb 1996). Recently, we described the characterisation of AtrA, a transcription factor required for maximum transcription of actII-ORF4, the cluster-situated regulator (CSR) of the actinorhodin biosynthesis genes in S. coelicolor. Like many of the genes that regulate actinorhodin production, atrA does not have an orthologue in published models for the regulation of streptomycin production by S. griseus. As part of our previous study, we found that S. coelicolor AtrA can bind in vitro to the promoter of strR (Uguru et al. 2005). This finding raised the possibility that the regulation of streptomycin production, which serves as an important model, may involve a homologue of S. coelicolor AtrA. The results presented here show that S. griseus carries a single close homologue of atrA and that expression of S. coelicolor AtrA (AtrAc) in S. griseus can affect streptomycin production by a mechanism that is independent of change in the AdpA step of the A-factor cascade. The possible biotechnological implications of our findings are discussed.

Materials and methods

Construction of S. griseus strains expressing atrA from S. coelicolor

Integrating plasmids pL646 and pL644, which constitutively express wild-type AtrA and an Arg 71 to Ala (R71A) mutant, respectively, were transferred by conjugation from Escherichia coli ET12567 (pUB307) (Flett et al. 1997) to S. griseus ATCC 12475 (Vallins and Baumberg 1985) using an established protocol (Kieser et al. 2000). These constructs were based on a pSET152-based integrating expression construct (Tilley 2003) that uses the constitutive ermE*p promoter (Schmitt-John and Engels 1992; Bibb et al. 1994) and the ribosome-binding site of the tuf1 gene (van Wezel et al. 2000). Exconjugants were selected with apramycin (50 μg ml−1) and the resulting strains were designated BH646, BH644 and BH152, respectively. Integration at the attB site in the S. griseus chromosome (Combes et al. 2002) was confirmed initially by a PCR assay using primers that hybridised to sites within attB (5′-CGG TGC GGG TGC CAG GGC) and the backbone of pSET152 (5′-TTC GGC GGC TTC AAG TTC GG), respectively.

Southern blotting

Total DNA was prepared as described previously (Kieser et al. 2000) from mycelia grown in YMPD liquid medium (Ohnishi et al. 1999) and cut using BamHI. After agarose gel electrophoresis, the DNA was transferred to a Zeta-Probe GT blotting membrane (BioRad) using a capillary blotting stack (Maniatis et al. 1982), and fixed to the membrane using a UV Stratalinker (Stratagene). The digoxigenin (DIG)-labelled DNA probe for the atrA gene was generated using the PCR DIG Probe Synthesis Kit (Roche). The primers were AtraAF (5′-GGA TCC TCA GGA TTC TCA TTG GTC GTC) and AtraAR (5′-GGA TCC GTG GCT ATG ACC AGC AGC AC). The membrane was prehybridised at 55°C for at least 30 min using the DIG Easy Hyb hybridisation buffer (Roche), denatured DIG-labelled probe was added and incubated continued overnight at the same temperature. After hybridisation, the membrane was washed twice with low stringency buffer (2 × SSC, 0.1% w/v SDS) at room temperature and twice with high stringency buffer (0.1 × SSC, 0.1% w/v SDS) at 68°C. At this temperature, only genes that have 80–100% sequence identity with the probe are bound (DIG Application Guide for Filter Hybridization, Roche). For the detection of probe, the membrane was washed and blocked using the DIG Wash and Block Buffer Set (Roche), incubated with anti-DIG-alkaline phosphatase antibody (Roche), and then incubated with a chromogenic substrate for alkaline phosphatase, Nitro-Blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt.

Bioassay of streptomycin

The bioassay of streptomycin essentially followed a method described previously (Horinouchi et al. 1984). The indicator was Bacillus subtilis 168 obtained from Dr. Keith Stephenson, University of Leeds. The specificity was checked by the parallel use of streptomycin-sensitive or -resistant E. coli. 245 × 245 mm2 bioassay dishes (Corning) containing 150 ml of Bennett’s medium in 2% (w/v) agar No. 1 (LAB M) were spotted at 3 cm intervals with 3 × 106 spores. The plates were incubated at 30°C for 2 days before being overlaid with 50 ml of Bennett’s soft agar containing 2.1 ml of a culture of the indicator at OD600 0.5, which had been grown in LB medium at 37°C. After another day of incubation at 30°C, the area of the ring of inhibition of indicator growth around each patch of S. griseus was measured. Standard conditions were used for the preparation and quantitation of spores (Kieser et al. 2000).

Analysis of transcript levels

RNA was isolated, quantitated, and reverse transcribed as described previously (Uguru et al. 2005). The RNA was isolated from S. griseus mycelia scraped from cellophane (AA Packaging, UK) laid on the surface of YMPD agar plates. The level of the strR transcript in samples from mycelial patches of different age was analysed in real time as described previously (Uguru et al. 2005) using primers strR_FOR (5′-GAG CAA GTC CGT GAG AGG TC) and strR_REV (5′-GAG GGA AGC AAT GAT TCG AC). As before (Uguru et al. 2005), the values were normalised using the level of hrdB mRNA as a control. The PCR reaction conditions were 95°C for 7 min followed by 45 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 40 s. The temperature at which fluorescence was measured at the end of each cycle was 83°C. The relative levels of several transcripts in samples from mycelial patches of the same age were analysed semi-quantitatively using end-point analysis. The primers for the amplification of segments of hrdB (127 bp), strB1 (123 bp), strD (125 bp) and adpA (130 bp) mRNA were 5′-TGG TCG AGG TCA TCA ACA AG with 5′-TGG ACC TCG ATG ACC TTC TC, 5′-GAC GGG TTC CAC GAC TAC TG with 5′-CAG CAG GAG GTC CTT GTA G, 5′-GTC GCC GAG ATA CAT GAT GA with 5′-ATG GTT CGA GAT TCG GAC TG, and 5′-CGA TCT CTG CCT CCA CAT AG with 5′-CTC CGG TAA AGA CCT GTC CA, respectively. The size of each amplicon is provided in the parentheses. The PCR conditions for end-product analysis were standard PCR reactions using PCR reaction buffer Mix J (Epicenter) and 10% (v/v) DMSO (Sigma). The reactions were done in 0.2 ml thin-wall tubes (BIOplastics) using a standard PCR machine with a heated lid (MiniCycler, MJ Research) and the final products were analysed by gel electrophoresis in TBE-buffered 2% (w/v) agarose. The reactions were incubated at 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 40 s.