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Molecular Genetics of Streptomycetes |
Department of Genetics, John Innes Centre, Norwich NR4 7UH, UK1
Departamento de Microbiología y Genética, Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Edificio Departmental, Campus ‘Miguel de Unamuno’, 37007 Salamanca, Spain2
Author for correspondence: Mervyn J. Bibb. Tel: +44 1603 452571. Fax: +44 1603 456844. e-mail: bibb{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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C31. These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a 
factor required for spore maturation, and of redD, a pathway-specific regulatory gene for the production of undecylprodigiosin, one of the four antibiotics made by S. coelicolor. While transcription of sigF appeared to be confined to developing and mature spore chains, transcription of redD occurred only in ageing substrate mycelium. A further plasmid derivative (pIJ8668) was made that lacks the C31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations. Keywords: Streptomyces coelicolor A3(2), green fluorescent protein (GFP), sigF, redD, tipAp
Abbreviations: EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; MCS, multiple cloning site; Thio, thiostrepton; tipAp, the Thio-inducible tipA promoter of Streptomyces lividans 66
This paper is dedicated to the memory of Kathy Kendrick, whose devotion to understanding the biology of Streptomyces was unsurpassed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The production of these molecules generally occurs in a growth-phase-dependent manner, and in agar-grown cultures it usually coincides with the onset of morphological development, a process that culminates in the formation of long chains of unigenomic spores that are the major means of dispersal to new environments. Morphological differentiation is initiated by the formation of aerial hyphae from a substrate mycelium that grows on, and into, a suitable growth medium. The aerial hyphae undergo a series of changes during which the multigenomic tip compartments subdivide into unigenomic prespore compartments by spatially regular and temporally synchronized multiple septation (Chater & Losick, 1996 ). During spore maturation, the spore wall thickens, and in most streptomycetes, spore pigment is deposited. This complex process of differentiation involves a co-ordinated programme of gene expression that promotes a variety of physiological and structural changes in both the vegetatively growing substrate mycelium and in the developing aerial hyphae. These changes include the onset of antibiotic biosynthesis, which generally appears to be confined to the older substrate mycelium (for reviews see Chater & Bibb, 1997 ; Chater, 1998 ; Kelemen & Buttner, 1998 ).
Streptomyces coelicolor A3(2) is by far the most genetically characterized streptomycete, and is currently the subject of a genome sequencing project (http://www.sanger.ac.uk/Projects/S_coelicolor). It produces at least four chemically distinct antibiotics, and undergoes a typical sporulation cycle (Hopwood et al., 1995 ). Many of the genes that are required for morphological and physiological differentiation in S. coelicolor have been identified, and a picture is beginning to emerge of the regulatory mechanisms that control each, and sometimes both, of these processes (Bibb, 1996 ; Chater, 1998 ). While several genes have been adapted for use as reporter systems for gene expression in streptomycetes, only the luciferase-encoding luxAB gene cassette of Vibrio harveyi has been applied to study the temporal and spatial expression of genes during development (Schauer et al., 1988 ). In many organisms, the green fluorescent protein (GFP) of the jellyfish Aequorea victoria has proved to be a particularly useful and sensitive reporter (Chalfie et al., 1994 ; Tsien, 1998 ). GFP is a 27 kDa protein with a cyclized tripeptide fluorophore (Ser65-Tyr66-Gly67 in the wild-type protein). Its detection does not require the application of any substrates or co-factors and can be achieved with a simple fluorescence microscope. Wild-type GFP absorbs UV and blue light, with a major peak of absorbance at 395 nm and a minor peak at 470 nm; it emits green light maximally at 509 nm, with a shoulder at 540 nm (Clontech Living Colors GFP application notes, 1996).
Earlier attempts to use wild-type GFP as a reporter for gene expression in S. coelicolor failed to reveal fluorescence even when the gene was placed on a multicopy (approx. 150 copies per chromosome) plasmid (M. S. B. Paget, personal communication). This may have reflected the relatively AT-rich nature of the wild-type GFP gene (38 mol% G+C compared to the estimated 74 mol% G+C of the S. coelicolor chromosome) and the consequent marked difference in codon usage between the GFP gene and its surrogate host. In addition, the wild-type GFP gene contains three TTA codons that may have also hindered synthesis of GFP. TTA codons occur rarely in streptomycete genes and may be translated in a developmentally regulated manner that limits expression of TTA-containing genes in vegetatively growing mycelium (Leskiw et al., 1991 ). Recently, a red-shifted variant of GFP was developed [EGFP (enhanced GFP)] that gives brighter fluorescence and higher levels of expression in mammalian cells (Clontech Living Colors GFP application notes, 1996; Haas et al., 1996 ; Cormack et al., 1996 ). EGFP contains two amino acid substitutions, Ser65 to Thr and Phe64 to Leu, that lead to a 35-fold enhancement of fluorescence over wild-type GFP when excited at 488 nm, and possesses excitation and emission maxima of 488 nm and 507 nm, respectively. This excitation maximum corresponds to the excitation wavelength of commonly used filter sets, and also corresponds to the wavelength emitted by the argon laser used in most confocal microscopes, resulting in brighter signals than those given by wild-type GFP. Perhaps more importantly for use in streptomycetes, the coding region of EGFP contains more than 190 silent nucleotide changes that were based on human codon usage preferences in an attempt to maximize translational efficiency in mammalian systems (Haas et al., 1996 ). Since this altered codon usage corresponds much more closely to that of streptomycete genes than does that of the wild-type GFP gene (only seven of the 239 codons present in the EGFP gene end in A or T compared with 163 out of 238 in the wild-type gene, and all three TTA codons have been removed), we assessed the use of EGFP as a reporter for the analysis of temporal and spatial gene expression in S. coelicolor.
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METHODS |
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) was the streptomycete used throughout these studies, and was grown and maintained using standard procedures (Hopwood et al., 1985 ). The EGFP gene was obtained from Clontech as pEGFP. Plasmids were constructed in Escherichia coli strain DH5
(Hanahan, 1983 ) using standard procedures (Sambrook et al., 1989 ), and introduced into the methylation-deficient E. coli strain ET12567 (dam dcm hsdS; MacNeil et al., 1992 ) containing the helper plasmid pUZ8002 (Paget et al., 1999a ) prior to conjugation into S. coelicolor M600. Integration of pIJ8600 derivatives at the C31 chromosomal attachment site of M600 was confirmed by Southern hybridization. Microscopy was carried out on cultures grown on SMMS agar (Floriano & Bibb, 1996 ).
Visualization of EGFP.
Fluorescence microscopy was carried out on a Zeiss Axiophot photomicroscope equipped with a FITC filter set, using ax20 objective for single colonies, and ax100 objective for examination of cultures grown on cover-slips. Confocal microscopy was carried out using a Leica TCS NT confocal microscope (488 nm argon laser,x63 objective 0·5 numerical aperture dry PL FLUOTAR) on cultures grown at 30 °C for 72 h (sigFp) or 60 h (redDp).
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RESULTS |
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) was first constructed. pIJ8600, which is a derivative of the conjugative and integrative pSET152 (Bierman et al., 1992 ), contains the pUC18 origin of replication and an apramycin-resistance gene, aac(3)IV, for maintenance and selection in E. coli, respectively. It contains an origin of transfer, oriT, from the plasmid RK2 which, with the assistance of an appropriate helper plasmid, allows mobilization of pIJ8600 into streptomycetes upon contact with E. coli. It possesses the C31 int gene and attP site, allowing insertion of the plasmid at the chromosomal C31 attachment site, using apramycin resistance for selection in streptomycetes. In addition, pIJ8600 contains the thiostrepton (Thio)-inducible tipAp promoter (Takano et al., 1995 ) located immediately upstream of a multiple cloning site (MCS), both of which are flanked by transcription terminators. The MCS (Fig. 1b) contains a unique NdeI site positioned downstream of the natural tipA ribosome-binding site, permitting efficient expression of cloned genes from their natural translational start codons.
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), and transferred to S. coelicolor strain M600 by conjugation. Exconjugants were selected on the basis of apramycin resistance. Plating of a mixture of M600(pIJ8655) and M600(pIJ8600) spores on SMMS agar plates containing 12 µg Thio ml-1 and observation of the resulting colonies by low-magnification fluorescence microscopy readily distinguished the EGFP-containing colonies from those harbouring the parental vector (Fig. 2a). For a more detailed analysis, spores of M600(pIJ8655) were inoculated adjacent to a cover-slip inserted at an angle of approximately 60° in an SMMS agar plate, and expression of the EGFP gene was induced by application of Thio (12 µl of a 1 mg ml-1 solution in water) to the developing mycelium. The results of induction were monitored by fluorescence microscopy of hyphae present on the extracted cover-slip. Induction with Thio at the time of inoculation resulted in fluorescence throughout the substrate and aerial hyphae, but not within mature spore chains (Fig. 2b). In contrast, application of Thio every 24 h for 4 d resulted in fluorescence throughout the mycelium, including the mature spores (Fig. 2b). This difference may reflect dissipation of the inducer prior to the formation of sporulation septa, or an active mechanism for restoration of the uninduced state of tipA expression that can be overcome by periodic addition of Thio. Interestingly, the level of fluorescence increased in an apparently linear fashion with the amount (1–12 µg) of Thio applied to the culture containing pIJ8655. Quantification of fluorescence emitted by cultures of the same strain grown on MS agar (Hobbs et al., 1989 ) for 24 h using a LabTech FLI fluorescence plate reader has confirmed this proportional relationship (P. Herron & P. Dyson, personal communication).
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); pIJ8660 differs from pIJ8630 in possessing translational stop codons in each of the three possible reading frames that precede the EGFP coding sequence, preventing the potentially deleterious formation of EGFP fusion proteins. In S. coelicolor, sigF encodes a factor that appears to play a late role in spore development. Compared with the wild-type strain, sigF mutants develop irregular, thin-walled, poorly pigmented, detergent-sensitive spores with apparently uncondensed DNA (Potú
ková et al., 1995 ). Consistent with this late function in sporulation, the appearance of sigF transcripts, monitored by S1 nuclease protection analysis, coincided with the appearance of sporulation septa in the aerial hyphae (Kelemen et al., 1996 ), although it was not clear whether this temporal control also reflected spatial regulation of sigF transcription. To address this question, a 0·4 kb PCR-derived BamHI–KpnI fragment containing the sigF promoter region (Kelemen et al., 1996 ) was inserted upstream of EGFP in pIJ8630, yielding pIJ8635 (Fig. 1a). Analysis by confocal microscopy revealed that expression of sigF did indeed appear to be limited to developing and mature spore chains, with no evidence of sigF transcription before sporulation septa formed (Fig. 3a) [additional analysis, using, for example, DAPI staining (Kwak & Kendrick, 1996 ), would be required to ascertain precisely when sporulation septa were formed with respect to sigF transcription].
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). redD plays a pivotal role in determining the onset of Red production, with its transcription increasing markedly upon entry into stationary phase in liquid culture (Takano et al., 1992 ). In contrast to the results obtained with the sigF promoter, when a 1·1 kb BamHI–KpnI fragment containing the redD promoter was cloned upstream of the EGFP gene in pIJ8630 and in pIJ8660, yielding pIJ8678 and pIJ8665, respectively (Fig. 1a), fluorescence was observed only in substrate hyphae and not in developing spore chains, nor in the hyphal compartments adjacent to them (Fig. 3b). Moreover, fluorescence was observed only when a proportion of the hyphae had lysed (apparent from the conspicuous presence, under transmission microscopy, of hyphal ghosts), suggesting that transcription of redD is confined to ageing substrate mycelium. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; E. Leibovitz, personal communication) and there seems little doubt that EGFP will prove to be a highly effective reporter, particularly for analysing both morphological and physiological differentiation, in this important group of micro-organisms. As well as the attP-containing vectors pIJ8630 and pIJ8660, a derivative of pIJ8660 (pIJ8668; Fig. 1) was made that lacks the C31 attachment site (attP) and int gene. pIJ8668 can be used to create a transcriptional fusion between the EGFP gene and a gene of interest at the latter’s native chromosomal locus by recombination between a cloned fragment that lacks the promoter of that gene and the homologous chromosomal sequence. There are many examples in the literature in which GFP has been fused to the N- or C-terminal end of a protein of interest; frequently, the biological activity of that protein and the fluorescence properties of GFP are retained, allowing in vivo localization of the fusion product. The development of vectors based on those described here that will permit the generation of EGFP-translational fusions for the analysis of many aspects of the biology of streptomycetes is envisaged. |
ACKNOWLEDGEMENTS |
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Received 23 February 1999;
revised 1 June 1999;
accepted 2 June 1999.
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H. A. Hussain and J. M. Ward Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts Appl. Envir. Microbiol., January 1, 2003; 69(1): 373 - 382. [Abstract] [Full Text] [PDF]
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M. D. Lefebre and M. A. Valvano Construction and Evaluation of Plasmid Vectors Optimized for Constitutive and Regulated Gene Expression in Burkholderia cepacia Complex Isolates Appl. Envir. Microbiol., December 1, 2002; 68(12): 5956 - 5964. [Abstract] [Full Text] [PDF]
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A. Sobczyk, A. Bellier, J. Viala, and P. Mazodier The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes Microbiology, June 1, 2002; 148(6): 1931 - 1937. [Abstract] [Full Text] [PDF]
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N. Ali, P. R. Herron, M. C. Evans, and P. J. Dyson Osmotic regulation of the Streptomyces lividans thiostrepton-inducible promoter, ptipA Microbiology, February 1, 2002; 148(2): 381 - 390. [Abstract] [Full Text] [PDF]
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E. Rodriguez, C. Banchio, L. Diacovich, M. J. Bibb, and H. Gramajo Role of an Essential Acyl Coenzyme A Carboxylase in the Primary and Secondary Metabolism of Streptomyces coelicolor A3(2) Appl. Envir. Microbiol., September 1, 2001; 67(9): 4166 - 4176. [Abstract] [Full Text] [PDF]
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J. Sun, A. Hesketh, and M. Bibb Functional Analysis of relA and rshA, Two relA/spoT Homologues of Streptomyces coelicolor A3(2) J. Bacteriol., June 1, 2001; 183(11): 3488 - 3498. [Abstract] [Full Text]
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W. P. Revill, M. J. Bibb, A.-K. Scheu, H. J. Kieser, and D. A. Hopwood {beta}-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A3(2) J. Bacteriol., June 1, 2001; 183(11): 3526 - 3530. [Abstract] |
; tipAp, the tipA promoter; tsr, Thio-resistance gene; aac(3)IV, apramycin-resistance gene selectable in E. coli and streptomycetes; ori pUC18, origin of replication from pUC18; oriT RK2, origin of transfer from plasmid RK2; int 
