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Keywords:

  • Sigma factor;
  • Differentiation;
  • Spore maturation;
  • Pigment production;
  • Gene disruption;
  • Streptomyces

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The Streptomyces aureofaciens sigF gene encodes a sigma factor. By integrative transformation, via double cross-over, a stable null mutant of sigF gene was obtained. This mutation appeared to have no obvious effect on vegetative growth, but affected the late stage of spore maturation. Microscopic examination showed that spores were deformed, and spore wall was thinner, compared with the wild-type spores. The spore pigment of sigF mutant was green, compared to wild-type grey-pink spore pigmentation. The plasmid-born wild-type sigF gene complemented the mutation after transformation of the mutant strain.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Gram-positive soil bacteria of the genus Streptomyces undergo a complex cycle of morphological differentiation, resulting in uninucleate spores characteristic by a species-dependent pigmentation [1]. The process is controlled at several levels, among which the heterogeneity of sigma factors of RNA polymerase plays an important role [2]. Two genes encoding sigma factors, having a function in the regulation of sporulation have been identified in Streptomyces. WhiG of S. coelicolor[3] and its homologue rpoZ in S. aureofaciens[4] have a role in the initiation of differentiation of aerial hyphae into spore chains. The second gene, sigF, was cloned in S. aureofaciens and S. coelicolor[5]. Disruption of the sigF gene in S. coelicolor affected late stages of sporulation. To investigate the function of sigF counterpart in S. aureofaciens, we attempted to disrupt the gene in the strain and to compare the phenotype to the sigF-disrupted strain of S. coelicolor.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial strains and plasmids

S. aureofaciens CCM3239 wild-type (ATCC 10762) was from Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia. S. lividans TK24 [6] was kind gift from Professor D.A. Hopwood (John Innes Institute, Norwich, UK), and plasmid pGM9 [7] from W. Wohllenben (University Bielefeld, Germany). E. coli SURE™ (Stratagene) was used in all cloning experiments. Plasmids used for Streptomyces transformation were passaged through the damdcmE. coli strain JM110 (Stratagene). E. coli plasmid pBluescript II SK+ was from Stratagene.

2.2Media, cultivation conditions, and transformation

For protoplasts preparation and DNA isolation, S. aureofaciens was cultured to late-exponential phase (24 h) in liquid TSB medium [6] containing 1% (w/v) maltose and 0.5% (w/v) glycine as described in [8]. Growth and transformation of S. lividans were carried out as described in [6]. Transformation of S. aureofaciens was performed as described in [8]. Conditions for E. coli growth and transformation were as described in [10]. Phenotype of mutants were analysed after growth on solid minimal MM medium [6] and rich Bennet medium [9].

2.3DNA isolation and Southern blot hybridization

Plasmid DNA was isolated from Streptomyces as described in [6] and from E. coli as described in [10]. Chromosomal DNA from wild-type and disrupted S. aureofaciens strains was isolated according to [6]. 1 μg of the chromosomal DNA was digested with appropriate restriction endonuclease, separated by electrophoresis in 0.8% (w/v) agarose gel in TBE, and transferred on Hybond N (Amersham) as described in [10]. The membrane was then hybridized with random primed [α-32P]dCTP-labelled DNA probe at 45°C in 50% (v/v) formamide as described in [10].

2.4Recombinant DNA techniques

All DNA manipulations in E. coli were performed as described in [10]. DNA fragments were isolated from agarose gels with Geneclean (BIO101, La Jolla, CA). The cassette containing S. azureus tsr gene conferring resistance to thiostrepton was prepared by inserting a 1.05 kb BclI fragment of plasmid pIJ486 [11] into the BamHI site of pBluescript SK. The neomycin resistance gene of Tn5 was cloned as 1.3 kb SmaI-HindIII fragment from pGM9 [7] in EcoRV-HindIII digested pBluescript SK, generating pAPHII1. Plasmid pRPO5-5L contained the 2.3 kb SacI-HindIII fragment (Fig. 1) containing the full-length S. aureofaciens sigF gene in pBluescript SK [5]. The gene was disrupted by inserting 1.05 kb PstI (blunt-ended)-NotI fragment containing tsr between SauI (blunt-ended) and NotI of pRPO5-5L to generate pRPO5-5P (Fig. 1). The 3.2 kb SacI (blunt-ended)-HindIII fragment of pRPO-5P was inserted in pAPHII1 cut with XhoI (blunt-ended) and HindIII to generate pRPO5-5S that was finally used for gene replacement of sigF.

image

Figure 1. (a) Chromosomal DNA comprising sigF of S. aureofaciens wild-type strain and the strain S. aureofaciens, sigF::Tsr2, disrupted via double cross-over. The 2.3 kb SacI-HindIII fragment of pRPO5-5L, and 3.2 kb SacI-HindIII fragment of pRPO5-5P, with flanking restriction sites from pBluescript SK polylinker, are shown by stippled boxes. The black bars below the maps represent probes used for Southern hybridization analysis. Probe 1, 1100 bp SacI-SauI fragment comprising upstream part of sigF; probe 2, 1050 bp SmaI fragment of pFK41 [8] containing the tsr gene. Restriction sites relevant to Southern hybridization experiment are indicated. (b) Southern hybridization analysis of chromosomal DNA from the gene replacement experiments. Experimental details are in Section 2. Lanes: 1, PvuII-digested S. aureofaciens CCM3239 wt DNA; 2, PvuII-digested DNA from the sigF-disrupted strain; 3, EcoRV-digested wt DNA; 4, EcoRV-digested disrupted DNA; 5, ClaI-digested wt DNA; 6, ClaI-digested disrupted DNA. Southern blot was probed with probe 1 comprising upstream part of sigF. The same blot was treated to remove radioactive probe 1 as described in [10] and probed with probe 2 comprising tsr gene under the same conditions. Lambda DNA-BstEII digest was used as the molecular mass standard. GenBank Acc. No. of S. aureofaciens sigF is L09565.

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The plasmid pGM-rpoX2, used for complementation of the sigF mutation, was prepared by inserting a 2.3 kb SacI (blunt-ended)-XhoI fragment from pRPO5-5L (Fig. 1), containing the sigF gene, in pGM9 cut with XbaI (blunt-ended)-XhoI.

2.5Electron microscopy

Plates after 5 days grown cultures were overlaid with 2% (w/v) agarose and 1 mm3 blocks were cut after agarose solidification. The blocks were washed in a buffer containing 0.1 M Na cacodylate (pH 7.2), fixed in the same buffer containing 2% (v/v) glutaraldehyde and 1% (w/v) OsO4, and further treated as described previously [12]. Cells were embedded in Spurr medium [13] and sectioned (40–120 nm thickness) on a NOVA3 ultramicrotome (LKB). Sections were examined in a Tesla T541 (Brno, Czechoslovakia) electron microscope.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Disruption of sigF in S. aureofaciens

We have previously cloned a sigma factor gene, sigF, in S. aureofaciens and S. coelicolor[5]. Disruption of the sigF gene in S. coelicolor affected the last stage in differentiation, resulting in white spores with much thinner spore cell wall [5]. To investigate the function of sigF in S. aureofaciens, we have disrupted the gene also in this strain. The chromosomal copy of sigF was inactivated by a double cross-over using a modification of the method for disruption of S. aureofaciens genes [8]. The thiostrepton resistance gene (tsr) was used to replace the internal part of sigF, deleting amino acids 8 to 87 (Fig. 1). Resulting plasmid pRPO5-5S (Section 2) contained an additional marker, kanamycin resistance gene, and lacked any sequence necessary for replication in S. aureofaciens. The plasmid was used to transform S. aureofaciens to thiostrepton resistance. Since pRPO5-5S was unable replicate in S. aureofaciens, thiostrepton-resistant transformants were expected to arise from homologous recombination between S. aureofaciens insert in the plasmid and corresponding region in chromosome. Two types of clones were expected; thiostrepton resistant and kanamycin resistant that might arise from a single cross-over; and thiostrepton resistant and kanamycin sensitive arising likely from a double cross-over between both flanking regions on both sides of the tsr gene, resulting in the replacement of the wild-type sigF gene by the disrupted allele. Twelve thiostrepton-resistant clones were identified. They were further analysed for thiostrepton resistance and kanamycin sensitivity that might indicate double cross-over event. Two kanamycin-sensitive clones were identified, and correct integration was confirmed by Southern blot hybridization (Fig. 1). Both clones had similar phenotype, and one clone, S. aureofaciens, sigF::Tsr2, was chosen for further study.

3.2Phenotypic analysis of the S. aureofaciens sigF-disrupted strain

Similar to S. coelicolor, sigF mutation did not affect vegetative growth. However, when grown on MM or Bennet media, S. aureofaciens, sigF::Tsr2 produced spores with green pigmentation in contrast to the wild-type grey-pink spore pigmentation (Fig. 2). This phenotype did not appear to be conditional. It was the same on rich Bennet medium, as on minimal MM medium, and it was not affected by the presence of various carbon sources (mannitol, glucose, maltose, glycerol). Similar to S. coelicolor sigF-disrupted strain, S. aureofaciens, sigF::Tsr2 produces smaller spores being much resistant to fragmentation. As shown in Fig. 3, electron microscopy of the mutant colony grown on solid medium revealed aberration in spore wall. The spore wall was much thinner (20–30 nm) than that of the wild-type (50–70 nm). However, there were no differences in chromosome condensation, which were shown in S. coelicolor sigF-disrupted strain. The results indicate a similar role of sigF in S. aureofaciens differentiation as shown in S. coelicolor[5]. The only significant difference between the wild-type and the sigF null mutant is in spore colour. S. coelicolor sigF mutant has a phenotype similar to whi producing spores with pale grey colour, as opposed to the wild-type spore pigmentation which is dark grey [5]. The green spore pigmentation of S. aureofaciens, sigF::Tsr2 indicates a different defect, likely in an intermediate stage of spore pigment production.

image

Figure 2. Sporulated S. aureofaciens wild-type and sigF-disrupted strains. Strains were grown 5 days on Bennet medium [9].

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image

Figure 3. Transmission electron microscopy of thin section of colonies of S. aureofaciens wild-type (A), and sigF-disrupted (B) strains. Strains were grown 5 days on Bennet medium [9]. Scale bar=0.5 mm.

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In S. coelicolor, the grey spore pigment is produced by the action of whiE, a complex locus encoding polyketide synthase proteins that are believed to be directly involved in the synthesis of the pigment [14]. The whiE gene cluster includes also a gene (ORFVIII) transcribed divergently from the previously identified ORFI-VII operon [15], and supposed to encode a hydroxylase-like gene product involved in late stages of spore pigment biosynthesis [16]. Disruption of the ORFVIII in S. coelicolor caused green spore colour, compared with the normal grey colour of wild-type spores [15]. A Southern blot hybridization with S. aureofaciens DNA at high stringency using a probe from internal part of S. coelicolor whiE locus (comprising ORFII-IV) clearly identified a homologous gene set also in this strain (data not shown). The green spore phenotype of the S. aureofaciens, sigF::Tsr2 indicates that the sigma factor encoded by sigF could directly or indirectly regulate expression of genes involved in spore pigmentation. Considering that S. aureofaciens also contains ORFVIII-homologous gene, based on the S. aureofaciens sigF disruption phenotype (green spore colour), this gene might be a candidate for sigF-encoded sigma factor, and green spore pigment might be a polyketide intermediate of spore pigment missing final hydroxylation. It is interesting that only the divergent whiE promoter (transcribed ORFVIII) is off in S. coelicolor sigF mutant (M.J. Buttner and G. Kelemen, personal communication). However, the mutant produced pale grey spores. It seems that there must be some differences in either the control of expression, or the enzymes involved in making the two spore pigments. To prove this hypothesis, cloning of the corresponding spore pigment regulatory region in S. aureofaciens is in progress.

3.3Complementation of the sigF mutation

To rule out the possibility that the mutant phenotype might be caused by a polar effect, we attempted to complement the mutation in S. aureofaciens sigF. With regard to the fact that the only plasmid able to replicate in S. aureofaciens is pGM9 [8], we have cloned the whole sigF gene with the upstream promoter region [17] in this plasmid (Section 2). Resulting plasmid, pGM-rpoX2, after isolation from S. lividans TK24, was used to transform S. aureofaciens, sigF::Tsr2. As shown in Fig. 2, transformed disrupted strain showed normal spore pigmentation and the spores appeared to be morphologically indistinguishable from wild-type in the light and electron microscopes (data not shown).

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

We would like to thank Mrs. Renáta Knirschová for excellent technical assistance. We are grateful to Prof. D.A. Hopwood for providing us with S. lividans TK24, W. Wohllenben for plasmid pGM9, and Mark J. Buttner for helpful comments on the manuscript. This work was supported by Grant 2/4007/97 from Slovak Academy of Sciences.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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