Department of Biological Sciences, CW405 Biological Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2E91
Department of Genetics, John Innes Centre, Colney, Norwich NR4 7UH, UK2
Author for correspondence: Brenda K. Leskiw. Tel: +1 780 492 1868. Fax: +1 780 492 9234. e-mail: brenda.leskiw{at}ualberta.ca
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ABSTRACT |
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Keywords: Streptomyces, differentiation, antibiotic production, anti-sigma factor antagonist, anti-sigma factor
The GenBank accession numbers for the sequences reported in this paper are AF134889 and AL035636
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INTRODUCTION |
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In Streptomyces coelicolor A3(2), the genetically most-studied streptomycete, many genes required for morphological and physiological differentiation have been identified. As a result of such studies we know that the regulatory mechanisms involve both pathway-specific and global regulatory genes (Champness, 2000 ). Of particular note are the bld (bald) genes, mutations in which often result in the failure to form both aerial hyphae and secondary metabolites. Thus the bld genes provide a key to understanding how the physiological and structural changes are initiated in the ageing substrate mycelium.
Several bld genes have been cloned, sequenced and characterized. This, together with functional analysis of several other genes, in which mutations give rise to a bald mutant phenotype but which had previously been given other gene designations (Chakraburtty & Bibb, 1997 ; Ma & Kendall, 1994
; Susstrunk et al., 1998
), is beginning to paint a picture whereby the onset of physiological and morphological differentiation is governed by extracellular and metabolic signals, and is regulated at the level of transcription, translation and post-translational modification. When S. coelicolor is grown on rich media, Willey et al. (1991)
showed that structural differentiation to form aerial mycelium depends on an extracellular protein, SapB. Although SapB production was impaired in all of the bld mutants tested, juxtaposition of pairs of bld mutants on the surface of agar plates resulted in the unidirectional restoration of both SapB and aerial-hyphae formation, suggesting that differentiation is governed by a hierarchical cascade of intercellular signals, with the bld genes themselves directly or indirectly responsible for the production of those signals (Willey et al., 1993
). Recent studies by Nodwell et al. (1996)
have provided biochemical evidence in support of this extracellular signalling model; the bldK locus encodes the subunits for an ATP-binding cassette family, oligopeptide permease that appears to be the importer of a bldJ (formerly bld261)-dependent, extracellular factor (Nodwell & Losick, 1998
). However, acceptance of this model is complicated by the observation that not all of the bld genes fit into the proposed hierarchy (Nodwell et al., 1999
; Willey et al., 1993
). Of particular note is bldB, which encodes a putative DNA-binding regulatory protein and mutations in which, along with many of the other bld mutations (bldA, -B, -C, -D, -G and -H), causes a deregulation of carbon utilization (Pope et al., 1996
). That these mutants activate the gal operon promoter under conditions where it is normally repressed, is consistent with the idea that a change in metabolic state is associated with the onset of aerial mycelium formation (Karandikar et al., 1997
; Nodwell et al., 1999
). This idea is further supported by the finding by Susstrunk et al. (1998)
that mutations in the adenylate cyclase gene, cya, which result in a classic bald phenotype, also cause a medium pH decrease, suggesting that the onset of differentiation involves a switch in metabolism to utilize organic acids released to the medium.
To formulate a model for the regulation of differentiation that takes into account all of these observations, it would be valuable to understand the function of all of the bld gene products. To this end we have cloned and sequenced the S. coelicolor bldG gene. bldG was first identified in a study in which mutagenized colonies were screened for blocks in the formation of aerial hyphae and antibiotic biosynthesis (Champness, 1988 ). Three of the mutants identified, C103, C107 and C101J, mapped to the same region of the chromosome and defined the bldG locus. Here we show that bldG encodes a protein product showing similarity to anti-sigma factor antagonists from Bacillus and Staphylococcus. Transcriptional analyses have also revealed that bldG and the ORF immediately downstream are co-transcribed. The deduced product of the downstream ORF shows some similarity to anti-sigma factors, suggesting that it and BldG might function as a regulatory pair governing the activity of an unknown sigma factor(s).
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METHODS |
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Escherichia coli host strains were DH5 (Gibco-BRL) and ET12567 (MacNeil et al., 1992
) (a gift from D. MacNeil, Merck Sharp & Dohme Research Laboratories). Media and culture conditions were as described by Sambrook et al. (1989)
.
Plasmid and bacteriophage vectors.
Streptomyces vector KC304 is a derivative of bacteriophage øC31 and contains the tsr (thiostrepton resistance) gene for vector selection; the vph (viomycin resistance) gene, flanked by BamHI sites, as a stuffer fragment for replacement by up to 6 kb of insert DNA; and the attint region to allow efficient integration at single copy number into the chromosomal att site for øC31. KC304 derivatives were manipulated as described by Hopwood et al. (1987) . Cosmid H5 (Redenbach et al., 1996
) was obtained from Helen Kieser, John Innes Centre, Norwich, UK. Prior to their use to transform S. coelicolor, the E. coliStreptomyces bifunctional vectors pSET152 (Bierman et al., 1992
) (NRRL B-14792) (containing a multiple-cloning site and replicon of pUC plasmids, the attint region of øC31 and the apramycin-resistance gene for vector selection in either E. coli or Streptomyces) and pKC1218 (Bierman et al., 1992
) (NRRL B-14790) (containing a multiple-cloning site and replicon of pUC plasmids, the SCP2* replicon and the apramycin-resistance gene for vector selection in either E. coli or Streptomyces) were replicated in the dam dcm host E. coli ET12567 using standard procedures (Sambrook et al., 1989
). Streptomyces plasmids were maintained by selection for resistance to thiostrepton (50 µg ml-1; a gift from S. Lucania, Bristol-Myers Squib, Princeton, NJ, USA) or Apralan [50 µg ml-1 and containing 50%, w/w, apramycin; Provel (Division of Eli Lilly Canada)]. E. coli plasmids pAU5 (Giebelhaus et al., 1996
) and pBluescript II SK/KS (Stratagene) were manipulated as described by Sambrook et al. (1989)
.
Isolation of a bldG103-complementing clone from an S. coelicolor phage library.
A library (generously provided by R. Passantino, Instituto di Biologia dello Sviluppo del Consiglio Nazionale dello Ricerche, Palermo, Italy) of S. coelicolor M145 DNA fragments in the Streptomyces phage vector KC304 was screened as previously described by Elliot et al. (1998) . In brief, the library screening involved spotting 5x20 µl aliquots of the phage-library suspension onto a lawn of S. coelicolor C103 mycelial fragments on the surface of each of two R2YE (Hopwood et al., 1985
) agar plates. The lawns were allowed to grow for 2 weeks and then the mycelium in and around the spotted areas was scraped from the surface of the plates, pooled and resuspended in sterile Milli-Q water. The mycelia were then homogenized to break the hyphae into small fragments, diluted and plated to give about 100 single colonies per plate on minimal medium (MM; Hopwood et al., 1985
) containing glucose as the carbon source and thiostrepton (50 µg ml-1) to select for phage-containing lysogens. Colonies showing aerial mycelium and pigmentation typical of the antibiotic-producing wild-type strain were sought as evidence of bldG complementation. Recombinant phages, containing the cloned bldG gene, were recovered from sporulating lysogens after CHCl3 fuming (to prevent transfer of viable spores) and replicated onto Difco nutrient agar plates with SNA (Soft Nutrient Agar) overlays containing S. lividans 1326 spores (Hopwood et al., 1985
) where free phages released from the lysogens resulted in plaque formation in the S. lividans lawn.
Subcloning and sequencing.
The 5·5 kb bldG-complementing fragment from KC741 (see Results) was removed from the øC31 vector as a
6·5 kb EcoRV fragment containing 1 kb of flanking øC31 vector DNA. The 6·5 kb blunt-ended fragment was ligated into EcoRV-digested pSET152 and the ligation mixture was used to transform E. coli DH5
. Digestion of the recombinant plasmid, designated pAU61, with HindIII allowed the subcloning of a
2·5 kb fragment that extended from the unique HindIII site in the cloned bldG-containing DNA rightwards to the HindIII site located in the vector polylinker (see Fig. 1
). The fragment was ligated into the HindIII site of pKC1218 and the recombinant plasmid, designated pAU63, was isolated after transformation of E. coli DH5
and selection for Apralan resistance. pAU63 was then passaged through E. coli ET12567 and used to transform protoplasts of S. coelicolor C103. Apralan-resistant transformants were selected and visually scored for their phenotype. The 2·5 kb complementing fragment was subcloned in both orientations into the HindIII site of pBluescript SK+ and recombinant plasmids were designated pAU64 and pAU65. Each of the pBluescript plasmids was used to generate a series of exonuclease III deletion derivatives by the method of Henikoff (1984)
using the Erase-a-Base system (Promega). Additional DNA sequence for the region downstream of the right-hand end of the clone was obtained by first subcloning an overlapping 2·3 kb BamHI fragment extending rightwards from the predicted bldG ribosome-binding site (see Fig. 1
). The BamHI fragment from the S. coelicolor cosmid H5 was shotgun cloned in both orientations into pBluescript KS+ and recombinant plasmids were identified by hybridization using an ORF3-specific oligonucleotide, JWA9 (5'-CGTCGACGAGCTGGAGG-3'), as the probe. As described above, the pBluescript plasmids, designated pAU66 and pAU67, were then used to generate a series of exonuclease III deletion derivatives. The DNA sequence for the entire insert was determined both by manual and automated (Applied Biosystems model 373A) sequence analysis. The Universal sequencing primer and all other specific primers were obtained from the Department of Biological Sciences Synthesis Service, University of Alberta.
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Complementation of bldG mutants.
A 917 bp fragment containing the bldG ORF and upstream promoter was generated by PCR using pAU64 as template and the oligonucleotides DBG12 [5'-GCGCGAATTCGTGCCGGTGGCGACGAC-3'; nt -450 to -434 and containing a non-homologous tail (underlined) with an EcoRI site] and DBG3 [5'-GCGCTCTAGAGTTCGACGGTGGCCATG-3'; complementary to nt 451467 and containing a non-homologous tail (underlined) with an XbaI site] as primers (nucleotide positions are relative to the bldG translation start). The amplified DNA was gel purified by the trough-purification method (Zhen & Swank, 1993 ), digested with EcoRI/XbaI and ligated into EcoRI/XbaI-digested pSET152. The resulting recombinant plasmid, designated pAU69, was isolated after transformation of E. coli DH5
and selection for Apralan resistance. After passage through E. coli ET12567, pAU69 was introduced into protoplasts of S. coelicolor bldG mutants C536, C107, C101J and C3b by protoplast transformation. Apralan-resistant transformants were scored for their morphological and pigmentation phenotype.
RNA isolation.
Streptomyces cultures were grown on cellophane discs (75 mm, 325P discs; Courtaulds Films) on the surface of R2YE agar as previously described (Leskiw et al., 1993 ). RNA was extracted essentially as described elsewhere (Hopwood et al., 1985
) except that mycelia were scraped directly from the cellophane discs into modified Kirby mix. The RNA was isolated at various time points as described in Results.
Northern blot analysis.
This was performed according to Williams & Mason (1985) . RNA (40 µg) was denatured with glyoxal and DMSO, and size fractionated by electrophoresis at 4 V cm-1 on a 1·25% agarose gel using a 10 mM Na2HPO4/NaH2PO4 (pH 7·0) recirculating buffer system. DNA molecular mass marker III (625 ng; Boehringer), treated in the same way, served as the size marker. Capillary transfer to a Hybond-N (Amersham) membrane was as described by Sambrook et al. (1989)
. For detection of bldG transcripts, the probe was an [
32P]dCTP random-primer-labelled 207 bp PCR product internal to bldG (the probe was generated by PCR using primers BKL63, 5'-GTGAGGCCGGTGATACG-3', and BKL64, 5'-GCCCAAGCTGCGTGAGC-3'). For detection of transcripts extending through the intergenic region and into the ORF located downstream of bldG, the probe was an [
32P]dCTP random-primer-labelled 428 bp PCR product internal to the downstream ORF [the probe was generated by PCR using primers DBG8, 5'-GCGCAAGCTTGTCCGTACCGCCCGTCT-3', and BKL83, 5'-GCGCAAGCTTGGGTGAATGCGGCGGTC-3', both containing 10 nt non-homologous extensions (underlined)]. Hybridization was performed overnight at 65 °C in a solution containing 50% formamide (Hopwood et al., 1985
) and 4x106 c.p.m. probe. After hybridization, the nylon filter was washed at the same temperature for 2x30 min in a solution containing 2xSSC (0·3 M NaCl, 0·03 M sodium citrate), 0·1% SDS and then 2x30 min in 0·2xSSC, 0·1% SDS. The signals were detected using a PhosphorImager (Molecular Dynamics model 445 SI). The molecular mass marker was visualized by hybridizing [
32P]dCTP random-primer-labelled marker DNA to the filter at 44 °C. As a control for RNA loading levels, a probe for 16S rRNA was hybridized to the same blot. The 16S rRNA probe was an oligonucleotide, 5'-CCGCCTTCGCCACCGGT-3', corresponding to a conserved region in Streptomyces 16S rRNA sequences. Hybridization and washing were performed at 55 °C without formamide according to Procedure B described by Hopwood et al. (1985)
.
S1 nuclease mapping of the bldG transcription-start site.
The probe for S1 nuclease mapping of bldG was generated by PCR amplification of a 264 bp fragment using pAU64 as the template. The primers were an 18-mer synthetic oligonucleotide, DBG14 (5'-CGTCAATTTCGCCACCGA-3'), corresponding to a sequence internal to the bldG ORF and a 27-mer synthetic oligonucleotide, JWA20 [5'-GCGCAA-GCTTGGGATCGATCGGGTCGG-3', corresponding to a region 194 nt upstream of the bldG start codon and containing a 10 nt non-homologous extension (underlined)]. The probes for S1 nuclease protection of the intergenic region between bldG and the downstream ORF were generated by PCR amplification of a 172 bp and a 401 bp fragment using pAU64 as template. The primers were a 17-mer synthetic oligonucleotide, JWA17 (5'-GGTACGGACGTGCTCGG-3'), corresponding to a sequence internal to the downstream ORF, and either a 27-mer synthetic oligonucleotide, JWA18 (5'-GCGCAAGCTTGCCACCGACTGACGACC-3') or a 27-mer oligonucleotide, BKL81 (5'-GCGGGAATTCGGTCGAGCTCGTGAACG-3'), corresponding to regions 121 nt and 350 nt upstream of the start codon for the downstream ORF and containing 10 nt non-homologous extensions (underlined). The amplified DNA was purified from a 2% agarose gel by the trough-purification method (Zhen & Swank, 1993 ). The 5' ends of the amplified DNA (about 2 pmol) were labelled with [
32P]ATP using T4 polynucleotide kinase. The probes, labelled at both ends, were used without treatment since the non-homologous extensions would be removed by the S1 nuclease treatment and would not result in the appearance of labelled, protected fragments (Leskiw et al., 1993
). The sequencing ladders for the bldG and intergenic region S1 nuclease mapping were generated by the dideoxy chain-termination method (Sanger et al., 1977
) using DBG14 and JWA17 as primers, and pAU64 as the template. For each S1 nuclease protection reaction, 40 µg RNA was hybridized to 50000 Cerenkov c.p.m. probe in formamide buffer as described by Hopwood et al. (1985)
except that glycogen (Roche) replaced the carrier tRNA. To control for RNA loading levels, the RNA samples were first subjected to Northern blot analysis using the 16S rRNA probe (see above) and aliquots showing equivalent signals were subsequently used for S1 nuclease mapping. The samples were run under standard conditions on a 6% polyacrylamide sequencing gel.
Primer-extension mapping of the bldG transcription-start site.
The primer was a 17-mer synthetic oligonucleotide, DBG15 (5'-GTCGACAGGGACAGGTC-3'), that was designed to hybridize immediately downstream from the bldG start codon and approximately 100 bp away from the proposed transcription-start point. Primer (50 pmol) was end-labelled with [32P]ATP as described above. Approximately 5 pmol of 32P-labelled primer and 40 µg RNA were dissolved in 1xSB buffer (60 mM NH4Cl, 10 mM Trisacetate, pH 7·4, 6 mM 2-mercaptoethanol) (Hartz et al., 1988
), denatured by heating to 90 °C for 5 min and annealed by transferring to 75 °C, slow cooling to 55 °C and then incubating at 55 °C for 1 h. The primer-annealed RNA was ethanol precipitated, washed with 80% ethanol and air-dried for 10 min. Extension of the primer was performed at 45 °C for 1 h in a solution of 1xSB buffer, 15 mM magnesium acetate, 3 mM dNTPs, 17·5 U RNAGuard (Amersham) and 12·5 U AMV reverse transcriptase (Roche). Loading dye (98% deionized formamide, 10 mM EDTA, pH 8·0, 0·025% xylene cyanol, 0·025% bromophenol blue) was added and the reaction was evaporated at 80 °C for 20 min. The entire reaction mixture was loaded onto a 6% polyacrylamide sequencing gel. A sequencing ladder was generated as described above using the same oligonucleotide as for the primer-extension reactions.
Computer-assisted sequence analysis.
General raw sequence handling was done using the GeneTool 1.0 program (BioTools). A version of the FRAME (Bibb et al., 1984 ) program modified to run on an Apple Macintosh (obtained from S. E. Jensen, University of Alberta, Canada) was used to detect putative ORFs. Similarities between deduced protein products and known proteins in the databases were detected using BLAST at the internet site http://ncbi.nlm.nih.gov. Multiple sequence alignments were generated using the PILEUP program of the Genetics Computer Group. Potential RNA secondary structures, together with
G values, were determined using the Mfold 2.3 program with the folding temperature set to 30 °C, the standard growth temperature used for these studies. The Mfold programs are available at the internet site http://www.ibc.wustl.edu/~zuker/rna/. Analysis of the ORF3 protein sequence for conserved bacterial histidine kinase domains was done using the Prosite ProfileScan server located at the internet site http://www.isrec.isb-sib.ch/software/PFSCAN_form.html.
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RESULTS |
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Sequencing and analysis of ORFs
The 5·5 kb DNA fragment containing bldG was subcloned from KC741 as a 6·5 kb EcoRV fragment containing 1 kb of flanking øC31 vector DNA. The fragment was introduced into pSET152, which integrates site specifically into the S. coelicolor chromosome at the øC31 attB site, generating pAU61. Further subcloning of a 2·5 kb HindIII fragment from pAU61 into pKC1218, a low-copy-number E. coliStreptomyces shuttle vector, generated pAU63. Introduction of pAU63 into the S. coelicolor C103 mutant restored apparently wild-type levels of both pigmented-antibiotic production and aerial-mycelium formation. Nucleotide sequencing of the 2·5 kb HindIII fragment revealed two partial (ORF1 and ORF3) and one complete ORF (ORF2) (Fig. 1
). BLAST analysis indicated that the predicted product of ORF2 resembles a group of anti-anti-sigma factor proteins that include RsbV from Staphylococcus aureus (Wu et al., 1996
) and SpoIIAA and RsbV of Bacillus subtilis (Kalman et al., 1990
; Dufour & Haldenwang, 1994
; Duncan et al., 1996
). Since ORF2 was the only complete ORF present on the subcloned 2·5 kb DNA fragment that was able to complement the bldG103 mutation, this ORF was designated bldG (GenBank accession no. AF134889). Since genes encoding anti-anti-sigma factors are typically encoded as the first gene in an operon also encoding a cognate anti-sigma factor and sigma factor (Kalman et al., 1990
), the remainder of the ORF3 sequence, as well as an additional
1 kb of downstream sequence was determined. Comparison with sequences in the databases revealed that the ORF3 protein product resembles anti-sigma factor proteins of B. subtilis, including SpoIIAB (Duncan & Losick, 1993
; Min et al., 1993
) and RsbW (Benson & Haldenwang, 1993a
); anti-sigma F and anti-sigma B proteins, respectively. Surprisingly, a sigma factor was not encoded in the sequence downstream of the putative anti-sigma factor. Instead, a partial ORF showing similarity to pyrophosphate synthases was located 339 nt downstream of the ORF3 stop codon. The long intergenic region, together with the existence downstream of the ORF3 stop codon of a sequence capable of forming, in RNA, a stable stemloop structure [
G=-21·3 kcal mol-1 (-89 kJ mol-1)] that might serve as a transcription-termination signal, suggested that the putative pyrophosphate synthase gene is not part of a bldG operon. The partial sequence of the divergently expressed ORF1 suggests that the protein product belongs to a group of bacterial RNA helicases. The sequences for bldG and the surrounding ORFs on cosmid H5 have been now been determined as part of the S. coelicolor genome sequencing project (http://www.sanger.ac.uk/Projects/S_coelicolor) and have been deposited under accession no. AL035636. It is clear from the sequence analysis that the 2·5 kb HindIII fragment present in our clone does not arise from contiguous chromosomal sequences and has come from the ligation of two non-contiguous Sau3AI fragments during library preparation. The non-contiguous DNA is shown as a hatched box in Fig. 1
.
Alignment of BldG and the ORF3 protein products with known proteins from the databases
The bldG gene encodes a 113 aa protein with end-to-end similarity to a number of anti-anti-sigma factors that regulate the activity of sigma factors responsible for stress-induced or growth-stage-specific transcription. BldG is most closely related to the Sta. aureus and B. subtilis RsbV proteins (38% and 40% identity, and 61% and 60% similarity, respectively), and to B. subtilis SpoIIAA (26% identity, 56% similarity). Alignment of BldG with RsbV and SpoIIAA proteins (Fig. 2a) reveals a highly conserved region of sequence surrounding the serine residue known to be phosphorylated on the B. subtilis SpoIIAA (Najafi et al., 1995
). Likewise, alignment of the ORF3 product with other anti-sigma factors revealed about the same degree of similarity to RsbW from B. subtilis and Listeria monocytogenes (28% identity, 43% similarity and 25% identity, 44% similarity, respectively), and to SpoIIAB from B. subtilis (26% identity, 42% similarity). These anti-sigma factors are encoded along with, and interact directly with, RsbV and SpoIIAA, respectively. Since both RsbW and SpoIIAB in B. subtilis have been shown to have kinase activity that regulates their interaction with either their cognate sigma factor or anti-anti-sigma factor by phosphorylation of the anti-sigma factor antagonist (Dufour & Haldenwang, 1994
; Min et al., 1993
), the ORF3 amino acid sequence was aligned with RsbW and SpoIIAB sequences to look for conserved kinase domains. As shown in Fig. 2b
, the ORF3 protein product contains only two of the five conserved amino acid residues that are thought to be important for ATP and magnesium binding in RsbW (Kang et al., 1996
) and SpoIIAB (Min et al., 1993
), and in bacterial histidine kinases in general (Stock et al., 1995
). Also, analysis of the ORF3 protein sequence using the Prosite ProfileScan Server did not reveal any conserved histidine kinase domains within the sequence. These findings suggest that the ORF3 protein product lacks the kinase activity that has been shown in B. subtilis to be important for the regulation of anti-anti-sigma factor and anti-sigma factor interactions.
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To confirm that the constructed J1501 bldG null mutant did not contain any other defects, pAU69, a pSET152-derivative containing a copy of bldG together with its promoter region, was introduced into bldG3b. The same complementation plasmid was also introduced into the bldG point mutants used in this study. pAU69 restored the wild-type phenotype to bldG3b as well as the C103, C107 and C536 strains; however, introduction of the plasmid had no effect on the phenotype of C101J.
Sequencing of bldG mutations
To map the location of the bldG mutation in C103, C107 and C536, as well as to confirm the absence of a mutation in the bldG coding region of C101J, the bldG ORF from the four bldG mutants was amplified by PCR and the resulting products were sequenced. As expected on the basis of the complementation studies, bldG101J did not have a mutation in the bldG coding or promoter region. Since the mutation in this strain maps to the same region of the chromosome as the mutations in C103, C107 and C536, it may reside in one of the nearby ORFs. Alternatively, the C101J strain may be a double mutant, making the genetic mapping data difficult to interpret and possibly misleading. As shown in Fig. 3, the bldG103 and bldG536 mutations both involve an AT substitution at the same base, introducing a stop codon that would generate a truncated 87 aa BldG protein. The bldG107 mutation, the substitution of an adjacent CT for AG, leads to an amino acid change of aspartate to glutamate at position 56, and of a serine to alanine at position 57 of BldG. The latter residue corresponds to one known to be phosphorylated in SpoIIAA by the SpoIIAB protein kinase. These data suggest that the C-terminal 26 aa of BldG, as well as the conserved region corresponding to the phosphorylation site on the B. subtilis SpoIIAA, are important for BldG function.
Although some bldG transcripts are monocistronic, some also include ORF3
Northern blotting was performed using RNA isolated from S. coelicolor J1501 to determine the size of the bldG transcript as well as the timing of expression. The experiments were performed at least twice using RNA from three different time courses, and representative results are shown in Fig. 4. The probe, a 207 bp fragment internal to bldG, hybridized to two RNA species with sizes of 600700 nt and 11001200 nt. The size of the smaller transcript is consistent with a monocistronic bldG transcript terminating in the intergenic region between bldG and the start of ORF3, whereas the larger transcript has a size comparable to that expected for a polycistronic transcript including both bldG and the downstream ORF3 gene. When a second blot, prepared at the same time and in the same manner as the first, was probed with a PCR-amplified probe internal to the putative anti-sigma factor-encoding ORF3 (Fig. 4
), only a band corresponding to the larger transcript was observed, and the same results were observed when a single blot was first hybridized with the bldG-specific probe, stripped and reprobed with the ORF3-specific probe (data not shown). The two transcripts were present at low levels during vegetative growth and at higher levels from 24 h post-inoculation, the time point corresponding to the appearance of aerial hyphae and pigmented antibiotics. Quantitative analysis of the two bands indicated that the smaller transcript is present at a two- to three-fold higher concentration than the larger transcript.
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DISCUSSION |
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Despite the sequence similarities between BldG and the deduced protein product of its co-transcribed, downstream ORF3 with proteins known to regulate the activity of certain sigma factors in Bacillus, we see differences between the way that the genes are organized and regulated in the two organisms. In contrast to both the spoIIA and rsb operons from Bacillus, a sigma factor is not encoded at the bldG locus. So far, there are no reports in the literature of genes for an anti-sigma factor antagonist and its anti-sigma factor lying at a distant location from the gene for the cognate sigma factor. If bldG and ORF3 do in fact encode an anti-anti-sigma/anti-sigma factor pair, this raises the possibility that the regulatory pair might regulate the activity of more than one sigma factor, or that they may regulate a particular sigma factor only in a subset of the conditions in which that sigma factor directs gene expression. The antibiotic- and aerial-mycelium deficient phenotype of bldG mutants then presents us with two possibilities: the regulatory pair could control one or more globally acting sigma factors that serve to activate transcription of both antibiotic and sporulation-specific genes; or alternatively, the pair could regulate two sigma factors, one of which activates transcription of antibiotic biosynthetic genes and one which activates sporulation-specific gene expression. Interestingly, of two sporulation-specific sigma factors so far identified in Streptomyces (Kelemen et al., 1996 ; Méndez & Chater, 1987
), one (
F) is of the subfamily regulated by homologues of BldG/ORF3 in Bacillus. Sigma factors recognizing antibiotic-biosynthetic-gene promoters have not been definitively identified. It is attractive to suggest that immobilization of the putative anti-sigma factor on an affinity column might lead us to one such sigma factor. A search of the S. coelicolor database reveals that there is no shortage of potential sigma factor targets. The Bacillus sigma factors that are regulated by anti-anti-sigma/anti-sigma factor pairs make up a subfamily of
70-like sigma factors (Lonetto et al., 1992
), and so far at least 9 sigma factors that would fall into this subfamily are found on the S. coelicolor chromosome; several of them (in addition to sigF) are not located next to bldG/ORF3-like gene pairs (Gabriella Kelemen, personal communication).
The C-terminal region of sigma factors in this subfamily shares homology with the DNA-binding region of a group of bacterial transcriptional-activator proteins (Kahn & Ditta, 1991 ; Lonetto et al., 1992
). Our analysis of the
F sequence using the Prosite ProfileScan server revealed similarity between region 4.2 near the
F C terminus and the helixturnhelix domain of LuxR-related DNA-binding proteins. This raises the alternative possibility that the BldG and ORF3 regulatory pair could target a DNA-binding protein rather than a sigma factor. However, this possibility seems unlikely since the candidate
F contact residues for SpoIIAB are not located in region 4.2 of
F. SpoIIAB contacts
F in three areas corresponding to conserved regions 2.1, 3.1 and 4.1 of
70-like sigma factors (Decatur & Losick, 1996
). So far, only two putative contact residues on SpoIIAB have been identified (Garsin et al., 1998
); and one of these two residues, R20, is conserved in the ORF3 gene product.
In addition to the absence of an operon-encoded sigma factor, the bldG operon differs from the Bacillus operons in the way that the expression of the genes is controlled. In the case of both the spoIIA and rsb operons, the genes for the anti-anti-sigma factor, the anti-sigma factor and the sigma factor are expressed as a single transcript, from either a H-dependent promoter in the case of spoIIA (Wu et al., 1991
) or a
A-dependent promoter in the case of rsb (Wise & Price, 1995
). Expression of the genes is also upregulated from a second promoter that is recognized by the operon-encoded sigma factor (Schuch & Piggot, 1994
; Wise & Price, 1995
). For the spoIIA operon, upregulation is dependent on the activity of a prespore-compartment-specific phosphatase, SpoIIE, that activates the SpoIIAA anti-anti-sigma factor by dephosphorylation, allowing the formation of SpoIIAA/SpoIIAB complexes and the liberation of
F (Duncan et al., 1995
; Wu et al., 1998
). Upregulation of
B occurs either in response to energy stress, where reduced ATP levels in the cell influence the phosphorylation state of the anti-anti-sigma factor, RsbV, or in response to environmental stress signals that activate a phosphatase that dephosphorylates RsbV and results in the liberation of
B (Voelker et al., 1995
).
For the bldG operon we also see a complex transcription pattern involving not only two different promoters, but also the generation of two differently sized bldG-containing transcripts. Based on the Northern analyses, the longer and less abundant of the two transcripts extends through bldG to include the downstream putative anti-sigma ORF3. The shorter, more strongly expressed transcript appears to terminate in the intergenic region, and includes only the bldG coding sequence, a situation not seen with either the spoIIA or rsb operons. Analysis of the intergenic sequence for potential RNA secondary structures revealed two inverted repeats that could give rise to stemloop structures with G values of -22·2 and -31·3 kcal mol-1 (-93 and -131 kJ mol-1) (see Fig. 3
), respectively, and either of which might play a role in mRNA stabilization or as a transcription-termination signal. Although a string of U residues is not present downstream of either inverted repeat, it is well documented that inverted repeats without poly (U) tails can act as terminators in Streptomyces (Deng et al., 1987
; Ingham et al., 1995
). The existence of the shorter transcript that hybridizes only to the bldG probe might suggest that a terminator is functional; however, an alternative mechanism for the generation of the shorter transcript could be endo- or exo-nucleolytic removal of the 3' ORF3-containing end of the longer transcript. Similarly, although a comparison of transcript abundance as seen by Northern analysis, primer-extension and S1 nuclease protection studies may suggest that the longer transcript arises from initiation at the more upstream promoter, and that transcripts initiating from the proximal bldG promoter terminate in the intergenic region, our analyses do not allow a definitive conclusion.
For both the Bacillus spoIIA and rsb operons, the genes appear to be translationally coupled such that they are expressed in equimolar concentrations (Magnin et al., 1997 ; Benson & Haldenwang, 1993b
; Kalman et al., 1990
). The lack of a promoter in the intergenic region between bldG and the downstream ORF3 encoding the putative anti-sigma factor suggests that these two genes might also be translationally coupled. However, the very long intergenic region means that there cannot be a straightforward mechanism of coupling that involves either closely spaced or overlapping start and stop codons to ensure a 1:1 stoichiometry. This, together with the added complication of a bldG monocistronic transcript that might lead to an excess of BldG, highlights the need to address the levels of the BldG and ORF3 proteins throughout the life cycle. By analysing the protein levels we should be able to answer questions about how the equilibrium between BldG and the ORF3 product is shifted at different stages of growth. Certainly, appearance of a transcript does not mean that the RNA is being translated to protein, a fact that warrants further investigation because of the expression of a putative RNA helicase from a divergently expressed promoter that overlaps the -10 region of the most upstream bldG promoter (J. Stoehr & B. K. Leskiw, unpublished).
The availability of the purified proteins and antibodies to those proteins will also help us to explore the role, if any, of the conserved phosphorylation site on BldG. Although mutation at this site does abolish BldG activity in the same way that Ser58 mutation in SpoIIAA does, the existence of a second mutation in this region in bldG107, together with the lack in the putative anti-sigma factor of conserved residues found in bacterial histidine protein kinases, makes it difficult to draw any conclusions.
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ACKNOWLEDGEMENTS |
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Received 14 April 2000;
revised 29 May 2000;
accepted 22 June 2000.