INTRODUCTION

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Members of the genus Streptomyces are filamentous soil bacteria that undertake a complex developmental process that culminates in the production of unigenomic spores (7, 8, 16). Colonies germinate from spores and grow into the soil by forming a branching network of multinucleoid hyphae termed the substrate mycelium. Morphological differentiation commences with the formation of the aerial mycelium, a fuzzy layer of specialized hyphae that project away from the surface of the colony into the air. These aerial hyphae later undergo septation to produce uninucleoid compartments that mature into pigmented spores, thereby completing the cycle. Morphological differentiation is associated with the production of secondary metabolites, such as pigments and antibiotics, and members of the Streptomyces genus are responsible for the production of a diverse array of such compounds, including the majority of the antibiotics currently in clinical use (7).

Mutant strains of Streptomyces coelicolor have been isolated that fail to initiate differentiation and do not form an aerial mycelium (for examples, see references 6, 22, and 27). These mutants exhibit a bald (bld) phenotype; the surface of mutant colonies remains smooth and does not accumulate the hairlike aerial hyphae. Whereas the corresponding bld genes have been identified from some of these mutant strains (2, 4, 11, 19, 24, 26, 31), it is clear that many genes that influence aerial mycelium formation remain to be uncovered (27). A striking property of bld mutants is their capacity to be rescued by extracellular complementation, a phenomenon that has been interpreted to indicate the existence of signaling molecules that help to regulate aerial mycelium formation under certain conditions (26, 32). But the nature of the signals and the genes involved in the signaling pathways are largely unknown.

As part of an effort to discover additional genes involved in aerial mycelium formation, we have been carrying out insertional mutagenesis with the transposon Tn5apr to generate mutants of S. coelicolor that exhibit a bld phenotype. Here we report the isolation of a mutant that cannot produce an aerial mycelium and shows delayed production of the blue-pigmented antibiotic actinorhodin. The mutant harbors an insertion in a previously uncharacterized gene that appears to encode a membrane-bound anti-sigma factor and is located adjacent to the gene for a putative member of the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors. We present evidence that the bld phenotype exhibited by the insertion mutant is an indirect consequence of the unregulated activity of the ECF sigma factor.

	MATERIALS AND METHODS

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Strains and growth conditions. S. coelicolor strain M145 (prototrophic, SCP1 SCP2) was used for mutant construction (18). Strains were grown on solid R2YE, mannitol soya flour, minimal medium (MM) with 1% (wt/vol) glucose or with 0.5% (wt/vol) mannitol, or Difco nutrient agar medium or in liquid yeast extract-malt extract medium (18) at 30°C with, as indicated (from Sigma), 25 µg of apramycin sulfate per ml, 200 µg of spectinomycin dihydrochloride per ml, or 50 µg of thiostrepton per ml.

Insertional mutagenesis of S. coelicolor. Insertional mutagenesis of M145 was carried out as described previously (13). Briefly, a plasmid library of S. coelicolor DNA in the pSpecoriT vector was subjected to in vitro transposition with Tn5apr. Transposon-disrupted plasmids were isolated and introduced into S. coelicolor M145 by conjugation from E. coli ET12567(pUB307). Exconjugants were selected with apramycin and visually screened for morphological defects leading to the isolation of apramycin-resistant (Apr^r), spectinomycin-sensitive (Spec^s), bald strain NY415. Linkage of the transposon insertion to the bald mutation in NY415 was confirmed by genomic DNA transformation (28), and the chromosomal location of Tn5apr in this strain was determined by sequencing of the transposon-flanking DNA (13).

Construction of a sigU-rsuA deletion strain A plasmid was constructed as follows for the deletion of sigU and rsuA and their replacement with tsr (conferring resistance to thiostrepton) in S. coelicolor M145. DNAs flanking the rsuA and sigU loci were amplified by PCR from M145 genomic DNA by using the primers 5'-CGAGCTCACCAGGCGTCCAGGAAGCCGGTGAA-3' and 5'-CTAGCTAGCAGGTCATGACCTTCGACGGGAA-3' and the primers 5'-CTAGCTAGCTTCATTGGCCACGACGGAAT-3' and 5'-CGGATATCTCGTCGTCCTCGTGCCGTCA-3' (restriction enzyme sites are underlined). The PCR products were digested with SacI/NheI and NheI/EcoRV, respectively, and cloned into the vector pSpec (13). The tsr gene, amplified by PCR with the primers 5'-CTAGCTAGCCGGCCACGACACCCCCATCGGCATCGCGT-3' and 5'-CTAGCTAGCGACCGGCGCCACACCGGCTTGCGCCGGCT-3' from plasmid pJR35 (laboratory collection), was then inserted into the NheI site to give the knockout construct.

Construction of plasmids containing sigU or rsuA and complementation experiments. Complementation plasmids containing the sigU or rsuA gene were constructed in modified versions of the vectors pKC1218 and pSET152 (3, 18). Both of these vectors contain a gene conferring resistance to apramycin that was removed by digestion with SacI and replaced with the aadA gene, which confers spectinomycin-streptomycin resistance. The aadA gene was amplified by PCR from the plasmid pSpec (13) by using primers 5'-CGAGCTCGGCTTGAACGAATTGTTAGAC-3' and 5'-CGAGCTCGCTTCGGTTTTCATGGCTTG-3'. These modified vectors, which confer spectinomycin-streptomycin resistance, were named pKC1218S and pSET152S.

Zone of growth inhibition assays. The sensitivity of S. coelicolor M145 or the sigU-rsuA::tsr strain to various chemicals was tested as follows. Spores of each strain (10⁷ CFU) were mixed in 4 ml of nutrient soft agar (Difco nutrient broth with 0.5% agar) and spread on nutrient agar plates. Glass microfiber filter disks (~10 mm in diameter) soaked in 20 µl of the compound being tested were then applied to the surface of the soft agar. The zone of growth inhibition was measured after 24 h of growth at 30°C.

	RESULTS

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A new bld mutant strain generated by insertional mutagenesis. By using an insertional mutagenesis protocol that relies on in vitro transposition (13), a mutant S. coelicolor strain, NY415, was isolated that did not initiate the morphological differentiation process normally undergone by this organism. NY415 exhibited a classic bald (bld) phenotype (16), failing to produce any aerial mycelium (Fig. 1A). NY415 also showed a severe delay in production of the blue-pigmented polyketide antibiotic actinorhodin. Whereas wild-type strain M145 produces large amounts of this pigment by 2 to 3 days of growth, blue pigmentation was not observed in NY415 until about 1 week of growth.

View larger version (46K): [in this window] [in a new window]   FIG. 1.   (A) Comparison of a wild-type S. coelicolor strain (M145), a bald strain (NY415) generated by transposon disruption of the gene rsuA (SCE59.12c), and a strain in which both rsuA and the upstream gene sigU (SCE59.13c) were deleted and replaced with a gene conferring thiostrepton resistance (sigU-rsuA::tsr). Strains were grown for 4 days at 30°C on R2YE rich medium. (Note the extracellular complementation of NY415 for aerial mycelium formation where this strain approaches the two morphologically normal strains.) (B) Reversal of the bald phenotype of strain NY415 by either deletion of the transposon-disrupted rsuA allele along with sigU (top) or by complementation (bottom). Comparison of NY415 (top left) to a Tsrr Aprs Specs strain (top right) generated by transformation of NY415 protoplasts with a knockout plasmid designed to remove genes rsuA and sigU and replace them with tsr. Comparison of strain NY415 transformed with the integrating vector pSET152S (bottom left) versus strain NY415 transformed with the same vector into which gene rsuA had been inserted (pSET152S-rsuA, bottom right). Growth was for 4 days at 30°C on R2YE medium. (C) Uncoupling of the sigma factor gene sigU from the downstream putative anti-sigma factor gene rsuA results in defects in morphological differentiation. Comparison of the wild-type strain M145 transformed with the low-copy-number vector pKC1218S (top left) and M145 transformed with the same vector containing the gene sigU (pKC1218S-sigU, top right). Note the slight delay in aerial mycelium formation here compared to that of the vector-only strain. Comparison of strain sigU-rsuA::tsr transformed with the integrating vector pSET152S (bottom left) and the same strain transformed with pSET152S containing gene sigU (pSET152S-sigU, bottom right). The presence of the sigma factor gene in the absence of its antagonist anti-sigma factor gene results in a severe bald phenotype. Growth was for 3 days (top) or 4 days (bottom) at 30°C on R2YE medium containing spectinomycin.

Extracellular complementation of the bld mutant strain. An extracellular signaling cascade has been proposed for the initiation of aerial mycelium formation on rich media based on observations of the ability of bld mutant strains to complement one another for aerial mycelium formation in a unidirectional manner (bldJ < bldK, bldL < bldA, bldH < bldG < bldC < bldD, bldM; 24, 26, 27, 32). The position of NY415 in this extracellular complementation cascade was assessed by plating this strain in close proximity to a number of previously isolated bld strains (data not shown).

The transposon insertion in the bld mutant disrupts a locus downstream of a putative ECF sigma factor gene. The site of the insertion in NY415 was determined by sequencing DNA flanking the transposon and searching for the corresponding sequence in the Sanger Centre S. coelicolor genome sequence database (www.sanger.ac.uk/Projects/S_coelicolor). The Tn5apr transposon was found to be inserted in the coding sequence of gene SCE59.12c (Fig. 2). The product of this gene was annotated by the Sanger Centre group as a putative membrane protein with a possible hydrophobic membrane-spanning region.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Organization of the S. coelicolor chromosome near the sigU and rsuA loci (SCE59.13c and SCE59.12c, respectively, by the nomenclature of the Sanger Centre genome sequencing project). Numbers on the scale above indicate the base pair positions of these genes in the E59 cosmid DNA sequence, and the Sanger Centre annotation is given below each gene. The chromosomal location of the Tn5apr insertion in strain NY415 is marked with a triangle (base pairs 13788 to 13796 of cosmid E59), and the region deleted and replaced with tsr in strain sigU-rsuA::tsr is indicated below. Finally, DNA regions that were PCR amplified and cloned into vectors pKC1218S and/or pSET152S for complementation experiments are also indicated.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Alignment of the N-terminal regions of RsuA and other proteins containing a consensus HXXXCXXC motif. The genes encoding these proteins are all located near, typically downstream and adjacent to, genes encoding members of the ECF family of sigma factors. All sequences are from S. coelicolor, except for Rv0736, which is from M. tuberculosis.

Aerial mycelium formation proceeds normally in strains in which both rsuA and sigU are deleted As transposon disruption of the putative anti-sigma factor gene rsuA elicits a bald phenotype, it was important to determine the phenotype of a strain in which both rsuA and the upstream ECF sigma factor gene, sigU, were disrupted. In the wild-type strain background, these genes were deleted and replaced with a gene conferring resistance to thiostrepton to give a sigU-rsuA::tsr strain. Unlike NY415, this strain exhibited normal morphological differentiation (Fig. 1A). The only apparent defect in the sigU-rsuA::tsr strain was a short delay in actinorhodin production that was very mild on R2YE medium but more noticeable on nutrient agar medium.

Expression of sigU uncoupled from rsuA elicits differentiation defects. To confirm that the presence of the sigU gene in the absence of its apparent antagonist rsuA results in a bald phenotype, the sigU-rsuA::tsr strain was transformed with an integrating plasmid containing only the sigma factor gene sigU (Fig. 2). The resulting transformants showed a phenotype identical to that of NY415they failed to generate an aerial mycelium and showed severe slowing of blue pigment production (Fig. 1C). Introduction of anti-sigma factor gene rsuA only into the sigU-rsuA::tsr background did not affect morphological differentiation (data not shown).

^U and the response to stress ECF sigma factors, as their name suggests, typically regulate a response to environmental stress(es) (23, 33). The ability of the sigU-rsuA::tsr strain to respond to several chemical stresses was therefore examined. The sensitivities of the M145 and sigU-rsuA::tsr strains to various compounds were compared by using a zone of growth inhibition assay. The two strains showed similar responses to oxidants (diamide, hydrogen peroxide, and cumene hydroperoxide), a reductant (dithiothreitol), metal ions (FeCl₃, CuCl₂, and ZnCl₂), EDTA, HCl, NaOH, sodium dodecyl sulfate, and lysozyme. Thus, the particular stress(es) to which ^U presumably responds remains to be uncovered. We note that while the wild-type and sigU-rsuA::tsr strains were able to grow well on nutrient agar supplemented with 0.5 M KCl, the bald mutant NY415 was unable to grow on this medium, suggesting that this strain is osmosensitive.

	DISCUSSION

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The initiation of aerial mycelium formation in S. coelicolor is a poorly understood process (16). A relatively small number of genes involved in this developmental pathway, the bld genes, have been characterized (2, 4, 11, 19, 24, 26, 31), and only recently have relationships between the activities of certain bld gene products been established (2, 10). Here we describe the identification and initial characterization of a new gene, rsuA, which is indirectly required for the formation of aerial hyphae, as well as for the appropriate timing of pigmented antibiotic production. This gene, located immediately downstream of an ECF sigma factor gene, appears to encode an anti-sigma factor. In the absence of this anti-sigma factor, unchecked activity of the corresponding sigma factor, ^U, blocks the normal developmental process, abrogating aerial mycelium formation and, thus, subsequent sporulation.

The RsuA anti-sigma factor gene product showed similarity in its N terminus to a recently defined anti-sigma factor family termed the ZAS (zinc-binding anti-sigma factor) family (15, 29). Members of this family, although they exhibit limited identity over their lengths, possess an absolutely conserved HXXXCXXC amino acid motif. In S. coelicolor, RsuA and at least 11 additional proteins encoded by genes located near ECF sigma factor genes share this conserved motif (Fig. 3), including the founding member of the ZAS family, RsrA.

RsrA is the cognate anti-sigma factor for ^R, a sigma factor demonstrated to direct the transcription of thioredoxin reductase and thioredoxin genes in response to thiol oxidation (29, 30). Biochemical studies have shown that RsrA binds to ^R under reducing conditions, thereby inhibiting transcription of the thioredoxin system (15). Conversely, under oxidizing conditions, intramolecular disulfide bonds form in RsrA and it loses the ability to bind to and inhibit ^R (15). Three cysteine residues in RsrA, including the two in the conserved HXXX CXXC motif, are required for this anti-sigma factor activity (29). In addition, RsrA has been shown to bind near stoichiometric amounts of zinc. The conserved HXXXCXXC residues have been proposed as possible zinc ligands (ergo, ZAS is the name of this anti-sigma factor family); however, this has not yet been demonstrated experimentally (29). Only one additional member of the proposed ZAS family, Rhodobacter sphaeroides ChrR, has been characterized (25). It was demonstrated that in ChrR, mutation of the second conserved motif cysteine residue to arginine abrogates binding to and inhibition of its cognate sigma factor (^E).

By analogy to the ^R-RsrA case, we anticipated that the ^U-RsuA pair might play a role in sensing and responding to the redox environment of the cell. However, we were unable to detect a convincing difference in a strain with these loci deleted, compared to wild-type S. coelicolor, in sensitivity to several oxidants and reductants. In mycobacteria, two ECF sigma factor genes located upstream of ZAS family anti-sigma factor genes (15), sigE and sigH, have been implicated in the oxidative stress response (12, 34). A Mycobacterium smegmatis strain in which sigE was disrupted showed a small decrease in survival compared to the wild type when exposed to a variety of stresses, including hydrogen peroxide (34), and an M. smegmatis strain with sigH disrupted, while showing normal susceptibility to hydrogen peroxide, was sensitive to cumene hydroperoxide (12). A sigH sigE double mutant was even more susceptible to cumene hydroperoxide, as well as heat shock (12). Similarly, perhaps the functions of rsuA and sigU overlap those of other members of the S. coelicolor ZAS and ECF sigma factor families (Fig. 3). Alternatively, RsuA and ^U may respond to a signal not directly related to oxidative stress, as previously suggested for other ZAS family members (29). For example, Bacillus subtilis sigW, which is found upstream of a ZAS family gene (15), has no apparent connection to an oxidative stress response (14) but has, instead, been shown to mediate resistance to the antibiotic fosfomycin (5).

The rsuA anti-sigma factor gene is the latest example of a growing number of sigma factor regulatory genes whose presence is required for the normal morphological differentiation of S. coelicolor. A strain in which the ^R regulatory gene rsrA was deleted formed an aerial mycelium but was unable to sporulate, producing straight, white aerial hyphae with little septation, whereas a rsrA sigR double mutant sporulated normally (29). A homolog of the B. subtilis general stress response factor ^B has recently been identified in S. coelicolor (^H), and the sigH gene is preceded by and cotranscribed with the prsH gene, whose product is homologous to the B. subtilis anti-sigma factors SpoIIAB and RsbW (17). A sigH mutant sporulated normally, but a prsH sigH double mutant exhibited a conditionally bald phenotype (17). This suggests that PrsH may regulate an additional sigma factor(s) whose uncontrolled activity disrupts development analogously to ^R and ^U. Finally, one of the classic bld loci (6), bldG, is now known to encode a protein that is homologous to anti-anti-sigma factors such as B. subtilis SpoIIAA and RsbV, which are antagonists of the anti-sigma factors SpoIIAB and RsbW, respectively (4). A candidate for the corresponding anti-sigma factor gene is present downstream of bldG. Presumably, and opposite to the other cases we have considered, a bldG mutation blocks aerial mycelium formation by preventing the activation of a sigma factor that is required for development. However, a cognate sigma factor for the bldG system, if it exists, is unknown (4).

If the sigU sigma factor gene is not required for development, why does deletion of its anti-sigma factor gene, rsuA, lead to a severe developmental (bald) phenotype? We presume that in the absence of its cognate anti-sigma factor, ^U is constitutively active and free to direct transcription of the genes under its control. Indeed, the trxC target gene of ^R has been shown to be transcribed constitutively and at a very high level in an rsrA mutant (29). One possible explanation for the block in aerial mycelium formation caused by a rsuA mutation is that unregulated and improperly active ^U competes in binding to core RNA polymerase with one or more sigma factors that are needed for development. Currently, only one sigma factor, the ECF sigma factor ^BldN, which directs the transcription of bldM, has been shown to be necessary for aerial mycelium formation (2). Perhaps the sigU-encoded sigma factor competes with ^BldN. However, the introduction of a low-copy-number vector (pKC1218S) containing bldN was insufficient to reverse the bald phenotype of NY415 (A. M. Gehring and R. Losick, unpublished data).

Another possible explanation for the bald phenotype of the rsuA mutant is that ^U directs the transcription of a gene(s) that inhibits aerial mycelium formation. We favor this explanation because it accounts for the fact that an rsuA mutation impairs differentiation at the stage of aerial mycelium formation whereas an rsrA mutation blocks development at the stage of spore formation. It is difficult to imagine how ^U and ^R could differentially compete with different sigma factors, one required for aerial mycelium formation and one required for spore formation. Rather, it seems more likely that ^U directs the transcription of a gene(s) that, when inappropriately expressed or expressed at high levels, causes a bald phenotype whereas ^R directs the transcription of a gene(s) whose product inhibits spore formation.

The developmental defects observed in rsuA and rsrA mutants imply an interconnection between development and the stress response in S. coelicolor. A role for ^R in responding to cytoplasmic thiol-disulfide stress is well established (29, 30), and the homologous ECF sigma factor ^U is presumably active in response to an as yet unidentified stress condition. This is in keeping with earlier evidence pointing to a connection between the regulation of stress and developmental pathways in S. coelicolor, such as the finding that transcription from the p2 promoter of the stress response sigma factor gene sigH is restricted to sporulating aerial hyphae and that BldD, a transcription factor also shown to regulate expression of the developmental genes bldN and whiG (10), represses transcription from the sigHp2 promoter in vegetative hyphae (17). Also, the catalase gene catB, which is required for protection against osmotic stress, has been shown to be necessary for aerial mycelium formation and proper secondary metabolite production (9). Given the increasing evidence that developmental events and the responses to cellular stress are intertwined in S. coelicolor, we suggest that stress-induced activation of ^U and ^R may be a physiologically significant mechanism by which to signal the cell to arrest development under conditions that are incompatible with aerial mycelium formation or sporulation.