mutant NP4, which was isolated by UV mutagenesis, showed a bald and wrinkled colony morphology because of ectopic septation in substrate hyphae and subsequent spore formation. The ectopic spores were the same as aerial spores in size, thickness of the spore wall, and shape, as determined by transmission and scanning electron microscopy, and in heat and lysozyme susceptibility. Mutant NP4 also formed abundant spores in liquid medium, whereas the parental strain IFO13350 rarely forms submerged spores under these conditions. The wall of the ectopic spores is supposed to be thicker than those of the submerged spores formed by several Streptomyces
spp., including S. griseus
), under specific conditions, because the spores of NP4 were resistant to lysozyme. We therefore assume that both on solid and in liquid medium, mutant NP4 forms two separate cross walls in the vegetative hyphae and matures each compartment into a spore indistinguishable from aerial spores in many aspects, as is observed in aerial spore formation in S. griseus
The frequency of septation in substrate mycelium in mutant NP4 is much higher than in S. coelicolor
A3(2) strains harboring multicopies of whiG
) or having a deletion of a region close to the glkA
). These strains form abundant aerial spores and only occasional septa in substrate mycelium and subsequent sporulation. The deprogrammed sporulation of NP4 implies that once septa are formed, even in substrate hyphae, in response to some signal, each compartment is inevitably destined to develop into a spore. In the substrate hyphae of the wild-type S. griseus
strain, some signals must block the commitment to septation and subsequent sporulation. A-factor does not release the block, because exogenous addition of an appropriate amount of A-factor to the substrate hyphae of the A-factor-deficient mutant HH1 causes no septum formation but normal formation of aerial spores. An excess amount of DasA appears to release the block, since introduction of pES1 into mutant HH1 results in ectopic septation.
Shotgun cloning of genes on the wild-type chromosome into a mutant is a useful approach for identifying the mutated gene and genes closely related to the mutant phenotype. We at first expected that the mutation(s) responsible for the ectopic sporulation of NP4 was in dasR or dasA, since the ectopic sporulation of mutant NP4 was completely reversed by dasR encoding a transcriptional factor belonging to the GntR family, and dasA made the wrinkled morphology of mutant NP4 more severe. However, nucleotide sequencing of the dasR-dasA region in mutant NP4 revealed no base changes.
On the basis of the observations that introduction of dasA into the wild-type strain caused ectopic sporulation and that the amount of dasA transcript was greater in mutant NP4 than in the wild type, we assume that NP4 has a mutation in the regulatory pathway to control the expression of dasA. Because ectopic sporulation appears to result solely from an increase in the amount of DasA, the regulatory pathway in mutant NP4 seems to lack the ability to repress dasA. The elevated expression of dasR in NP4 suggests that the putative regulatory pathway controls dasR too. The increase in the amount of DasR, which may repress not only dasA but also some other genes involved in programmed development, is a possible explanation for the difference in timing of ectopic septation between the wild-type strain carrying dasA and mutant NP4; an increase in the amount of only DasA in the same background as in the wild type results in early commitment of septation, whereas an increase in the amount of DasR during early growth and the existence thereafter (Fig. ) bring forth a different background, resulting in septation at the programmed time.
Is DasRABC involved only in sugar import as an ABC transporter, as predicted by the homology of each of the components? The gene organization dasR-dasA-dasB-dasC
and their predicted functions are the same as those for the maltose and cellobiose/cellotriose import systems in Streptomyces
) and cebR-cebE-cebF-cebG
), respectively. In addition, a gene encoding a glucosidase-like protein is encoded downstream of all three of these gene clusters. Although no ATP-binding proteins as ABDs are encoded in the vicinity of the das
operon, the absence of the gene encoding ABD also holds for the malEFG
operons. As pointed out by van Wezel et al. (64
), MsiK or an MsiK-like ATP-binding protein (24
), which is encoded elsewhere on the chromosome and homologous to the ATP-hydrolyzing subunit MalK in the maltose import system in E. coli
), may serve as a general ATP-hydrolyzing subunit for “orphan” ABC transporters. MalK is an essential component in the E. coli
maltose import system, forming MalEFGK2
The effect of glucose on the ectopic sporulation of mutant NP4 and on the wild type containing multicopies of dasA tempted us to speculate that DasA recognizes and binds to glucose or a glucose derivative and imports it via the Das system. However, dasA is developmentally regulated, and its transcription is enhanced just after commitment of aerial mycelium formation and during spore formation. This means that DasA is not produced until the glucose in the medium is almost consumed. Conceivably, DasA binds a certain sugar compound other than glucose which is needed for septation in aerial hyphae at the programmed point. The bald phenotype of the ΔdasA mutant suggests an additional role of DasA in aerial mycelium formation, although we have no plausible explanation for it. In addition, the bald phenotype, but accompanied with occasional septation in substrate hyphae, of the ΔdasR mutant suggests that the concentration of DasA controlled by dasR is critical for aerial mycelium formation.
In considering the function of DasA, we would like to point out the multiple functions of some substrate-binding proteins of the ABC transporters. For example, ChvE is a multifunctional glucose/galactose-binding protein which participates in the uptake of specific monosaccharides, chemotaxis to these sugars, and virulence gene induction in Agrobacterium
. For induction of the virulence genes to form crown gall tumors, monosaccharide-bound ChvE interacts with the periplasmic region of VirA, a sensor kinase in the VirA-VirG two-component signal transduction system (46
). For chemotaxis, ChvE is supposed to interact with chemotaxis receptors such as Tar and Trg (33
). The maltose-binding protein MalE of E. coli
, the oligopeptide-binding protein OppA of E. coli
, and the galactose-binding protein MglB of Salmonella enterica
serovar Typhimurium are other examples that function as a chaperone for protein folding and protection from stress in the periplasm, in addition to their function in import and chemotaxis (49
). These examples, together with the developmentally regulated expression of dasA
and involvement in septum formation of DasA, present a possibility that substrate-bound or free DasA interacts with other regulatory proteins in the membrane, thus commencing a regulatory pathway for morphological development.
The ectopic septation and subsequent sporulation of S. griseus
triggered solely by overexpression of dasA
was independent of A-factor, because the A-factor-deficient mutant HH1 harboring pES1 showed ectopic sporulation. This is consistent with the observation that the wild-type strain harboring pES1 formed ectopic septa at day 1, when the concentration of A-factor is still low (Fig. and ). In S. griseus
, A-factor at a critical concentration triggers aerial mycelium formation and streptomycin biosynthesis by binding a repressor-type receptor protein, ArpA, and dissociating it from the promoter region of adpA,
encoding a transcriptional activator (45
). A-factor is produced in a growth-dependent manner (20
One of the targets of AdpA is adsA,
encoding an extracytoplasmic function sigma factor, ςAdsA
, essential for the initiation of aerial mycelium formation (70
). AdpA and AdsA supposedly activate many structural genes required for aerial mycelium formation. A-factor thus determines the timing of programmed and ordered development by acting as a master switch for turning on many genes at several hierarchic regulatory steps. The ectopic spores which are triggered by an excess amount of DasA and independently of A-factor germinate at the same frequency as aerial spores, although they are sensitive to lysozyme and heat because of a thinner spore wall. The difference in lysozyme and heat resistance of the aerial spores and ectopic spores of the wild-type harboring multicopies of dasA
implies that some of the gene products necessary for the architecture of aerial spores are absent in the maturation of the ectopic spores.
Comparison of transcription of genes necessary for normal morphological and physiological development in the wild-type, A-factor-controlling background and in the dasA
-overexpressing background will reveal the difference in the genetic network between the programmed septation in aerial hyphae and ectopic septation in substrate hyphae triggered by an excess of DasA. It will also be useful to study their counterparts in different Streptomyces
spp. S. coelicolor
A3(2) contains a very similar gene cluster, open reading frames CAB94616 to -94619, in cosmid SC7E4 (www.sanger.ac.uk/Projects/S_coelicolor/
), each gene of which shows 33 to 91% identity to the corresponding gene in the dasRABC