ShyA, a membrane protein for proper septation of hyphae in Streptomyces

The life cycle of Streptomyces involves the formation of filamentous substrate and aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to produce a chain of unigenomic spores. A gene, shyA, which influences several aspects of this growth, was isolated and partially characterized in Streptomyces coelicolor. The gene product is a representative of a well-conserved family of small actinomycete proteins. The shyA mutant sporulates normally but displays hyper septum formation and altered spore-chain morphology. Biochemical separation experiments and immunofluorescence staining demonstrated that the shyA gene product locates at cell membranes. Moreover, yeast two-hybrid screen and GST-pull-down assay showed that ShyA can interact with itself. Altogether, ShyA belongs to a new family of membrane-associated proteins which plays a role in morphological differentiation in actinomycetes.     

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library of Streptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_(TH4) as probe. The experiments revealed that this DNA fragment consists of saw D gene and a 1.4 kb Pvu Ⅱ fragment which can accelerate mycelium formation of S. ansochromogerms. The nucleofide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was desiguated as samfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded by hppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) in Rhodococcus globerulus. The function of samfR gene was studied using strategy of gene disruption, and the resulting samfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped     

Cloning and analysis of a gene cluster from Streptomyces coelicolor that causes accelerated aerial mycelium formation in Streptomyces lividans.

We describe the cloning and analysis of two overlapping DNA fragments from Streptomyces coelicolor that cause aerial mycelium to appear more rapidly than usual when introduced into Streptomyces lividans on a low-copy-number plasmid vector. Colonies of S. lividans TK64 harboring either clone produce visible aerial mycelia after only 48 h of growth, rather than the usual 72 to 96 h. From deletion and sequence analysis, this rapid aerial mycelium (Ram) phenotype appears to be due to a cluster of three genes that we have designated ramA, ramB, and ramR. Both ramA and ramB potentially encode 65-kDa proteins with homology to ATP-dependent membrane-translocating proteins. A chromosomal ramB disruption mutant of S. lividans was found to be severely defective in aerial mycelium formation. ramR could encode a 21-kDa protein with significant homology to the UhpA subset of bacterial two-component response regulator proteins. The overall organization and potential proteins encoded by the cloned DNA suggest that this is the S. coelicolor homolog of the amf gene cluster that has been shown to be important for aerial mycelium formation in Streptomyces griseus. However, despite the fact that the two regions probably have identical functions, there is relatively poor homology between the two gene clusters at the DNA sequence level.     

RNA polymerase sigma factor that blocks morphological differentiation by Streptomyces coelicolor

The filamentous bacterium Streptomyces coelicolor undergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation. Genes required for the initiation of aerial mycelium formation have been termed bld (bald), describing the smooth, undifferentiated colonies of mutant strains. By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a bld mutant harboring an insertion in a previously uncharacterized gene, SCE59.12c, renamed here rsuA. The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin. The rsuA gene encodes an apparent anti-sigma factor and is located immediately downstream of SCE59.13c, renamed here sigU, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors. The absence of rsuA in a strain that contained sigU caused a block in development, and the overexpression of sigU in an otherwise wild-type strain caused a delay in aerial mycelium formation. However, a strain in which both rsuA and sigU had been deleted was able to undergo morphological differentiation normally. We conclude that the rsuA-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded by sigU. We also conclude that the sigU-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage.     

Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface

The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (DeltardlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the DeltardlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.