A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.     

In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation. Received: 17 July 1997 / Accepted: 20 September 1997     

Effects of increased and deregulated expression of cell division genes on the morphology and on antibiotic production of streptomycetes

This paper describes the effects of increased expression of the cell division genes ftsZ, ftsQ, and ssgA on the development of both solid- and liquid-grown mycelium of Streptomyces coelicolor and Streptomyces lividans. Over-expression of ftsZ in S. coelicolor M145 inhibited aerial mycelium formation and blocked sporulation. Such deficient sporulation was also observed for the ftsZ mutant. Over-expression of ftsZ also inhibited morphological differentiation in S. lividans 1326, although aerial mycelium formation was less reduced. Furthermore, antibiotic production was increased in both strains, and in particular the otherwise dormant actinorhodin biosynthesis cluster of S. lividans was activated in liquid- and solid-grown cultures. No significant alterations were observed when the gene dosage of ftsQ was increased. Analysis by transmission electron microscopy of an S. coelicolor strain over-expressing ssgA showed that septum formation had strongly increased in comparison to wild-type S. coelicolor, showing that SsgA clearly influences Streptomyces cell division. The morphology of the hyphae was affected such that irregular septa were produced with a significantly wider diameter, thereby forming spore-like compartments. This suggests that ssgA can induce a process similar to submerged sporulation in Streptomyces strains that otherwise fail to do so. A working model is proposed for the regulation of septum formation and of submerged sporulation.     

A new genus of the Actinoplanaceae: Planobispora,gen. nov.

Summary A new species of a new genus of the Actinoplanaceae is described, for which the name Planobispora longispora gen. nov. sp. nov. is proposed. The organism is a typical mesophilic, aerobic actinomycete, producing a filamentous growth which is differentiated into a vegetative and an aerial mycelium. The new organism is characterized by the formation of sporangia only on the aerial mycelium and by containing a longitudinal pair of motile spores.     

Effect of Pamamycin-607 on Secondary Metabolite Production by Streptomyces spp.

The effect of the aerial mycelium-inducing compound, pamamycin-607, on antibiotic production by several Streptomyces spp. was examined. Exposure to 6.6 μM pamamycin-607 stimulated by 2.7 fold the puromycin production by Streptomyces alboniger NBRC 12738, in which pamamycin-607 had first been isolated, and restored aerial mycelium formation. Pamamycin-607 also stimulated the respective production of streptomycin by S. griseus NBRC 12875 and that of cinerubins A and B by S. tauricus JCM 4837 by approximately 1.5, 1.7 and 1.9 fold. The antibiotic produced by Streptomyces sp. 91-a was identified as virginiamycin M₁, and its synthesis was enhanced 2.6 fold by pamamycin-607. These results demonstrate that pamamycin-607 not only restored or stimulated aerial mycelium formation, but also stimulated secondary metabolite production.     

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2)

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network     

Influence of the culture medium composition on the excreted/secreted proteases from <Emphasis Type="Italic">Streptomyces violaceoruber</Emphasis>

Streptomyces violaceoruber produces two different classes of mycelium, the substrate and the aerial mycelium. Since proteases have been associated with morphological turnover processes in other Streptomyces species, the presence of excretory/secretory proteolytic activities was investigated here in S. violaceoruber culture supernatants. Various polypeptide bands, with apparent molecular masses ranging from 40 to 180 kDa, were detected in soy trypticase broth (STB) culture media supernatants following 72 h of growth, using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Zymograms showed the presence of five proteolytic enzymes (Spvio1–5), which migrated as bands of 167.7, 130.7, 110.7, 48.3 and 40.9 kDa, respectively. The characterization of these proteases by specific inhibitors showed that Spvio1–4 belong to the serine protease group and Spvio5 corresponds to a cysteine protease. Additionally, Spvio2 and 5 were inhibited by a mixture of EDTA and EGTA, indicating that both require divalent cations. The protease pattern obtained in STB enriched with glucose was identical to that obtained in STB. However, Spvio3 and 4 were absent when nitrogen was added to the culture medium. Cell death was fluorescently detected following 72 h of S. violaceoruber growth in STB and in STB that was enriched with glucose. On the contrary, no cell death was detected in nitrogen-enriched STB media. Additionally, the formation of the aerial mycelium was impaired in solid cultures of STB media enriched with nitrogen. These results demonstrate that the composition of the media influences the morphological turnover of the colony and the pattern of excreted/secreted proteases from S. violaceoruber, and suggest that Spvio3 and 4 are involved in the aerial mycelium formation.     

Developmental mutants ofStreptomyces coeruleorubidus,a producer of anthracyclines: Isolation and preliminary characterization

Abstbact The wild strainStreptomyces coeruleorubidus JA 10092 was found to segregate into two spontaneous morphological variants (spo-1 andbld-1) with a different ability to form aerial mycelium in media with glucose as the main carbon source. Six new types of developmental mutants were obtained from the bald variantbld-1 after treatment with mutagens (UV light, γ radiation, nitrous acid) and after natural selection Formation of the aerial mycelium was fully suppressed in thebld-2 type growing on media both with glucose and with starch. The other types were bald only on starch media, forming the aerial mycelium on media with glucose; typesspo-2, spo-3, spo-4 andspo-5 differed in size, shape and surface structure of spores, the typewhi formed asporogenous aerial hyphae.     

6-Methylsalicylic Acid Production in Solid Cultures of Penicillium patulum Occurs Only When an Aerial Mycelium Is Present

When Penicillium patulum was grown on Czapek-Dox agar, 6-methylsalicylic acid was produced as an aerial mycelium was forming. Nutrients were often plentiful in the medium when biosynthesis began. If the formation of an aerial mycelium was prevented by growing the fungus between two sheets of dialysis membrane, no 6-methylsalicylic acid was produced even when nutrients were completely consumed. If the upper sheet of dialysis membrane was stripped off cultures of the latter type, an aerial mycelium formed; concomitantly, 6-methylsalicylic acid biosynthesis was observed. We conclude that 6-methylsalicylic acid was produced only by P. patulum colonies that possessed an aerial mycelium.     

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.     

Streptomyces genes involved in aerial mycelium formation

Abstract Cloning of genes as suppressors of the aerial mycelium-defective phenotype of Streptomyces griseus HH1 resulting from A-factor-deficiency has led to the identification of several genes, including amfR, amfAlamfB, amfC , and orf1590 . These genes are involved in aerial mycelium formation independent of secondary metabolic function. Among these, AmfR which belongs to the family of response regulators of two-component regulatory systems and AmfA/AmfB similar to ATP-dependent membrane translocators are analogous to the multicomponent phosphorelay and the Spo0K system, respectively, both of which are required for the initiation of sporulation in Bacillus subtilis . Involvement of a protein serine/threonine kinase in aerial mycelium formation is also suggested, because the Streptomyces coelicolor A3(2) afsK gene encoding a 'eukaryotic'-type protein kinase reverses the aerial mycelium-defective phenotype of strain HH1, independent of secondary metabolic function.     

Initiation of aerial mycelium formation in Streptomyces

In the past two years, the isolation of extracellular factors involved in the initiation of aerial mycelium formation, the identification of metabolic defects in certain developmental mutants, and the characterisation of three further bld genes and several gamma-butyrolactone receptor genes have led to new ideas about the mechanisms that initiate aerial mycelium formation in Streptomyces. The emerging picture suggests the integration of numerous signals from both inside and outside the cell.     

TheStreptomyces aureofaciens homologue of thewhiB gene is essential for sporulation; Its expression correlates with the developmental stage

In previous experiments, aStreptomyces aureofaciens gene highly similar to the sporulation-specificwhiB gene ofStreptomyces cœlicolor was identified. By intergrative transformationvia double cross-over, a stable null mutant of thewhiB-homologous gene ofS. aureofaciens was obtained. The disruption blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores, producing white aerial hyphae without septation. Expression of thewhiB gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared fromS. aureofaciens in various developmental stages. Two putative promoters were identified upstream of thewhiB coding region. The stronger promoter,whiB-P2, was induced at the beginning of aerial mycelium formation, and the weaker promoter,whiB-P1, was expressed fairly constantly during differentiation. No differences in the expression of thewhiB promoters were detected in anrpoZ-disruptedS. aureofaciens strain. The promoter bearing DNA fragment was inserted into the promoter-probe vector pARC1 to produce an expression pattern consistent with the results of direct RNA analysis.     

Reduction of Methionine Sulfoxide by Plants

A relaxed (rel) mutant was found among thirty spontaneous thiopeptin-resistant isolates of Streptomyces antibioticus strain 3720, an actinomycin-producing strain, which showed severely reduced ability to accumulate ppGpp during a nutritional shift-down. The pool size of GTP decreased markedly in the parental strain, but to a lesser extent in the rel mutant. The rel mutant did not show the induction of an enzyme, phenoxazinone synthase, which is involved in the biosynthesis of actinomycin. No negative effect of the rel mutation was observed on a constitutive enzyme, kynurenine formamidase, which also plays a role in actinomycin synthesis. The mutant also failed to produce melanin, but still retained the ability to form aerial mycelium and spores, although the onset of the formation of aerial mycelium was markedly delayed. Neither the phenoxazinone synthase activity nor the kynurenine formamidase activity was affected by ppGpp in vitro. It is suggested tha the stringent response (ppGpp) may be generally essential for the induction of enzymes involved in secondary metabolism.     

Sporulation-inducing factor inStreptomyces avermitilis

The excretion of a diffusible factor was detected in sporulating, avermectin-producing strain ofStreptomyces avermitilis. The factor induced the formation of aerial mycelium and spores by a nonsporulating mutant.     

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3′,5′ monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 μM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co-ordinate antibiotic biosynthesis.     

Comparative structural characterization of material extractable from aerial sheath of Streptomyces roseoflavus var. roseofungini

1. Tubular-like structures were regularly revealed in the surface sheath of the aerial mycelium of the parent strain of Streptomycetes roseoflavus var. roseofungini. In their shape and dimensions these structures were highly reminiscent of those massive accumulation of which was earlier reported to occur in cultures of dedifferentiated nocardioform “fructose” mutant of the same parent strain.
2. The tubular-like structures of the aerial mycelium sheath were shown to be markedly susceptible to brief acetone washing, undergoing almost complete desintegration.
3. On addition of water to crude acetone extract of the aerial mycelium precipitation and possible selfassembly of a spectrum of various structures occurred (folded scaly, bubble-like, spout-like). Some among structures so produced were reminiscent of those found in spectra of structures observed in reconstruction experiments with tubules from the mutant as well as of structures found in the aerial sheath of the parent strain and some other actinomycetes. Similarity in subunit structure of above materials was also noticeable.

The presence, in the sheath of aerial mycelium, of material with a tendency to selforganization is discussed in relation to the possible involvement of selfassembly processes in the formation of the surface sheath.     

Intracellular protease activity during the growth of Streptomyces coelicolor

Multiple intracellular proteases were produced by Streptomyces coelicolor throughout growth as surface cultures. Zymography revealed two constitutive, gelatinolytic proteases of approximate molecular masses 32.5 and 36.5 kDa. In addition, transient expression of a large (183.5 kDa) protease preceded aerial mycelium formation and following this, during sporulation, an additional protease of mass 27.5 kDa was produced.     

Colony development in Streptomyces carpinensis: a streptomycete with substrate mycelium spores

In this paper we present an ultrastructural study of spore formation in aerial vs. substrate mycelia of Streptomyces carpinensis. Both mycelia initiated spore formation at nearly the same time of colony development but exhibited different patterns of spatial localization of sporulation: spore formation took place throughout the aerial mycelium whereas in the substrate mycelium was confined to a narrow zone at the bottom of the colony. The ultrastructural changes leading to spore formation, however, were quite similar in both mycelia, differing only with respect to the outer components of the sporal wall. Spores formed in the aerial mycelium were covered by a thin sheath whereas the spores formed in the substrate mycelium were covered by an amorphous electron-dense material.