Age-dependent differential responses ofSaprolegnia hyphal tips to a helical growth-inducing factor in the agar substitute,gellan

Saprolegnia ferax produces more-or-less straight, subapically branched, hyphae when growing in liquid or agar-solidified media, with abundant aerial mycelium on the latter. In Contrast, the same medium solidified with gellan gum induced helical growth with reduced branching and almost no aerial mycelium. Helical growth induction was gellan concentration-dependent, peaking at 0.4–0.6% (w/v), when about 60% of tips were helical. Gellan-induced helices showed concentration-dependent inhibition by agarose and polyethylene glycol. Colonies on gellan-agarose, where helices were inhibited, reverted to having aerial mycelium, whereas those on gellan-polyethylene glycol did not. Branches on helical hyphae were initially linear, but converted to helical growth after about 2 h of extension. This transition was often marked by a branch, thus branch and helix competency appeared to be related. Germinating cysts took twice as long as hyphal inocula before producing helical hyphae, reinforcing the suggestion that helix competence was age-related.Achlya, but notPhytophthora, also showed gellan-induced helical growth and aerial mycelium suppression. These results showed (a) that morphogenic regulators of hyphal growth responded to gelling agents, probably high-molecular-weight polysaccharides, (b) that all growing hyphal tips were not equivalent, and (c) that hyphal tips underwent age-related changes in their response to the environment. The gellan-related differences in aerial mycelium mimic hydrophobin-based mycelium behavior and may thus indicate environmental regulation of hydrophobin production.     

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.     

A decrease in GTP content is associated with aerial mycelium formation in Streptomyces MA406-A-1

Aerial mycelium formation by Streptomyces sp. MA406-A-1, a formycin-producing strain, was suppressed by the presence of excess nutrient. In such suppressed cultures, decoyinine, which specifically inhibits GMP synthetase, initiated the formation of aerial mycelium at concentrations which only partially inhibited growth. The intracellular GTP pool of organisms growing in liquid culture markedly decreased on the addition of decoyinine. Decoyinine was also effective in initiating aerial mycelium formation of three other Streptomyces spp. examined. Regardless of the successful initiation of aerial mycelium formation, the ability of the cells to produce antibiotics (formycin or actinomycin D) did not increase, but decreased, on the addition of decoyinine. It is concluded that aerial mycelium formation by Streptomyces results from a decrease in the pool of GTP (or GDP), whereas antibiotic synthesis results from a different signal(s).     

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.     

A membrane-like structure envelops substrate mycelium during colony development in Streptomyces

Abstract Colonies of Streptomyces antibioticus were studied by transmission and scanning electron microscopy. The micrographs show that substrate mycelium growth takes place among an intercellular material and this mycelium is covered by a surface film. This structure could be a boundary between the aerial mycelium and the substrate mycelium.     

Changes in ribosomal proteins during colony development in Streptomyces.

The structure and functionality of the ribosomal subunits of the substrate and the aerial mycelium of Streptomyces antibioticus were compared. Using SDS-PAGE and HPLC, several differences between the ribosomal protein pattern from both stages of development were observed, including a clear decrease in the L7/L12 content of the aerial mycelium. The activity of the aerial mycelia ribosomes was also decreased when compared with that of the substrate mycelium. This effect was more pronounced in the 50S subunit. These results suggest that during cell differentiation in Streptomyces important changes occur at the ribosomal level, particularly in the transition from the substrate to the aerial mycelium.     

Streptomyces fragmentosporus,ein neuer thermophiler Actinomycet

Zusammenfassung Ein neuer thermophiler Actinomycet, Streptomyces fragmentosporus wird beschrieben. Kennzeichnend für die Art sind gabelig bis rechtwinklig verzweigte Sporophoren mit kurzen, geraden Ketten aus locker gestellten, stacheligen Fragmentationssporen und die Bildung von Fragmentationssporen im Substratmycel. Neben den Sporophoren werden lange Lufthyphen entwickelt, die teils Sporophoren tragen und gewöhnlich basipetal in Sporen zerfallen. Das Luftmycel ist weiß. Substrat-und Luftsporen sind polymorph. Die Art wächst nur auf wenigen komplexen Nährböden, Pigmente oder Melanin werden nicht gebildet. Streptomyces fragmentosporus ist am nächsten mit der mesophilen Art Streptomyces ramulosus verwandt. Die vergleichende morphologische Untersuchung beider Arten zeigte eine große Übereinstimmung in der Differenzierung des Substrat-und Luftmycels.

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Streptomyces fragmentosporus, a new thermophilic actinomycete

Summary The new thermophilic species Streptomyces fragmentosporus is described. The organism is characterized by short, straight, often rectangularly branched aerial sporophores with loosely arranged fragmentation spores, and by the production of fragmentation spores in the substrate mycelium. Beside the sporophores long aerial hyphae are developed which may bear sporophores, and usually fragmentate into spores in basipetal direction. The spores are polymorph and bear small spines.Good growth and production of aerial mycelium was obtained only on manure- and hay-yeast-agar. No pigments or melanin were formed, and no antibiotic activity was observed. The optimum temperature was 50 to 60° C. Streptomyces fragmentosporus is most closely related to the mesophilic Streptomyces ramulosus. It differs from that species mainly by the colour of the aerial mycelium, the absence of pigments, and by the spiny spores which are connected in the chains by ± long parts of hyphal remnants.In a comparative morphological study Streptomyces ramulosus was found to produce abundantly short, ± rectangularly branched sporophores as reported by Shirling and Gottlieb (1968), and occasionally long aerial hyphae bearing sporophores as described by Ettlinger and coworkers (1958). In contrast to the latter authors we observed a production of spores also in these long hyphae like in Streptomyces fragmentosporus.

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Die Untersuchung wurde durch eine Sachbeihilfe der Deutschen Forschungsgemeinschaft unterstützt.     

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.     

Measurement and simulation of the morphological development of filamentous microorganisms

Growth of Streptomyces tendae was investigated in submerged culture. Images of several mycelia were analyzed by means of an image-processing system. The studies revealed that tip growth angles and branching outgrowth angles could be regarded as normally distributed. Based on these results, a random model for directional growth of hyphal tips as well as directional growth of branches is proposed. This model shows curved elongation of hyphal tips, so that the morphological development of a mycelium up to the formation of a pellet is predicted, similar to that observed in nature.     

Expression analysis of the spi gene in the pock-forming plasmid pSA1.1 from Streptomyces azureus and localization of its product during differentiation

The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.     

Relationship between response to and production of the aerial mycelium-inducing substances pamamycin-607 and A-factor

Respectively, exogenous pamamycin-607 and A-factor restored or stimulated aerial mycelium formation in 30 (67%) and 6 (13%) of 45 Streptomyces strains, and both restored or stimulated it in 5 strains (11%). Pamamycin-607 production was detected in 3 of those strains that responded to pamamycin-607. These findings indicate that pamamycin-607 acts on the common regulatory system for aerial mycelium formation in Streptomyces spp. but is not a universal autoregulator. Increased or decreased antibacterial production occurred in 5 strains in association with aerial mycelium formation by pamamycin-607 or A-factor.     

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens

A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.     

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.     

An essential GTP-binding protein functions as a regulator for differentiation in Streptomyces coelicolor

The Streptomyces coelicolor obg gene, which encodes a putative GTP-binding protein of the Obg/Gtp1 family, was characterized. The obg gene was essential for viability. Introduction of multiple copies of obg into wild-type S. coelicolor suppressed aerial mycelium formation. A single amino acid substitution at any of six positions was introduced into the GTP binding site of Obg, and the mutated proteins were expressed in wild-type cells. Obg^P168 → V exerted a more accentuated suppressive effect on aerial mycelium formation than did the wild-type Obg protein. In contrast, Obg^G171 → A accelerated the development of aerial mycelium. These results show that Obg protein functions as a pivotal regulator for the onset of cell differentiation through its ability to bind GTP. Western analysis revealed that expression of obg is regulated in a growth phase-dependent manner, indicating a sharp decrease just after onset of aerial mycelium development or at the end of vegetative growth. Obg was a membrane-bound protein as determined by immunoelectron microscopy.     

Tissue-specific glycogen branching isoenzymes in a multicellular prokaryote, Streptomyces coelicolor A3(2)

In the overtly differentiated colonies of Streptomyces coelicolor A3(2), discrete phases of glycogen synthesis are found at the vegetative/aerial mycelium boundary (phase I) and in the immature spore chains at aerial hyphal tips (phase II). We have characterized two S. coelicolor glgB genes encoding glycogen branching enzyme, which are well separated in the genome. Disruption of glgB I led to the formation of abnormal polyglucan deposits at phase I, with phase II remaining normal, whereas disruption of glgB II interfered specifically with phase II deposits, and not with those of phase I. Thus, each branching enzyme isoform is involved in a different phase of glycogen synthesis. This situation contrasts with that in simple bacteria, which typically have a single set of enzymes for glycogen metabolism, and more closely resembles that in plants.     

Evidence of heterokaryosis in streptomyces coelicolor A3(2)

Summary Hyphae from mixed cultures of complementary auxotrophs of Streptomyces coelicolor A3(2) did not grow on minimal media (MM) when fertility plasmids (SCP1 and SCP2) were missing in both strains. The addition of one part per cent of complete medium (CM) to MM allowed growth of vigorous colonies among the tiny colonies of the parental types. The former, amounting to 1%–10% of the total population, turned out to be heterokaryons. They could be propagated on the same medium by plating of hyphal fragments. When five parts per cent of CM were added to MM, beside the heterokaryotic colonies vigorous spindles of aerial mycelium were formed whenever complementary colonies overlapped. When the SCP1 and SCP2 plasmids were present in one or both parents a clear constraint on the outburst of heterokaryotic aerial mycelium was observed.     

Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ~20 μm h⁻¹, which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.