Alkaliphilic Soil Actinomycetes

The actinomycete complex of alkaline soils was found to be dominated by alkaliphilic streptomycetes, which showed maximal radial rates of colony growth at pH 8. At pH values of 7 and 10, the growth of these streptomycetes was poor. Alkaliphilic streptomycetes can be morphologically differentiated from other actinomycetes based on their high radial rates of colony growth and increased spore formation in alkaline media as compared to neutral media.     

Alkaliphilic soil actinomycetes

The actinomycete complex of alkaline soils was found to be dominated by alkaliphilic streptomycetes, which showed maximal radial rates of colony growth at pH 8. At pH values of 7 and 10, the growth of these streptomycetes was poor. Alkaliphilic streptomycetes can be morphologically differentiated from other actinomycetes based on their high radial rates of colony growth and increased spore formation in alkaline media as compared to neutral media.     

The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins

Streptomycetes form hydrophobic aerial hyphae that eventually septate into hydrophobic spores. Both aerial hyphae and spores possess a typical surface layer called the rodlet layer. We present here evidence that rodlet formation is conserved in the streptomycetes. The formation of the rodlet layer is the result of the interplay between rodlins and chaplins. A strain of Streptomyces coelicolor in which the rodlin genes rdlA and/or rdlB were deleted no longer formed the rodlet layer. Instead, these surfaces were decorated with fine fibrils. Deletion of all eight chaplin genes (strain DeltachpABCDEFGH) resulted in the absence of the rodlet layer as well as the fibrils at surfaces of aerial hyphae and spores. Apart from coating these surfaces, chaplins are involved in the escape of hyphae into the air, as was shown by the strong reduction in the number of aerial hyphae in the DeltachpABCDEFGH strain. The decrease in the number of aerial hyphae correlated with a lower expression of the rdl genes in the colony. Yet, expression per aerial hypha was similar to that in the wild-type strain, indicating that expression of the rdl genes is initiated after the hypha has sensed that it has grown into the air.     

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.     

Mycelial differentiation and spore formation by Streptomyces brasiliensis in submerged culture.

Streptomyces brasiliensis ATCC 23727 showed extensive sporulation when cultured in a liquid medium containing galactose and glutamic acid as carbon and nitrogen sources. Sporogenic hyphae formed under these conditions were morphologically similar and developmentally equivalent to aerial hyphae and metamorphosed into chains of spores by following a sequence of ultrastructural changes similar to that observed during growth on solid media. In addition, our electron microscopy study revealed two previously unrecognized aspects of hyphal development in streptomycetes: the formation of sporogenic hyphae was always preceded by changes in the structure of the nucleoid, and the sheath that characteristically covered these hyphae was not deposited coincidently with wall formation in the apical growing portion of the hypha.     

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.     

Cell division and DNA segregation in Streptomyces: how to build a septum in the middle of nowhere?

Streptomycetes are antibiotic-producing filamentous microorganisms that have a mycelial life style. In many ways streptomycetes are the odd ones out in terms of cell division. While the basic components of the cell division machinery are similar to those found in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, many aspects of the control of cell division and its co-ordination with chromosome segregation are remarkably different. The rather astonishing fact that cell division is not essential for growth makes these bacteria unique. The fundamental difference between the cross-walls produced during normal growth and sporulation septa formed in aerial hyphae, and the role of the divisome in their formation are discussed. We then take a closer look at the way septum site localization is regulated in the long and multinucleoid Streptomyces hyphae, with particular focus on actinomycete-specific proteins and the role of nucleoid segregation and condensation.     

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.     

Localization of growth and secretion of proteins in Aspergillus niger

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.     

Morphogenic effects of ramihyphin A in filamentous fungi

Ramihyphin A at subfungistatio concentrations stimulates ramification of hyphae of filamentous fungi. Stimulation of terminal ramification of hyphae that can be observed particularly in phytopathogenic fungi is most frequent. Hyphae ofMicrosporon canis, Trichophyton mentagrophytes, Blastomyces dermatitidis, Coccidioides immitis andHistoplasma capsulatum ramify intensively laterally. Stimulation of the lateral ramification was observed inMonilia fructigena, Penicillium marneffei andPenicillium chrysogenum. The antibiotic induces also formation of vesicular structures in phytopathogens. Due to the substantial ramification of hyphae, both terminal and lateral, the growth of colonies is interrupted. The addition of the antibiotic to a growing colony ofBotrytis cinerea induces dichotomic ramification of terminal hyphae after 3 h of growth. Lateral hyphae begin to grow later and further ramify dichotomically. Dense bundles of ramified hyphae are formed after 24 h due to the unbalanced ramification and the colony no longer increases its size.     

Defense Reactions in Host and Nonhost Plants Against the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.)

Growth and development of two races of the soybean rust fungus (Phakopsora pachyrhizi Syd.) were compared on host and nonhost plants. Both groups had several lines of defense, each of which could stop a part of attacking uredospores. Germ tubes and appressoria were produced equally well on hosts and nonhosts. A reduced formation of penetration hyphae contributed to the resistance of nonhosts and resistant host genotypes. In the epidermal cells of wheat and barley leaves, lower frequencies of penetration hyphae coincided with the production of papillae-like structures which were not penetrated. The last line of defense of all nonhosts was localized in the epidermal cell where the growth of the penetration hyphae was arrested definitively. The colony development in these species was suppressed completely. In highly resistant host genotypes the last defense reaction occurred later as a hypersensitive cell collapse which interrupted the growth of the rust colony.     

Roles of superoxide dismutase and catalase of Staphylococcus xylosus in the inhibition of linoleic acid oxidation

Streptomyces brasiliensis ATCC 23727 showed extensive sporulation when cultured in a liquid medium containing galactose and glutamic acid as carbon and nitrogen sources. Under such conditions, glycogen and trehalose are accumulated in the hyphae coinciding with spore formation. The results reported here suggest that glycogen accumulated in sporogenic hyphae is converted into trehalose during the final period of spore maturation. Glycogen is also accumulated in the hyphae when S. brasiliensis is cultured under conditions which did not support sporulation. Under such conditions, however, glycogen degradation is not accompanied by accumulation of trehalose. This suggest that the conversion of glycogen into trehalose might be a sporulation-specific event in streptomycetes.     

Streptomycetes: a new model to study cell death.

Colonies of streptomycetes are now viewed as multicellular entities containing morphologically and biochemically differentiated cell types which have specific functions and precise spatial relationships to one another. Like multicellular organisms, colony development in streptomycetes is also maintained by a tight balance between cell proliferation and cell death processes. This review describes the current state of knowledge concerning cell death in streptomycetes.     

SapB and the chaplins: connections between morphogenetic proteins in Streptomyces coelicolor

Morphogenesis in the streptomycetes features the differentiation of substrate-associated vegetative hyphae into upwardly growing aerial filaments. This transition requires the activity of bld genes and the secretion of biosurfactants that reduce the surface tension at the colony-air interface enabling the emergence of nascent aerial hyphae. Streptomyces coelicolor produces two classes of surface-active molecules, SapB and the chaplins. While both molecules are important for aerial development, nothing is known about the functional redundancy or interaction of these surfactants apart from the observation that aerial hyphae formation can proceed via one of two pathways: a SapB-dependent pathway when cells are grown on rich medium and a SapB-independent pathway on poorly utilized carbon sources such as mannitol. We used mutant analysis to show that while the chaplins are important, but not required, for development on rich medium, they are essential for differentiation on MS (soy flour mannitol) medium, and the corresponding developmental defects could be suppressed by the presence of SapB. Furthermore, the chaplins are produced by conditional bld mutants during aerial hyphae formation when grown on the permissive medium, MS, suggesting that the previously uncharacterized SapB-independent pathway is chaplin dependent. In contrast, a bld mutant blocked in aerial morphogenesis on all media makes neither SapB nor chaplins. Finally, we show that a constructed null mutant that lacks all chaplin and SapB biosynthetic genes fails to differentiate in any growth condition. We propose that the biosurfactant activities of both SapB and the chaplins are essential for normal aerial hyphae formation on rich medium, while chaplin biosynthesis and secretion alone drives aerial morphogenesis on MS medium.     

In vitro evidence of mycoparasitism of the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria laccata</Emphasis> against <Emphasis Type="Italic">Mucor hiemalis</Emphasis> in the rhizosphere of <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Mucor hiemalis f. hiemalis in co-culture, and in the rhizosphere of in vitro-grown Pinus sylvestris seedlings were investigated by light- and scanning electron-microscopy. In co-culture, mycelial growth away from the L. laccata colony reduced the number of aerial hyphae at the contact zone and increased the density and compactness of the mycelium-characterized gross morphology of the saprobic fungus. Although the growth of M. hiemalis was suppressed, no penetration of M. hiemalis hyphae after the colony was entered by L. laccata was observed. Instead, dense coiling of L. laccata hyphae around sporangiophores, overpowering them and causing them to disappear, was quite common. On nonmycorrhizal roots, sporangiospores germinated heavily and formed long hyphae for 2 days post inoculation, whereas their germination was totally inhibited on mycorrhizal roots. At 3 days after inoculation, only sporangia were seen with mycelial mats firmly attached to the roots by the mantle hyphae, whereas some remnants of sporangiophores, ruptured sporangial walls and degraded hyphae of M. hiemalis were overgrown by the mantle hyphae. During the next 3 days, the mantle-hyphae-invading sporangia formed short, thin branches that grew directly towards individual spores, tapering off upon contact.     

Hyphal tip growth in Achlya bisexualis. I. Distribution of 1,3-{beta}-glucans in elongating and non-elongating regions of the wall

We have approached the problem of hyphal tip growth by comparing the cell wall composition of elongating and non-elongating regions of the hyphae of Achlya bisexualis. To ensure that we could distinguish between elongating and non-elongating hyphae, light microscopic observations were used to determine the rates of elongation under growing and non-growing conditions. When elongation was measured in 10 min intervals it was found to consist of fluctuating periods of fast and slow growth rates, in the form of cycles. Even under our growing conditions, however, a very small number of hyphae in a colony are not elongating. SEM analysis revealed that elongating hyphae have tapered apices, whereas non-elongating hyphae have a rounded apex. The major matrix wall components, 1,3-β-glucans, were localized with an indirect immunogold technique specific for these polymers. This method resulted in their localization to all regions of both elongating and non-elongating hyphae, including the apex.     

Surface Structure of Streptomycete Spores as Revealed by Negative Straining and Freeze-Etching

The application of a simple negative staining technique and freeze-etching has revealed detailed information on the species-specific surface structure of spores and aerial hyphae of streptomycetes.     

Structural Proteins Involved in Emergence of Microbial Aerial Hyphae

Filamentous fungi and filamentous bacteria (i.e., the streptomycetes) belong to different kingdoms that diverged early in evolution. Yet, they adopted similar lifestyles. After a submerged feeding mycelium has been established, hyphae grow into the air and form aerial structures from which (a)sexual spores can develop. These spores are dispersed and can give rise to a new mycelium. Some of the key processes involved in the formation of aerial hyphae by these microbes appear to be very similar. In both cases molecules that lower the surface tension are secreted into the aqueous environment, thereby enabling hyphae to grow into the air. Aerial hyphae are then covered with a hydrophobic film. In fungi, this film is characterized by a mosaic of parallel rodlets, while similar rodlets have also been observed on aerial structures of filamentous bacteria. Although the erection of aerial hyphae in both filamentous fungi and filamentous bacteria is dependent upon (poly)peptides that are structurally unrelated, they can, at least partially, functionally substitute for each other.     

小麦条锈菌吸器母细胞入侵菌丝现象的进一步研究

应用电镜技术对小麦条锈菌吸器母细胞入侵自身菌丝的现象进行了研究,观察发现,在吸器母细胞与寄主细胞和菌丝细胞同时相接触的情况下,入侵栓可在与寄主细胞接触处形成,也可在与菌丝接触处形成;在菌落中心部位,吸器母细胞虽然未与寄主细胞接触,但同样可在与菌丝细胞接触处产生入侵栓;吸器母细胞在与菌丝接触处形成的人侵栓,其超微结构正常,并且可侵入到菌丝细胞壁内,但是未能穿透菌丝细胞壁。本文观察结果表明,小麦条锈菌吸器母细胞和人侵栓形成所需诱导因子可能是物理接触作用,而不涉及到化学作用,并且该病菌与寄主间的识别作用可能发生在入侵栓形成之后。     

The actinobacterial signature protein ParJ (SCO1662) regulates ParA polymerization and affects chromosome segregation and cell division during Streptomyces sporulation

Bacterial chromosome segregation usually involves cytoskeletal ParA proteins, ATPases which can form dynamic filaments. In aerial hyphae of the mycelial bacterium Streptomyces coelicolor, ParA filaments extend over tens of microns and are responsible for segregation of dozens of chromosomes. We have identified a novel interaction partner of S. coelicolor ParA, ParJ. ParJ negatively regulates ParA polymerization in vitro and is important for efficient chromosome segregation in sporulating aerial hyphae. ParJ-EGFP formed foci along aerial hyphae even in the absence of ParA. ParJ, which is encoded by sco1662, turned out to be one of the five actinobacterial signature proteins, and another of the five is a ParJ paralogue. We hypothesize that polar growth, which is characteristic not only of streptomycetes, but even of simple Actinobacteria, may be interlinked with ParA polymer assembly and its specific regulation by ParJ.