Development Of Aerial Mycelium Research Articles

LOX1- and PLP1-dependent transcriptional reprogramming is essential for injury-induced conidiophore development in a filamentous fungus

ABSTRACT Fungi use oxylipins (oxidized lipids) as signaling molecules to induce asexual development. These molecules play an essential role in the response to wounding, exerting a protective effect against plant pathogens, and are part of the inflammatory process in animals. However, the physiological and molecular mechanisms triggered by oxylipins that lead to asexual development in fungi are not well understood. Using a genetic approach, mass spectrometry, and phenotypic analysis, we describe the functional role of a patatin-like phospholipase ( plp1 ) and a unique lipoxygenase ( lox1 ) in the response to injury in the model fungus Trichoderma atroviride. lox1 and plp1 are co-expressed and regulated by damage signaling and sensing components. Phenotypic analysis revealed an essential defect in the emergence of aerial hyphae in the lox1 and plp1 null mutant strains, blocking injury-induced conidiation. In addition, functional loss analysis demonstrated that both genes are essential for wound-associated linoleic acid-derived oxylipin 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) production and the transcriptional reprogramming required for conidiation. T. atroviride requires LOX1 and PLP1 to induce transcription factors involved in asexual development such as brlA , hox2 , and azf1 homologs at the early stages of the response and at a later stage to activate lipid metabolism and the structural proteins involved in aerial mycelium emergence. Our study shows how the cooperative function of lox1 and plp1 , during the response to wounding, regulates the molecular and physiological processes of damaged-sensitized cells that lead to reproductive aerial mycelium development and consequently, ensure survival through asexual reproduction. IMPORTANCE In addition to being considered a biocontrol agent, the fungus Trichoderma atroviride is a relevant model for studying mechanisms of response to injury conserved in plants and animals that opens a new landscape in relation to regeneration and cell differentiation mechanisms. Here, we reveal the co-functionality of a lipoxygenase and a patatin-like phospholipase co-expressed in response to wounding in fungi. This pair of enzymes produces oxidized lipids that can function as signaling molecules or oxidative stress signals that, in ascomycetes, induce asexual development. Furthermore, we determined that both genes participate in the regulation of the synthesis of 13-HODE and the establishment of the physiological responses necessary for the formation of reproductive aerial mycelium ultimately leading to asexual development. Our results suggest an injury-induced pathway to produce oxylipins and uncovered physiological mechanisms regulated by LOX1 and PLP1 to induce conidiation, opening new hypotheses for the novo regeneration mechanisms of filamentous fungi.

ОЦЕНКА ФУНГИЦИДНОЙ АКТИВНОСТИ ПРЕПАРАТОВ ДЛЯ ПРЕДПОСЕВНОЙ ОБРАБОТКИ СЕМЯН ОЗИМОЙ ПШЕНИЦЫ

The massive use of one-component fungicides has caused the problem of the emergence of resistant forms of phytopathogens. The development of multicomponent plant protection products is one of the ways of dealing with such a situation. The aim of the study is to determine the effectiveness of multicomponent chemical dressing agents in suppressing the most common fungal pathogens and seed infections in vitro. This work was performed in 2020 in the Federal Research Center for Biological Plant Protection (Krasnodar) in the laboratory of the creation of microbiological plant protection products and a collection of microorganisms. Fungi of the Fusarium, Microdochium, Bipolaris, Alternaria, Penicillium, Rhizoctonia, Septoria, Pyrenophora genera were used as test objects. The fungicidal activity of chemical preparations against economically significant phytopathogens causing root rot was evaluated in laboratory conditions by the method of serial dilutions followed by sowing on a nutrient medium. In addition, the effectiveness of the preparations was investigated by the method of treated seeds phytoexamination according to GOST 12044–93. The preparations, which included triazoles and phenylpyrroles, had the strongest inhibitory effect (100 %) on pathogens of Rhizoctonia, helminthosporiosis and septoria. The preparation containing compounds of the group of triazoles and strobilurins suppressed the growth of Rhizoctonia solani only. The preparations’ inhibitory effect on a number of phytopathogenic fungi was manifested in growth retardation, absence of aerial mycelium development and mycelium pigmentation. Concerning the causative agents of Fusarium root rot, the two-component preparations efficacy ranged from 51 % to 74.4 %; three-component one – from 42.9 % to 84.7 % depending on the species of the genus Fusarium. Presowing seed treatment made it possible to inhibit the growth of fungi of the genera Fusarium, Mucor, Aspergillus; significantly inhibit the development of the fungus Alternaria (spread in the experimental variant – 1.7 %, in the control variant – 46.7 %). From the data obtained, it can be concluded that multicomponent preparations based on triazoles and phenylpyrroles are highly effective in suppressing a wide range of phytopathogens and can be recommended for presowing seed treatment.

Coding the α-subunit of SNF1 kinase, Snf1 is required for the conidiogenesis and pathogenicity of the Alternaria alternata tangerine pathotype

To well cope with various external carbon sources, fungi have evolved an adaptive mechanism to overcome the adversity of carbon source deficiency. The sucrose non-fermenting (SNF1) protein kinase mainly mediates the utilization of non-fermentable carbon sources. In this study, we determined the function of Snf1, coding the α-subunit of SNF1 kinase, in the phytopathogenic fungus Alternaria alternata via analyzing the Snf1 deletion mutants (ΔAasnf1). Aasnf1 is required for growth, development of aerial mycelium, and conidiation. Results of pathogenicity test showed that ΔAasnf1 induced smaller lesions on detached citrus leaves. Moreover, in the carbon utilization assay, ΔAasnf1 showed growth inhibition on the minimal medium supplemented with polygalacturonic acid, sucrose or alcohol as the only carbon source. Compared to the wild type, ΔAasnf1 also exhibited stronger resistance to cell wall stressors of sodium dodecyl sulfate and congo red. In conclusion, Aasnf1 played important roles in the carbon utilization, vegetative growth, conidiation, cell wall functions and pathogenicity of A. alternata. This study is the first report on the functions of Aasnf1 and our results suggest that Snf1 is critical for the conidiogenesis and pathogenesis of the A. alternata tangerine pathotype.

Descripción de un caso de pitiosis cutánea equina y su diagnóstico mediante diversas técnicas

Pythiosis is a disease widely distributed worldwide caused by Pythium insidiosum. It is transmitted and propagated in semi-aquatic environments, mainly affecting equines, canines, felines, cattle and humans. In Paysandu County, Uruguay, in the month of May 2017, a case of pitiosis was registered in a 24-year-old Creole breed female equine. An extensive circular lesion located in the ventral abdominal area was surgically removed. Macroscopically, it was observed granulation tissue, multifocal areas of necrosis with suppurative hemorrhagic content and yellowish-white pasty to chalky concretions. At the histopathological examination with HE in superficial and deep dermisthere were severe proliferation of connective tissue, neovascularization, multifocal areas of necrosis surrounded by infiltration of polymorphonuclears, mainly eosinophils, and macrophages. Inside of the areas of necrosis there was presence of hyphae stained positively by the argentic Grocott stain. Positive immunostaining was obtained for P. insidiosum. In the culture of the fresh material, colonies of whitish color and radial growth were isolated with the development of aerial mycelia after 48 hours. The isolation of P. insidiosum was confirmed by the induction of zoospores and the characterization of them. The ITS region (ITS1, 5.8S, ITS2) was amplified by the nested PCR reaction and in a second reaction the gene specific for P. insidiosum from the ITS1 region was amplified. The sequence obtained was compared with that registered in GenBank confirming a 100% homology with P. insidiosum.

Neurospora crassa NADPH Oxidase NOX-1 Is Localized in the Vacuolar System and the Plasma Membrane

The NADPH oxidases (NOX) catalyze the production of superoxide by transferring electrons from NADPH to O2, in a regulated manner. In Neurospora crassa NOX-1 is required for normal growth of hyphae, development of aerial mycelium and asexual spores, and it is essential for sexual differentiation and cell-cell fusion. Determining the subcellular localization of NOX-1 is a critical step in understanding the mechanisms by which this enzyme can regulate all these different processes. Using fully functional versions of NOX-1 tagged with mCherry, we show that in growing hyphae NOX-1 shows only a minor association with the endoplasmic reticulum (ER) markers Ca2+-ATPase NCA-1 and an ER lumen-targeted GFP. Likewise, NOX-1 shows minor co-localization with early endosomes labeled with YPT-52, a GTPase of the Rab5 family. In contrast, NOX-1 shows extensive co-localization with two independent markers of the entire vacuolar system; the vacuolar ATPase subunit VMA-1 and the fluorescent molecule carboxy-DFFDA. In addition, part of NOX-1 was detected at the plasma membrane. The NOX-1 regulatory subunit NOR-1 displays a very different pattern of localization, showing a fine granular distribution along the entire hypha and some accumulation at the hyphal tip. In older hyphal regions, germinating conidia, and conidiophores it forms larger and discrete puncta some of which appear localized at the plasma membrane and septa. Notably, co-localization of NOX-1 and NOR-1 was mainly observed under conidial cell-cell fusion conditions in discrete vesicular structures. NOX functions in fungi have been evaluated mainly in mutants that completely lacked this protein, also eliminating interactions between hyphal growth regulatory proteins NOR-1, the GTPase RAC-1 and the scaffold protein BEM-1. To dissect NOX-1 roles as scaffold and as ROS-producing enzyme, we analyzed the function of NOX-1::mCherry proteins carrying proline 382 by histidine (P382H) or cysteine 524 by arginine (C524R) substitutions, predicted to only affect NADPH-binding. Without notably affecting NOX-1 localization or protein levels, each of these substitutions resulted in lack of function phenotypes, indicating that NOX-1 multiple functions are all dependent on its oxidase activity. Our results open new interpretations to possible NOX functions, as components of the fungal vacuolar system and the plasma membrane, as well as to new vacuolar functions.

Discovery of Actinomycetes from Extreme Environments with Potential to Produce Novel Antibiotics.

IntroductionAntimicrobial-resistant pathogens pose serious challenges to healthcare institutions and health of the public. Thus, there is an urgent need for the discovery of new and effective antimicrobial agents. Microorganisms that exist in extreme environments such as those with high salinity or alkalinity, are known as extremophiles, and include various species of actinomycetes. The goal of this study is to discover novel antibiotics from extremophiles found in Kazakhstan that are effective against drug resistant pathogens.MethodsSoil from extreme environments of Kazakhstan was collected, and pure cultures of actinomycetes were isolated and cultured in modified Bennett’s broth with either high concentrations of salt or high pH to mimic extreme environments. Extracts obtained from selected actinomycetes strains were used to test for antimicrobial activity against Staphylococcus aureus, Escherichia coli and Aspergillus niger.ResultsA total of 5936 strains of extremophile actinomycetes were isolated; from these, 2019 strains were further isolated into pure cultures. Of these, 415 actinomycetes strains that demonstrated antagonistic antibacterial activities were selected. These actinomycetes were further classified into groups and subgroups based on their responses to different culture conditions. Antimicrobial antagonism activity for some of the actinomycetes strains was dependent on culture conditions and development of aerial mycelia under extreme conditions.ConclusionWe identified several interesting candidate extracts with putative antimicrobial activities against several strains of drug resistant pathogens. Our research of the actinomycetes’ ability to produce antibiotics in the near-natural conditions provides a great opportunity to assess their biodiversity and distribution in the Central Asian region and to develop new methodological approaches to the screening of new antimicrobial agents.

The SCO1731 methyltransferase modulates actinorhodin production and morphological differentiation of Streptomyces coelicolor A3(2)

Streptomyces coelicolor is a Gram-positive microorganism often used as a model of physiological and morphological differentiation in streptomycetes, prolific producers of secondary metabolites with important biological activities. In the present study, we analysed Streptomyces coelicolor growth and differentiation in the presence of the hypo-methylating agent 5′-aza-2′-deoxycytidine (5-aza-dC) in order to investigate whether cytosine methylation has a role in differentiation. We found that cytosine demethylation caused a delay in spore germination, aerial mycelium development, sporulation, as well as a massive impairment of actinorhodin production. Thus, we searched for putative DNA methyltransferase genes in the genome and constructed a mutant of the SCO1731 gene. The analysis of the SCO1731::Tn5062 mutant strain demonstrated that inactivation of SCO1731 leads to a strong decrease of cytosine methylation and almost to the same phenotype obtained after 5-aza-dC treatment. Altogether, our data demonstrate that cytosine methylation influences morphological differentiation and actinorhodin production in S. coelicolor and expand our knowledge on this model bacterial system.

Effect of Tricyclazole on morphology, virulence and enzymatic alterations in pathogenic fungi Bipolaris sorokiniana for management of spot blotch disease in barley.

Bipolaris sorokiniana synthesizes the 1,8-dihydroxynaphthalene (DHN) melanin via pentaketide pathway and promotes the development of aerial mycelia and conidia. A melanin biosynthesis inhibitor Tricyclazole (TCZ), brought changes when applied at 5-100μgml(-1) concentration in the colony morphology, radial growth, mycelia weight, melanin content, antioxidant enzymes (SOD and CAT) and extracellular hydrolytic enzymes (cellulase, pectinase, amylase and protease) in black, mixed and white isolates of B. sorokiniana. A significant alteration was recorded in antioxidant enzymes in black and mixed isolates; however, non-significant alteration was recorded in white isolate. Isolates of B. sorokiniana exposed to 100µgml(-1) TCZ showed significantly increased formation of superoxide radical (O 2 (·-) ) and hydrogen peroxide (H2O2)·H2O2 was detected significantly high in hyphae and conidia while, O 2 (·-) was found primarily in the conidia. Microscopic results suggest that TCZ damages not only the cell wall but also the cell membrane. The foliar application of TCZ (25, 50 and 100µgml(-1)) decreases the area under disease progress curve, lesion development and spore formation on barley leaves thereby reducing potential for the disease development. In conclusion TCZ influences the pathogenic ability by damaging the cell structure of hyphae and conidia and also alters the antioxidant enzyme levels in B. sorokiniana. TCZ may therefore,works against to pathogen for better management of spot blotch disease in barley infected with B. sorokiniana.

Streptomycin-induced expression in Bacillus subtilis of YtnP, a lactonase-homologous protein that inhibits development and streptomycin production in Streptomyces griseus.

Bacillus subtilis induces expression of the gene ytnP in the presence of the antimicrobial streptomycin, produced by the Gram-positive bacterium Streptomyces griseus. ytnP encodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium in S. griseus.

Implication of stringent response in the increase of mutability of the whiG and whiH genes during Streptomyces coelicolor development

In Streptomyces ambofaciens, genetic instability occurring during aerial mycelium development gives rise to white mutant papillae on colonies. Pig-pap mutants deriving from such papillae are unable to sporulate and devoid of the large genome rearrangement usually observed in the other Streptomyces mutants that genetic instability generated. Pig-pap mutants frequently harboured discrete mutations affecting the whiG gene. Furthermore, it has been established that the production of papillae dramatically increased when S. ambofaciens was grown under an amino acid limitation. In this work, we tested the implication of the stringent response, induced during an amino acid limitation, in the production of white papillae in Streptomyces coelicolor, a species which is phylogenetically close to S. ambofaciens. First, we showed that S. coelicolor produced mutant papillae and that this production was increased under an amino acid limitation. Secondly, we showed that the Pig-pap mutants generated both with and without amino acid limitation frequently exhibited mutations in whiH or whiG genes. Finally, we observed that a relA mutant of S. coelicolor, which was unable to elicit the stringent response under an amino acid limitation, was also unable to produce papillae. The restoration of the ability to elicit the stringent response also restored the papillae production. These papillae gave rise to Pig-pap mutants displaying the same characteristics as Pig-pap mutants spontaneously appearing on wild-type colonies. Altogether, these results show that whatever the underlying mechanism, the stringent response is involved in the production of white papillae in S. coelicolor.

Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria

We conducted a comparative study of the effects of alpha-amino-gamma-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of gram-positive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10(-5)-10(-3) M HSL or 10(-5)-10(-4) M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10(-4)-10(-3) M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10(-4) to (1.5-2.5) 10(-4) M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10(-4) M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1cm2 disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075-0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions on the microbial community level.

Genetic instability of whiG gene during the aerial mycelium development of Streptomyces ambofaciens ATCC23877 under different conditions of nitrogen limitations

In Streptomyces ambofaciens, white papillae that genetic instability events generate during aerial mycelium growth, give rise to Pig-pap mutants which are unable to sporulate and devoid of large genome rearrangement. Knowing that genetic and environmental factors can influence the number of papillae per colony, we investigated the effect of nutrient limitated conditions of growth on the formation of white papillae. We observed that under nitrogen limitation and, most particularly, under amino acid limitation, the number of papillae per colony dramatically increased. Most of the Pig-pap mutants deriving from such papillae displayed a mutation in the whiG gene, which encodes the sigma factor σ whiG which is absolutely required for the sporulation process. In most cases, the mutation led to a loss of function. We showed that the Pig-pap mutants deriving from papillae appearing under usual growth conditions also frequently displayed null mutation of whiG too. As the whiG mutation ratio among the Pig-pap mutants isolated with or without nitrogen limited conditions did not change, the results described in this paper suggest that the production of papillae could constitute a response of S. ambofaciens to an amino acid limitation.

MEDS and PocR are novel domains with a predicted role in sensing simple hydrocarbon derivatives in prokaryotic signal transduction systems

We identify two conserved domains in diverse bacterial and archaeal signaling proteins. One of them, the MEDS domain, is typified by the DmcR protein from Methylococcus and the other by the PocR protein of Salmonella typhi. We provide evidence that both these domains are likely to sense simple hydrocarbon derivatives and transduce downstream signals on binding these ligands. The PocR ligand-binding domain is shown to contain a novel variant of the fold found in PAS and GAF domains. The MEDS domain is present in both methylotrophs and complex methanogens, and both the MEDS and PocR domains show a lineage-specific expansion in the latter organisms, suggesting a role in sensing their principle growth substrates. The MEDS domain is also found in the negative regulators of the sigma factor SigB in actinomycetes, including pathogens like Mycobacterium tuberculosis. Hence it is possible that these sigma factors, involved in aerial mycelium development and stress response in the actinomycetes, might be under the regulation of as yet uncharacterized small molecules. aravind@ncbi.nlm.nih.gov.

Duplicated gene clusters suggest an interplay of glycogen and trehalose metabolism during sequential stages of aerial mycelium development in Streptomyces coelicolor A3(2).

DNA sequencing and operon disruption experiments indicate that the genes glgBI and glgBII, which code for the two developmentally specific glycogen branching enzymes of Streptomyces coelicolor A3(2) each form part of larger duplicated operons consisting of at least four genes in the order pep1-treS-pep2-glgB. The sequences of the TreS proteins are 73% identical (93% similar) to that of an enzyme that converts maltose into trehalose in Pinmelobacter, a distantly related actinomycete; and the Pep1 proteins show relatedness to the alpha-amylase superfamily. Disruptions of each operon have spatially specific effects on the nature of glycogen deposits, as assessed by electron microscopy. Upstream of the glgBI operon, and diverging from it, is a gene (glgP) that encodes a protein resembling glycogen phosphorylase from Thermatoga maritima and a homologue in Mycobacterium tuberculosis. These three proteins form a distinctive subgroup compared with glycogen phosphorylases from most other bacteria, which more closely resemble the enzymes from eukaryotes. Diverging from the glgBII operon, and separated from the pep1 gene by two very small ORFs, is a gene (glgX) encoding a probable glycogen debranching enzyme. It is suggested that most of these gene products participate in the developmentally modulated interconversion of immobile, inert glycogen reservoirs, and diffusible forms of carbon, both metabolically active (e.g. glucose-1-phosphate generated by glycogen phosphorylase) and metabolically inert but physiologically significant (trehalose).

Expression analysis of obg, the gene controlling morphological development of Streptomyces griseus IFO 13189.

The expression of the obg gene of S.griseus, which codes for a GTP-binding protein and controls morphological development of this organism, was studied by Western and Northern blot analyses. Western analysis revealed that Obg protein level sharply decreases just after the onset of aerial mycelium development or at the end of vegetative growth on agar plate cultures, indicating that expression of obg is regulated in a growth phase-dependent manner. The obg gene was found to be transcribed as a mRNA of 1.7 kb and its transcription was cut off just prior to the decrease of Obg protein, indicating that obg gene expression is controlled at the transcription level.

Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54.

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi-preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H-, 13C-NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation: ANALOGIES WITH THE BIOCHEMICAL PROCESSES DESCRIBED IN LATE STEPS OF EUKARYOTIC APOPTOSIS

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.

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.

Streptofactin, a novel biosurfactant with aerial mycelium inducing activity from Streptomyces tendae Tü 901/8c

Streptomyces tendae Tü 901/8c produces an extracellular hydrophobic peptide, termed streptofactin. The compound was isolated from conditioned agar medium and characterized. Streptofactin is a mixture of structurally related peptides with the major components ranging in size from 1003 m/ z to 1127 m/ z. Extracellular application of the purified peptide restored the capacity to form aerial mycelium in mutants defective in morphological development. Surface activity measurements revealed that streptofactin decreased the surface tension of water from 72.0 mN m −1 to 39.4 mN m −1 and achieved a critical micelle concentration value of 36 mg l −1. The properties of streptofactin suggest that it plays a structural role in aerial mycelium development and supports the erection of aerial hyphae by lowering the surface tension of water films enclosing the colonies. A model for aerial mycelium formation which represents a novel mechanism for the adaption to environmental changes in Streptomyces is proposed.

Streptofactin, a novel biosurfactant with aerial mycelium inducing activity fromStreptomyces tendaeTü 901/8c

Streptomyces tendae Tu 901/8c produces an extracellular hydrophobic peptide, termed streptofactin. The compound was isolated from conditioned agar medium and characterized. Streptofactin is a mixture of structurally related peptides with the major components ranging in size from 1003 m/z to 1127 m/z. Extracellular application of the purified peptide restored the capacity to form aerial mycelium in mutants defective in morphological development. Surface activity measurements revealed that streptofactin decreased the surface tension of water from 72.0 mN m−1 to 39.4 mN m−1 and achieved a critical micelle concentration value of 36 mg l−1. The properties of streptofactin suggest that it plays a structural role in aerial mycelium development and supports the erection of aerial hyphae by lowering the surface tension of water films enclosing the colonies. A model for aerial mycelium formation which represents a novel mechanism for the adaption to environmental changes in Streptomyces is proposed.