Mycelium differentiation and antibiotic production in submerged cultures of Streptomyces coelicolor

Despite the fact that most industrial processes for secondary metabolite production are performed with submerged cultures, a reliable developmental model for Streptomyces under these culture conditions is lacking. With the exception of a few species which sporulate under these conditions, it is assumed that no morphological differentiation processes take place. In this work, we describe new developmental features of Streptomyces coelicolor A3(2) grown in liquid cultures and integrate them into a developmental model analogous to the one previously described for surface cultures. Spores germinate as a compartmentalized mycelium (first mycelium). These young compartmentalized hyphae start to form pellets which grow in a radial pattern. Death processes take place in the center of the pellets, followed by growth arrest. A new multinucleated mycelium with sporadic septa (second mycelium) develops inside the pellets and along the periphery, giving rise to a second growth phase. Undecylprodigiosin and actinorhodin antibiotics are produced by this second mycelium but not by the first one. Cell density dictates how the culture will behave in terms of differentiation processes and antibiotic production. When diluted inocula are used, the growth arrest phase, emergence of a second mycelium, and antibiotic production are delayed. Moreover, pellets are less abundant and have larger diameters than in dense cultures. This work is the first to report on the relationship between differentiation processes and secondary metabolite production in submerged Streptomyces cultures.     

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.     

Physiologische Selektion auf kältebehandelten Oberflächenkulturen von Streptomyces rimosus

Zusammenfassung Overflächen- und Submerskulturen von Streptomyces rimosus zeigen nach Kältebehandlung einen raschen Vitalitätsverlust. Physiologische Untersuchungen an flüssigen und festen Nährubstraten haben ergeben, daß die Ausreifung des Mycels von einer erheblichen Alkalisierung der Medien begleitet wird; in den Flüssigkeitskulturen werden pH-Werte, von über 8,5 erreicht. Submerse Folgepassagen solcher alkalischer Aufbewahrungskulturen zeigen in Abhängigkeit von der Zeitdauer der Kältewirkung physiologische Veränderungen, die auf synthetischen Medien als gestörter Kohlenhydratstoffwechsel zu fassen sind. In Übereinstimmung mit einschlägigen Literaturangaben unterliegen die reifen Streptomycetenkulturen der Wirkung ihres selbstgebildeten Antibioticums, die entweder eine Selektion stoffwechselabweichender Organismen auslöst oder die den Stoffwechsel der Resistenten erst während der Aufbewahrungszeit prägt. In diesem Falle wäre der abweichende Stoffwechselzustand über mehrere Folgekulturen im Mycel manifestiert worden.

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Physiological selection in cold-treated surface cultures of Streptomyces rimosus

Summary Surface and submerged cultures of Streptomyces rimosus show a rapid loss of viability after storage at + 4°C. Physiological tests with fluid and solid nutrient substrates indicate that as the mycelium ripens, the medium becomes strongly alkaline (fluid cultures show a pH of over 8,5). Subsequent submerged passages of such alkaline stored cultures show physiological changes dependent on the duration of the cold treatment which identified on synthetic media as disturbances of the carbohydrate metabolism.From the literature it is known that cultures of ripe Streptomycetes are affected by their own antibiotic. This either causes a selection of organisms with unusual metabolism, or affects the metabolism of the resistant strains during cold storage. In this case, the deviant metabolism should persist in the mycelium through several culture passages.

     

Antibiotic Production of Hyphal Fractions of Streptomyces griseus: II. Streptomycin Production of Different Fractions Obtained by Density Gradient Centrifugation

The streptomycin-producing activity (SPA) of hyphal fractions from washed mycelium of submerged cultures of Streptomyces griseus strain 52-1, as obtained by density gradient centrifugation, was investigated. Activity of the various fractions differed strongly in intensity. The highest SPA was evident in the unfractionated mycelium. A synergistic effect upon SPA was found in the interaction of cultures of different ages, and a 55% increase in yield was obtained by mixing the 48- and 72-hr cultures. A synergistic effect occurred in all combinations studied. By use of fractions obtained from 72-hr mycelium for inoculation, differences in streptomycin production were noted. Some inoculum fractions yielded a greater amount of streptomycin (36%) than the unfractionated mycelial inoculum.     

Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients.

Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis.     

Improvement of transglutaminase production by extending differentiation phase of Streptomyces hygroscopicus: mechanism and application

Streptomyces transglutaminase (TGase) is an important industrial enzyme that catalyzes cross-linking of proteins. It is secreted as a zymogene and then is activated by proteases under physiological conditions. Although the activation process of TGase has been well investigated, the physiological function of TGase in Streptomyces has not been revealed. In this study, physiological function of TGase from Streptomyces hygroscopicus was found to be involved in differentiation by construction of a TGase gene interruption mutation strain (Δtg). The mutant Δtg showed an absence of differentiation compared with the parent strain. Furthermore, the production of TGase was found to be increased with the extending growth arrest phase of mycelium in submerged cultures. Thus, to enhance yield of TGase, the mycelium differentiation of Streptomyces was regulated via low temperature stress in a 3-L stirred-tank fermenter. The production of TGase increased by 39 % through extending the growth arrest phase for 4 h. This study found that TGase is involved in Streptomyces differentiation and proposed an approach to improve TGase production by regulation of mycelium differentiation in submerged cultures.     

Spontaneous variability ofStreptomyces glomeratus,a producer of the anthracycline antibiotics beromycins

Spontaneous variants of the beromycin-producing strainStreptomyces glomeratus 3980 were divided into five groups (A-E) according to increasing antibiotic activity. The most active variants (group E) differed from the other types and the wild strain by a suppressed ability to produce aerial mycelium and melanoid pigment and by an increased production of propionic acid. Strains with a 12-fold higher antibiotic production capacity (with respect to strain 3980) were obtained by selection of superior segregants from submerged cultures of the E type.     

Quantitative characterization of cellular differentiation of Streptomyces ambofaciensin submerged culture by image analysis

Image analysis methods were developed for light and epifluorescence microscopy of Streptomyces ambofaciens undergoing differentiation in submerged culture. Grey level images were obtained with an integration controlled CCD camera and allowed three parameters to be measured: occurence of empty zones in mycelium, number of septations, mycelium thickness.     

Endospore formation by Streptomyces avermitilis in submerged culture

The capability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycles on synthetic medium CP1 with glucose under phosphate limitation, and (2) in autolysing cell suspensions of high density obtained by tenfold concentration of a stationary-phase culture grown in a synthetic medium resuspended in phosphate buffer (pH 7.2) with 0.2% CaCl2. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to pertain to the main species-defining features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in a surface culture.     

Formation and dispersion of mycelial pellets of Streptomyces coelicolor A3(2)

The pellets from a culture of Streptomyces coelicolor A3(2) that were submerged shaken were disintegrated into numerous hyphal fragments by DNase treatment. The pellets were increasingly dispersed by hyaluronidase treatment, and mycelial fragments were easily detached from the pellets. The submerged mycelium grew by forming complexes with calcium phosphate precipitates or kaolin, a soil particle. Therefore, the pellet formation of Streptomyces coelicolor A3(2) can be considered a biofilm formation, including the participation of adhesive extracellular polymers and the insoluble substrates.     

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).     

Cell-bound and extracellular carboxylesterases from Streptomyces: hydrolytic and synthetic activities

AIM: The distribution of cell-bound and extracellular carboxylesterases was investigated among the genus Streptomyces using 420 strains. METHODS AND RESULTS: Primary screening was carried out on solid media using tributyrin, triolein and Tween 60 as current substrates. Eleven representative strains were selected and grown in submerged cultures for evaluating their cell-bound and extracellular hydrolytic activity independently on various naphthyl and aliphatic esters. The best lipolytic strain was lyophilized and used as dry mycelium for catalysing the synthesis of various aliphatic esters in heptane, with molar conversions ranging from 28 to 78% after 3 days. CONCLUSIONS: Carboxylesterase activities can easily be found among the Streptomyces, often being cell-bound and also employable for catalysing esterification in organic solvent. SIGNIFICANCE AND IMPACT OF THE STUDY: A wide screening among Streptomyces, a genus poorly studied for the production of carboxylesterases, has allowed the selection of several strains with interesting enzymatic activities to be used in commercially valuable biotransformation.     

Monoclonal Antibody to Single-Stranded DNA Is a Specific and Sensitive Cellular Marker of Apoptosis

The most widely used histochemical marker of apoptosis (in situend labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis.     

Regulation of biosynthesis of secondary metabolites

The biosynthetic activity of mutants with altered morphological and biochemical characteristics, obtained from two strains ofStreptomyces aureofaciens by means of physical and chemical mutagens, was studied. The majority of mutants formed pigments different from pigments of the parent strains, which did not usually give fluorescence in UV-light and, in addition, differed as to the solubility in organic solvents. The production of further secondary metabolites was investigated chromatographically in the extracts from mycelium and in the fermentation fluid of submerged cultures. According to the results of chromatographical analysis, the obtained mutants can be divided into 12 metabolic types. In most of them the production of substances was found which are different both mutually and from the metabolites of parent strains in physical and chemical properties. A direct correlation was observed between the character of colonies and biosynthetic properties of mutants.     

Enhanced Production of Ganoderic Acids and Cytotoxicity of Ganoderma lucidum Using Solid-Medium Culture

Submerged cultures of Ganoderma lucidum are used to produce fungal mycelium, which is used as a functional food and in the production of various triterpenoids, including ganoderic acids (GAs). Specific culture approaches that produce fungal mycelium with high levels of GAs and good biological activity are critical in the functional food industry. In this study, a solid-medium culture approach to producing mycelium was compared to the submerged culture system. Production of GAs, biomass, intracellular polysaccharides, and cytotoxicity of the cultured mycelium were compared as between solid and submerged culture. Growing G. lucidum strains on solid potato dextrose agar medium increased biomass, the production of ganoderic acid 24 (lanosta-7,9(11), 24-trien-3α-o1-26-oic acid), GAs, and total intracellular polysaccharides as compared to fungi grown in submerged culture. Triterpenoid-enriched methanol extracts of mycelium from solid-medium culture showed higher cytotoxicity than those from submerged culture. The IC(50) values of methanol extracts from solid-medium culture were 11.5, 8.6, and 9.9 times less than submerged culture on human lung cancer cells CH27, melanoma cells M21, and oral cancer cells HSC-3 respectively. The squalene synthase and lanosterol synthase coding genes had higher expression on the culture of solid potato dextrose medium. This is the first report that solid-medium culture is able to increase GA production significantly as compared to submerged culture and, in the process, produces much higher biological activity. This indicates that it may be possible to enhance the production of GAs by implementing mycelium culture on solid medium.     

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 novel Streptomyces gene, samR, with different effects on differentiation of Streptomyces ansochromogenes and Streptomyces coelicolor

A 1.4-kb DNA fragment from Streptomyces ansochromogenes accelerated mycelium formation of S. ansochromogenes when present on a multicopy plasmid. The DNA fragment contains one complete open reading frame, designated samR, encoding a protein with 213 amino acids that contains a likely DNA-binding helix-turn-helix motif close to its N-terminus. The deduced SamR protein resembles the product of the hppR gene, which is involved in the regulation of catabolism of 3-(3-hydroxyphenyl) propionate in Rhodococcus globerulus. A samR disruption mutant was constructed that presented a bald phenotype and failed to form aerial hyphae and spores. We suggest that samR plays an important role in the emergence of aerial hyphae from substrate mycelium. An almost identical gene of Streptomyces coelicolor was also subjected to gene disruption. Surprisingly, the mutant was able to develop an aerial mycelium, but it remained white and deficient in sporulation instead of forming gray spores.     

Gene transfer between streptomycetes in soil.

The growth and activity of Streptomyces violaceolatus and Streptomyces lividans was studied in soil under controlled conditions. The life cycle was followed under differing nutrient regimes and the fate of plasmid- and phage-borne genes determined by direct and indirect techniques. Methods were developed for the direct monitoring of plasmid DNA extracted from soil which allowed differentiation of the cellular location of plasmid DNA between mycelium and spores. In a dynamic, nutrient-fed soil microcosm, inoculants survived poorly, but a specific stage was defined by direct and indirect methods when the inoculants were most active and this correlated with the detection of gene transfer events. Plasmid transfer, phage infection and lysogeny only occurred to a significant extent within this stage at days 15-17 during a 60-day incubation. Estimates based on plasmid DNA recovery indicated that viable counts underestimated spore and mycelial propagules by a factor of greater than 100.     

A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin

A relaxed (rel) mutant was found among 70 thiopeptin-resistant isolates of Streptomyces coelicolor A3(2) which arose spontaneously. The ability of the rel mutant to accumulate ppGpp during Casamino acid deprivation was reduced 10-fold compared to the wild-type. Analysis of the ribosomal proteins by two-dimensional PAGE revealed that the mutant lacked a ribosomal protein, tentatively designated ST-L11. It was therefore classified as a relC mutant. The mutant was defective in producing A-factor and the pigmented antibiotic prodigiosin, in both liquid and agar cultures, but produced agarase normally. Production of actinorhodin, another pigmented antibiotic, was also abnormal; it appeared suddenly in agar cultures after 10 d incubation. Although aerial mycelium still formed, its appearance was markedly delayed. Whereas liquid cultures of the parent strain accumulated ppGpp, agar cultures accumulated only trace amounts. Instead, a substance characterized only as an unidentified HPLC peak accumulated intracellularly in the late growth phase, just before aerial mycelium formation and antibiotic production. This substance did not accumulate in mutant cells. It was found in S. lividans 66 and S. parvulus, but not in seven other Streptomyces species tested. The significance of these observations, and the relationship of the mutant to earlier rel isolates of Streptomyces is discussed.     

复合诱变选育绿色产色链霉菌抑菌高活性菌株

以一株绿色产色链霉菌xzte06菌株为出发菌株,依次通过软X射线、低能离子束诱变、紫外线照射和微波诱变手段对菌株进行复合诱变,得到一株对产气荚膜梭状芽胞杆菌具有较强抑制活性的菌株W26菌株,且该菌株的遗传特性稳定。对菌株进行抑菌试验发现,该菌株液体深层发酵菌丝体的内酮提取物的抑菌活性与出发菌株相比提高了101.42%。