Growth Of Streptomycetes Research Articles

Влияние источников азота в питательной среде на рост и целлюлазную активность стрептомицетов

Biomass accumulation and cellulase activity were determined in two promising soil isolates during liquid phase cultiva-tion depending on organic (peptone, casein hydrolysate) and mineral (NH4Cl, NaNO3, NH4NO3, (NH4)2SO4, KNO3) nitrogen sources. The only source of carbon in the medium was straw (1ob.%). Using two-factor analysis of variance, it was found that the production of cellulase complex enzymes is equally influenced by the nitrogen source (F= 225,92; p<0,0001), the producer strain (F=205,98; p<0,0001), the interaction of these factors (F=225,06; p< 0,0001). Inorganic nitrogen sources were the most preferred for cellulase production: NH4NO3 provided enzymatic activity in Streptomyces sp. Mb 4-2 is 912,82±60,40 units/10 min /g, and NH4Cl in Streptomyces sp. 1.3 NH4Cl is 531,37±27,25 units/10 min /g, which is 58–481 % and 34,5–409% more, respectively, than on other tested nitrogen sources. The accumulation of biomass during growth was more dependent on the nitrogen source (F= 65,47; p<0,0001) than on the strain of streptomycetes (F=18,65; p<0,0001). The interaction of these factors did not have a significant effect on the accumulation of biomass (F=1,11; p=0,38). Peptone and casein hyd-rolysate became the best nitrogen source for the growth of streptomycetes, providing an average of 19 % more biomass than inorganic nitrogen sources. It is shown that the optimization of the nutrient medium by the nitrogen source makes it possible to regulate the growth rates of streptomycetes-producing cultures and achieve maximum cellulase yield. The results obtained are of interest for the development of new environmentally friendly technological processes using streptomycetes in the production of cellulase complex enzymes and in the destruction of cellulose-containing agricultural waste.

Dynamics of Pellet Fragmentation and Aggregation in Liquid-Grown Cultures of Streptomyces lividans.

Streptomycetes are extensively used for the production of valuable products, including various antibiotics and industrial enzymes. The preferred way to grow these bacteria in industrial settings is in large-scale fermenters. Growth of streptomycetes under these conditions is characterized by the formation of complex mycelial particles, called pellets. While the process of pellet formation is well characterized, little is known about their disintegration. Here, we use a qualitative and quantitative approach to show that pellet fragmentation in Streptomyces lividans is initiated when cultures enter the stationary phase, which coincides with a remarkable change in pellet architecture. Unlike young pellets, aging pellets have a less dense appearance and are characterized by the appearance of filaments protruding from their outer edges. These morphological changes are accompanied by a dramatic increase in the number of mycelial fragments in the culture broth. In the presence of fresh nutrients, these fragments are able to aggregate with other small fragments, but not with disintegrating pellets, to form new mycelial particles. Altogether, our work indicates that fragmentation might represent an escape mechanism from the environmental stress caused by nutrient scarcity, with striking similarities to the disassembly of bacterial biofilms.

Aggregation of germlings is a major contributing factor towards mycelial heterogeneity of Streptomyces.

Streptomycetes are filamentous bacteria that produce numerous valuable compounds, including the majority of clinically used antibiotics. At an industrial scale, most of these compounds are produced in bioreactors. Growth of streptomycetes under these conditions is characterized by the formation of complex mycelial particles, whose sizes follow a bimodal distribution. Given the correlation between specific productivity and morphology, this size heterogeneity poses a potential drawback in industry. Recent work indicates that mycelial morphology is controlled by a number of genes that encode proteins required for the synthesis of cell surface-associated glycans. Using a quantifiable system based on fluorescent markers, we here show that these glycans mediate aggregation between germlings and young mycelia, yielding mycelial particles that originate from many different individuals. We also demonstrate that at later time points aggregation between distinct particles is no longer detectable. Notably, the absence of the corresponding glycan synthases yields mycelia that are homogeneous in size, identifying mycelial aggregation as a driving factor towards size heterogeneity. Given that aggregation is widespread within streptomycetes and can also occur between different Streptomyces strains, our work paves the way to improve Streptomyces as a cell factory for the production of known metabolites, but possibly also to discover new ones.

Effect of distant interactions on growth and development of streptomycetes

Regulation of streptomycete growth and development by distant interactions of physical nature was shown using a vial-in-vial experimental setup, providing physical isolation of the inducer and detector cultures. Some effects of distant interaction were observed with Streptomyces netropsis proliferating submerged culture as an inducer and a surface culture of the same strain as a detector. The growth rate of the detector strain doubled. Spore germination was stimulated, as was indicated by a 30% increase in the number of colony-forming units (CRU) in the detector culture plated as a spore suspension. The phase variation spectrum also changed, with an increased share of the colonies of the morphotype predominant under the standard conditions.

Growth of streptomycetes in soil and their impact on bioremediation

The impact of the extremely heavy metal resistant actinomycete Streptomyces mirabilis P16B-1 on heavy metal mobilization/stabilization, phytoremediation and stress level of plants was analyzed in the presence and absence of Sorghum bicolor in sterile microcosms containing highly metal contaminated or control soil. For control, a metal sensitive S. lividans TK24 was used. The metal contents with respect to the mobile and specifically adsorbed fractions of the contaminated soil were considerably decreased by addition of both, living and dead biomass of the strains, with the heavy metal resistant S. mirabilis P16B-1 showing considerably higher impact. Both strains could grow in control soil, while only S. mirabilis P16B-1 formed new tip growth in the metal contaminated soil. A plant growth promoting effect was visible for S. mirabilis P16B-1 in contaminated soil enhancing the dry weight of inoculated Sorghum plants. Thus, metal resistant strains like S. mirabilis P16B-1 are able to enhance phytoremediation of heavy metal contaminated soils.

Exchange of a Conjugative Plasmid At Different Soil Moisture Levels Between Streptomyces Species Colonizing Artificial Soil Aggregates

Spores of a Streptomyces lividans strain (donor) bearing the recombinant conjugative plasmid pIJ303, which codes for thiostrepton resistance, and spores of a plasmid-free Streptomyces parvulus strain (recipient) were added together to finelysieved, sterile silt loam soil. This bulk soil was adjusted to either 20%, 35% or 45% of the soil’s water holding capacity. To this inoculated bulk soil were added sterile, nutrientamended, artificial soil aggregates. After incubation to allow streptomycete growth and aggregate colonization, both the sieved bulk soil and the aggregates were assayed for numbers of transconjugants by spreadplanting on a thiostrepton – agar selective medium. This allowed estimation of parental ability to colonize nutrient – rich soil sites at different soil moisture levels, and comparison of plasmid exchange frequency in the nonamended bulk soil versus the nutrient – rich soil aggregates. Plasmid exchange was detected only on aggregates of about 20% water holding capacity or less (4.2% wt/wt H20). The soil moisture threshold for heterotrophic streptomycete activity appeared to lie between 2.87% and 4.2% wt/wt H20 for the nutrientamended soil aggregates.

Dynamics of spore germination and mycelial growth of streptomycetes under low humidity conditions

At extremely low values of moisture pressure (−96.4 MPa; aw 0.50), the spores of xerotolerant streptomycetes (Streptomyces odorifer and S. rubiginosohelvolus) germinated, their germ lengths increased, and lateral branching of the mycelium was observed after 5 days of incubation in a thin layer of agarized nutrient medium. At −22.6 MPa (aw 0.86), the mycelium begins to branch after a 2-day incubation; over a 5-day incubation at −2.8 MPa (aw 0.98), it goes through a reproduction cycle, which culminates in spore formation. The mathematical model approach enabled us to elucidate the behavioral patterns of Streptomyces spores in a thin layer of agarized nutrient medium at low humidity levels. The dynamics of spore germination is governed by the exponential law, which allows calculation of the average duration of the period a before spore germination, as well as the time needed for 50% of viable spores to germinate.

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 approximately 20 microm 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.

Characterization of the sporulation control protein SsgA by use of an efficient method to create and screen random mutant libraries in streptomycetes.

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function.

Spore Germination and Mycelial Growth of Streptomycetes at Different Humidity Levels

This study is the first to show the ability of streptomycetes to develop at a very low humidity level. All of the streptomycetes studied produced growth at low humidity (aw 0.86 and 0.67). This capacity was most markedly pronounced in Streptomyces odorifer, whose spores were capable of germinating, and mycelial germs increased in length, at the air humidity aw 0.50. The formation of lateral branches (mycelium branching) at this humidity was noted only in single S. odorifer germs and only after 72 h of incubation. Study of streptomycete growth on an agarized medium with different osmotic pressures, created by various glycerol concentrations in the medium, showed that, at aw 0.67, the spores of all the streptomycetes studied germinate, producing mycelial germs but not microcolonies. The ecological significance of mycelial prokaryotes in soil microbial communities that develop and function under conditions of extremely low humidity is discussed.

Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms.

The growth and activity of introduced (S. lividans TK24 pIJ673 and S. lividans TK23) and indigenous (S. griseus CAG17) streptomycete strains in soil was studied, under controlled conditions. The effect of environmental parameters such as temperature, soil water content and nutrient availability on the growth and activity of these strains, was studied using a highly dynamic fed-batch soil microcosm system. Using this new system, repeated cycles of active streptomycete growth were achieved, allowing long-term investigation of metabolic activity, plasmid stability and conjugative plasmid transfer. In long-term experiments, respiration rates and enzyme activity patterns matched the pattern of germination/sporulation cycles of the inoculants. In situ hybridisation, using fluorescently labelled oligonucleotides, also proved the presence of metabolically active streptomycete mycelia in sterile soil. Plasmid stability under varying temperatures and selective pressure was studied using the above system. In both sterile and non sterile amended antibiotic containing soil, no intraspecific transfer of plasmid pIJ673 from S. lividans TK24 to S. griseus CAG17 was detected. The soil microcosm system used, though, permitted detection of intraspecific conjugative transfer of this plasmid from S. lividans TK24 to S. lividans TK23 in soil.

Isolation and Sequencing of the rho Gene from Streptomyces lividans ZX7 and Characterization of the RNA-dependent NTPase Activity of the Overexpressed Protein

The gene for transcription termination factor Rho was isolated from Streptomyces lividans ZX7. It encoded a 77-kDa polypeptide (Rho 77) with considerable homology to known Rho factors. An atypical hydrophilic region of 228 residues was found within the N-terminal RNA-binding domain. Only Rho from Micrococcus luteus and Mycobacterium leprae (closely related GC-rich Gram-positive bacteria) had an analogous sequence. Rho 77 was overexpressed in Escherichia coli and purified using an N-terminal hexahistidine-tag. Rho 77 displayed a broad RNA-dependent ATPase activity, with poly(C) RNA being no more than 4-fold more effective than poly(A). This contrasts with the ATPase activity of Rho from E. coli which is stimulated primarily by poly(C) RNA. Rho 77 was a general RNA-dependent NTPase, apparent Km values for NTPs were: GTP 0.13 mM, ATP 0.17 mM, UTP 1.1 mM, and CTP >2 mM. Rho 77 poly(C)-dependent ATPase activity was inhibited by heparin, unlike the E. coli Rho. The antibiotic bicyclomycin inhibited the in vitro RNA-dependent ATPase activity of Rho 77, did not inhibit growth of streptomycetes but delayed the development of aerial mycelia. N-terminal deletion analysis to express a truncated form of Rho (Rho 72, 72 kDa) indicated that the first 42 residues of Rho 77 were not essential for RNA-dependent NTPase activity and were not the targets of inhibition by heparin or bicyclomycin.

Screening of compounds stimulating spore formation and mycelial growth of pock-forming plasmid-carrying strains in Streptomyces azureus

Sporulation-stimulating compounds were screened by using mutant PK100a of Streptomyces azureus ATCC 14921, in which spore formation was markedly inhibited by the pock-forming plasmid pSA1.1. On agar media, cysteine, bacitracin, glutathione and β-NAD induced the formation of coloured spores or spore mass of strain PK100a. These compounds also stimulated the spore formation of the wild-type and its plasmid-cured (good spore-forming) strains and the growth of aerial mycelia of these three strains. This screening method appears to be an effective method for the screening of substances that stimulate spore formation and mycelial growth of streptomycetes or that overcome the inhibitory effect of pock-forming plasmids.

Effects of potential plant CS 2 emissions on bacterial growth in the rhizosphere

Carbon disulfide (CS 2) is a volatile, flammable, foul-smelling liquid with bacteriostatic, fungicidal, nematicidal and insecticidal properties. Some plants emit CS 2. The present study was designed to determine if CS 2 emissions from Mimosa pudica could inhibit the rhizosphere growth of six different bacterial species. Albizia julibrissin, a plant that does not emit CS 2 and is closely related to M . pudica, was used as a negative control. Two pseudomonads, streptomycetes and rhizobia were each inoculated at a concentration of 10 5 colony-forming units (CFU) plant −1 into assemblies with and without a gnotobiotically grown M. pudica or A. julibrissin plant. Roots of M. pudica and A. julibrissin had a variable effect on bacterial growth. Neither plant inhibited pseudomonad nor rhizobial growth on the rhizoplane or in the rhizosphere, but both plants inhibited streptomycete growth on the rhizoplane and in the rhizosphere. However, the inhibition of streptomycete growth was temporary and after 7 days, little difference in streptomycete numbers was observed between assemblies with a plant and assemblies without a plant but with an added carbon source. Similar results were observed in a competition study between a rhizobium and a streptomycete on the rhizoplane and in the rhizosphere of M. pudica except the streptomycete counts did not recover to the same extent after 7 days. In CS 2 studies, the growth of all six bacterial species was inhibited by 155 ng CS 2 ml −1, and with two exceptions, to a lesser extent by 1.9 ng CS 2 ml −1. It is unlikely that CS 2 inhibited streptomycete growth because the streptomycetes were inhibited by A. julibrissin (which does not emit CS 2), and little relationship was observed between the inhibition of streptomycete growth in assemblies with M. pudica plants and in assemblies with CS 2. It is likely that other compounds on the rhizoplane and in the rhizosphere of M. pudica and A. julibrissin are responsible for the inhibition of streptomycete growth.

Distribution of actinomycetes in near-shore tropical marine sediments

Actinomycetes were isolated from near-shore marine sediments collected at 15 island locations throughout the Bahamas. A total of 289 actinomycete colonies were observed, and all but 6 could be assigned to the suprageneric groups actinoplanetes and streptomycetes. A bimodal distribution in the actinomycete population in relation to depth was recorded, with the maximum numbers occurring in the shallow and deep sampling sites. This distribution can be accounted for by a rapid decrease in streptomycetes and an increase in actinoplanetes with increasing depth and does not conform to the theory that actinomycetes isolated from marine sources are of terrestrial origin. Sixty-three of the isolated actinomycetes were tested for the effects of seawater on growth. Streptomycete growth in nonsaline media was reduced by 39% compared with that in seawater. The actinoplanetes had a near obligate requirement of seawater for growth, and this is presented as evidence that actinomycetes can be physiologically active in the marine environment. Problems encountered with the enumeration of actinomycetes in marine sediments are also discussed.

Study of the early growth and branching of Streptomyces tendae by means of an image processing system

A method is described for directly observing submerged growth of streptomycetes, which can be easily adapted for investigations of filamentously growing fungi. The use of an image processing system allowed a fast and accurate calculation of important morphological parameters. Most of the images can be analysed on-line. Some results are presented for early growth and branching of Streptomyces tendae. It seems that mechanisms for growth of streptomycetes were the same for both solid and liquid media.

Potential sites of geosmin production by streptomycetes in and around reservoirs

Water from several oligotrophic reservoirs of the North West Water Authority at Longridge, Lancashire, supported only sparse growth of streptomycetes isolated from the reservoirs. Growth was enhanced by nutrient‐amendment of the water and one common isolate, identified as Streptomyces albidoflavus, produced geosmin in water which was supplemented with sufficient concentrations of available carbon, nitrogen and phosphorus. The minimum concentrations required for geosmin production were higher than those recorded in natural reservoir water. Synthesis of geosmin in water also required a minimum temperature of about 15dEC. Sterilized samples of reservoir sediment, sediment extract, plant debris from banks and surrounding soil also supported geosmin production by S. albidoflavus. Both bank debris and exposed sediment developed earthy odours without inoculation. The structure of reservoir banks influenced the quantity of plant growth and accumulation of litter, and therefore also the potential for contamination of reservoir water by geosmin. The potential of these various sites of geosmin production to contaminate reservoir water is assessed.

Effect of osmotic potential on streptomycete growth, antibiotic production and antagonism to fungi

The growth of eighteen isolates of Streptomyces spp. on agars, the osmotic potentials of which were controlled by the addition of various solutes, showed that nine isolates were able to grow at −100 bars and two of these grew at −150 bars. This suggests that the water requirements of soil streptomycetes are almost as diverse as those of soil fungi. In general, linear growth and sporulation declined with decreasing osmotic potential, but one isolate grew optimally at potentials between −5 and −10 bars. Another isolate formed maximum amounts of a water-diffusible antibiotic per unit colony area at potentials between −20 and −35 bars. This appears to be the first report of osmotic potential influencing the production of antibiotic by Streptomyces. The increased antibiotic production at moderate potentials was associated with inhibition of Pythium ultimum, Rhizoctonia solani and a Curvularia sp. by the streptomycete at these potentials on osmotic agar plates. The possible ecological significance of this phenomenon in soil is discussed.

Studies on the ecology of actinomycetes in soil IV. Observations on the form and growth of streptomycetes in soil

The behaviour of streptomycetes in soil was studied by electron microscopy, fluorescence microscopy, soil stripping and maceration techniques. Results were used to build up a picture of the behaviour of these organisms in natural soil. All results obtained supported the suggestion that streptomycetes exist in soil for much of the time as spores. Periods of active growth are discontinuous in space and time, making their detection by any method difficult.

Morphology of submerged growth of streptomycetes as a taxonomic aid. I. Morphological development of Streptomyces aureofaciens in agitated liquid media.

In a study of the submerged growth morphology of 145 different streptomycetes in A C Broth, the species Streptomyces aureofaciens was observed to represent an unusual type. It was characterized by profuse mycelial segmentation, transformation of disarticulated fragments of segmented mycelium into chains of thickened arthrospores, and formation of filiform appendages on the arthrosporic cells giving an insectival appearance to these elements. This unusual morphology was determined to be a consistent feature in 110 strains of S. aureofaciens studied, hence suggesting a useful taxonomic aid in identification of this species. Preliminary studies of multiple strains of many different Streptomyces species indicate that other useful taxonomic features based on morphology of submerged growth are found in a significantly large part of the genus.