Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.     

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.     

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.     

Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to A factor.

I investigated the significance of the intracellular accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and of the coordinated decrease in the GTP pool for initiating morphological and physiological differentiation of Streptomyces griseus, a streptomycin-producing strain. In solid cultures, aerial mycelium formation was severely suppressed by the presence of excess nutrients. However, decoyinine, a specific inhibitor of GMP synthetase, enabled the cells to develop aerial mycelia in the suppressed cultures at concentrations which only partially inhibited growth. A factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone) added exogenously had no such effect. Decoyinine was also effective in initiating the formation of submerged spores in liquid culture. The ability to produce streptomycin did not increase but decreased drastically on the addition of decoyinine. This sharp decrease in streptomycin production was accompanied by a decrease in intracellular accumulation of ppGpp. A relaxed (rel) mutant was found among 25 thiopeptin-resistant isolates which developed spontaneously. The rel mutant had a severely reduced ability to accumulate ppGpp during a nutritional shift-down and also during postexponential growth and showed a less extensive decrease in the GTP pool than that in the rel+ parental strain. The rel mutant failed to induce the enzymes amidinotransferase and streptomycin kinase, which are essential for the biosynthesis of streptomycin. The abilities to form aerial mycelia and submerged spores were still retained, but the amounts were less, and for both the onset of development was markedly delayed. The decreased ability to produced submerged spores was largely restored by the addition of decoyinine. This was accompanied by an extensive GTP pool decrease. The rel mutant produced A factor normally, indicating that synthesis of A factor is controlled neither by ppGpp nor by GTP. Conversely, a mutant defective in A-factor synthesis accumulated as much ppGpp as did the parental strain. It was concluded that morphological differentiation of S. griseus results from a decrease in the pool of GTP, whereas physiological differentiation results from a more direct function of the rel gene product (ppGpp). It is also suggested that A factor may render the cell sensitive to receive and respond to the specified signal molecules, presumably ppGpp (for physiological differentiation) or GTP (for morphological differentiation).     

Surface hydrophobicity, viability and efficacy in biological control of Penicillium oxalicum spores produced in aerial and submerged culture

The surface hydrophobicity, viability and biocontrol ability of Penicillium oxalicum spores, produced either in aerial or submerged culture, were characterized. A phase distribution test showed that spores produced in both methods of culture were highly hydrophobic, but those produced in aerial culture were more hydrophobic. Spores stored fresh at either 4 or 25 degrees C retained a high viability (80%) after 27 weeks of storage, although aerial spores survived better. Freeze-drying severely affected viability, especially of submerged spores. Biocontrol ability against Fusarium oxysporum f. sp. lycopersici was studied in the growth chamber. Aerially- produced spores were more effective than submerged ones. Aerially-produced P. oxalicum spores appeared to have more advantages than those produced by submerged culture, in relation to both viability and efficacy. These results demonstrate that physiological changes occur depending on production conditions which significantly influences quality of the biocontrol agent.     

Expression of the chaplin and rodlin hydrophobic sheath proteins in Streptomyces venezuelae is controlled by σ(BldN) and a cognate anti-sigma factor, RsbN

The chaplin and rodlin proteins together constitute the major components of the hydrophobic sheath that coats the aerial hyphae and spores in Streptomyces, and mutants lacking the chaplins are unable to erect aerial hyphae and differentiate on minimal media. We have gained insight into the developmental regulation of the chaplin (chp) and rodlin (rdl) genes by exploiting a new model species, Streptomyces venezuelae, which sporulates in liquid culture. Using microarrays, the chaplin and rodlin genes were found to be highly induced during submerged sporulation in a bldN-dependent manner. Using σ(BldN) ChIP-chip, we show that this dependence arises because the chaplin and rodlin genes are direct biochemical targets of σ(BldN) . sven3186 (here named rsbN for regulator of sigma BldN), the gene lying immediately downstream of bldN, was also identified as a target of σ(BldN) . Disruption of rsbN causes precocious sporulation and biochemical experiments demonstrate that RsbN functions as a σ(BldN) -specific anti-sigma factor.     

Ultrastructure and properties of Paecilomyces lilacinus spores

Strains of the filamentous soil fungus Paecilomyces lilacinus are currently being developed for use as biological control agents against root-knot, cyst, and other plant-parasitic nematodes. The inoculum applied in the field consists mainly of spores. This study was undertaken to examine the size, ultrastructure, and rodlet layers of P. lilacinus spores and the effect of the culture method on structural and functional spore properties. A rodlet layer was identified on aerial spores only. Other differences noted between aerial spores and those produced in submerged culture included the size and appearance of spores and thickness of spore coat layers when examined with transmission electron microscopy. The two spore types differed in UV tolerance, with aerial spores being less sensitive to environmentally relevant UV radiation. Also, viability after drying and storage was better with the aerial spores. Both spore types exhibited similar nematophagous ability.     

Emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168

Abstract The emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168 was examined. Resistance to an organic solvent (toluene), heat and lysozyme were included for comparison. A sequential development of resistance was observed, with toluene resistance occuring early on in sporulation (stages III and IV), thermal resistance at early stage V, lysozyme resistance at middle stage V and glutaraldehyde resistance arising late in stage V. Studies with sporulation mutants also indicate that glutaraldehyde resistance is acquired even later than lysozyme resistance and may therefore possibly be considered as a very late marker event for sporulation, characterizing late stages of B. subtilis 168 spore formation.     

Endospore formation by <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> in submerged culture

The ability 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 cycle on synthetic medium CP1 with glucose under phosphate limitation and (2) in autolysing cell suspensions of high density obtained by tenfold concentration in phosphate buffer (pH 7.2) with 0.2% CaCl₂ of stationary-phase cells grown in synthetic medium. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to retain the main species-specific 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 surface cultures.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 204–214.Original Russian Text Copyright © 2005 by Filippova, Gorbatyuk, Poglazova, Soina, Kuznetsov, El-Registan.     

Starvation-independent sporulation in Myxococcus xanthus involves the pathway for β-lactamase induction and provides a mechanism for competitive cell survival

Myxococcus xanthus is a Gram-negative, soil-dwelling bacterium with a complex life cycle which includes fruiting body formation and sporulation in response to starvation. This developmental process is slow, requiring a minimum of 24–48 h, and requires cells to be at high cell density on a solid surface. It is known that, in the absence of starvation, vegetatively growing cell suspensions can form 'glycerol spores' when exposed to high levels of glycerol, usually 0.5 M. The cells differentiate from rods to resistant spheres rapidly (2–4 h) and synchronously. We have found that the chromosomally encoded β-lactamase of M. xanthus can be induced by numerous β-lactam antibiotics as well as by non-specific inducers including glycine and many D -amino acids. In addition, D -cycloserine, phosphomycin, and hen egg-white lysozyme also induce β-lactamase in this bacterium. Unexpectedly, agents which induce β-lactamase can induce 'glycerol spores'; all of the agents tested which induce glycerol spores (glycerol, DMSO, ethylene glycol) also induce β-lactamase. During the induction of sporulation, β-lactamase activity increases, reaching a peak during the morphological transition from rod-shaped cells to spherical spores. These spores are viable and resistant to many treatments which disrupt vegetatively growing rods but are not as resistant as fruiting body spores. The concomitant induction of β-lactamase and starvation-independent sporulation suggests that these processes share a common signal-transduction pathway. These results also suggest that starvation-independent sporulation may be an adaptation of cells in order to resist agents that damage peptidoglycan structure and therefore threaten cell survival.     

Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes.

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.     

Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

Rapid Identification of Viable Bacterial Spores Using a Fluorescence Method

A method has been devised to differentiate viable and nonviable bacterial spores. “Germination-like” changes are initiated in spores with performic acid and lysozyme. The germinated spores are stained with aqueous acridine orange, a fluorescent dye. Nonviable spores fluoresce lemon-green and viable spores orange-red. It is proposed that with the use of a membrane filter resistant to performic acid and lysozyme, the method may be used for spore enumeration in foods in about 4 hr compared to conventional plating methods, which usually require up to 72 hr.     

Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.     

Hybridization of A-factor deficient mutants of Streptomyces griseus by means of fusion of protoplasts

Characteristics of 6 A-factor deficient mutants of S. griseus are presented. The common feature of the mutants was impairment of sporulation, formation of aerial mycelium and streptomycin synthesis. Pair-by-pair hybridization of the mutants was performed with protoplast fusion followed by regeneration. 9 pair couplings of the mutants were performed. In 3 of them sporulating recombinants were detected. The antibiotic production level in 70 hybrids was different and ranged from 0 to 1700 micrograms/ml. The morphological features of the colonies and the number of the spores formed were also different. The common feature of all the 70 sporulating hybrid strains was recovery of synthesis of A-factor, an endogenic regulator of S. griseus development. Therefore, in the A-factor deficient mutants impairment of A-factor synthesis was induced not by the plasmid elimination, as was suggested, but by mutation of separate genes.     

Sporulation of Streptomyces venezuelae in submerged cultures

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.     

In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation. Received: 17 July 1997 / Accepted: 20 September 1997     

Effects of media composition on submerged culture spores of the entomopathogenic fungus, Metarhizium anisopliae var. acridum, Part 1: Comparison of cell wall characteristics and drying stability among three spore types

Metarhizium anisopliae var. acridum (IMI 330189) can produce at least three spore types in vitro; blastospores, submerged conidia, and aerial conidia, as defined by culturing conditions, sporogenesis, and spore morphology. This study compares morphological characteristics (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), and performance (germination rate and drying stability) among these three spore types. Submerged conidia and aerial conidia both possessed thick, double-layered cell walls, with hydrophobic regions on their surfaces. However, in contrast to aerial conidia, submerged conidia have: (1) a greater affinity for the lectin concanavalin-A; (2) more anionic net surface charge; and (3) a less distinct outer rodlet layer. Blastospores were longer and more variable in length than both submerged conidia and aerial conidia, and had thinner single-layered cell walls that lacked an outer rodlet layer. Also, blastospores had a greater affinity than either conidia type for the lectin, wheat germ agglutinin. Blastaspores lacked hydrophobic regions on their surface, and had a lower anionic net surface charge than submerged conidia. In culture, blastospores germinated the fastest followed by submerged conidia, and then aerial conidia. Survival of submerged conidia and aerial conidia were similar after drying on silica gel, and was greater than that for blastospores. We provide corroborating information for differentiating spore types previously based on method of production, sporogenesis, and appearance of spores. These physical characteristics may have practical application for predicting spore-performance characteristics relevant to production and efficacy of mycoinsecticides.