Aerial Mycelium Formation Research Articles

Sporulation without aerial mycelium formation on agar medium by Streptomyces bikiniensis HH1, an A-factor-deficient mutant

Streptomyces bikiniensis HH1, an A-factor-deficient mutant that did not form aerial mycelium on agar medium unless supplemented with A-factor, produced spores abundantly within colonies. The spores formed on reproductive branches morphologically similar to aerial hyphae except that they did not emerge from the surface of the colonies. The spores were morphologically more heterogeneous than those formed when A-factor was added.

Genetic Studies into Musty Odor Production by Actinomycetes

Musty taste and odor is a serious problem for drinking water. Much data regarding its occurrence are being accumulated. However, we may be able to solve this problem by inquiring into the nature of secondary metabolism through the mevalonic acid pathway in actinomycetes and/or cyanobacteria that yield musty smelling compounds. Therefore, genetic analysis is applied to look for new solutions. By investigating Streptomyces sp. with the protocol to recover plasmid DNA, it was difficult to get the infinitesimally small covalently closed circular (ccc) plasmid. On the other hand, Streptomyces strains which have been cured, which implicates plasmids, lost phenotypic characters such as musty odor production with aerial mycelium and pigment formation. DNA in Streptomyces are unstable and often cause amplification and deletion. The existence of giant linear plasmids was detected in musty smelling strains using pulsed – field gel electrophoresis.

Additional copies of the actII regulatory gene induce actinorhodin production in pleiotropic bld mutants of Streptomyces coelicolor A3(2)

In S. coelicolor there are mutants (named bld) which fail to form aerial mycelium and which are also pleiotropically blocked for synthesis of several antibiotics, including the blue compound actinorhodin. Using a high copy-number vector, a DNA fragment was cloned which restored actinorhodin production, but not aerial mycelium formation, to bldA, D, F, G and H mutants. Subcloning and Southern blotting revealed that the fragment was from the region (actII) that regulates actinorhodin biosynthesis. The ability to elicit actinorhodin production and to complement an actII mutant was localized to a 1·3 kb fragment. These results indicate that the structural genes for actinorhodin biosynthetic enzymes do not contain targets for the bldA, D, F, G and H gene products, and suggest that the corresponding bld genes control actinorhodin synthesis principally via a single gene in the actII regulatory region.

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.

Characterization of a family of multimeric ccc molecules of amplified chromosomal DNA in Streptomyces ambofaciens DSM 40697

A spontaneous mutant strain of Streptomyces ambofaciens DSM 40697, a producer of spiramycin, defective in pigment and aerial mycelium formation was isolated. The DNA analysis revealed the presence of an amplified DNA sequence (ADS). This ADS was characterized, localized on the chromosome within the AUD6 region and a rearrangement on one side of the ADS was detected. Experiments led to the detection and characterization of an extrachromosomal multimeric ccc (covalently closed and circular) DNA molecule family. The monomeric form presented a size similar to the previous ADS and all multimeric forms were homologous with it.

Characterization of a family of multimeric ccc molecules of amplified chromosomal DNA inStreptomyces ambofaciensDSM 40697

A spontaneous mutant strain of Streptomyces ambofaciens DSM 40697, a producer of spiramycin, defective in pigment and aerial mycelium formation was isolated. The DNA analysis revealed the presence of an amplified DNA sequence (ADS). This ADS was characterized, localized on the chromosome within the AUD6 region and a rearrangement on one side of the ADS was detected. Experiments led to the detection and characterization of an extrachromosomal multimeric ccc (covalently closed and circular) DNA molecule family. The monomeric form presented a size similar to the previous ADS and all multimeric forms were homologous with it.

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.

Butalactin, a new butanolide antibiotic. Taxonomy, fermentation, isolation and biological activity.

Butalactin, [2-(4',5'-epoxy-hex-2'(E)-en)oyl-2-hydroxy-3-hydroxymethyl-2, 3-(Z)-butanolide] is a new antibiotic produced by Streptomyces sp. HIL Y-86,36923. Taxonomically, the producing organism most closely resembles Streptomyces corchorusii. The strain also produces cineromycin B. Though butalactin is structurally related to 'signal molecules' such as A-factor, the anthracycline inducing factors and the virginiae butanolides, it does not show inducing activity for antibiotic production or aerial mycelium formation in the indicator strain. Butalactin possesses a weak antibiotic activity against Gram-positive and Gram-negative bacteria.

“Definitive senescence” in stock cultures of basidiomycetous wood-decay fungi

For 7 cultures of basidiomycetous wood-decay fungi the decline of the following vitality marks were recorded over a 6-year period: (i) Consumption of sterilized beech wood substrate within a time unit as an indicator of the overall vigorousness of the fungus, (ii) decline of the competitive saprophytic ability (kratovirulence), which enables the fungus to colonize natural nonsterile substrates, (iii) capacity for sexual reproduction, (iv) several morphological features of the fungal colony. The reference strains kept growing at + 1 … 2°C on fertilized wood dust substrate retained in 5 of 7 cases all of their juvenility marks, although the wood-decay capacity gradually dropped back to 65 to 34% that of the initial value. The substrains maintained on liquid still cultures at 23°C lost their vitality marks after a period of continuous decline rather abruptly. With the loss of kratovirulence and fruiting capacity the strains were no longer viable under field conditions, and with the drop of the sterile wood-decay capacity to less than 3% by dry weight the formation of aerial mycelium and melanin pigments virtually ceased. In several fungal species the colonies segregated into coexisting sectors of extreme senescence and comparative juvenility, and a spread of senescence to the juvenile sectors did not occur. It is concluded that stock cultures of wood-decay fungi are subject to progressive senescence even under optimum conditions of strain preservation. Their current vitality should be examined in a sterile wood decay test before they are used in biotechnology or research.

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.

The level of a transcript required for production of a Streptomyces coelicolor antibiotic is conditionally dependent on a tRNA gene

In Streptomyces coelicolor A3(2), bldA mutants are conditionally defective in aerial mycelium formation and fail to synthesize all four antibiotics produced by bldA+ strains. Previous studies showed that bldA specifies the tRNA for the rarely used leucine codon UUA. Here we describe experiments examining the abundance in a bldA mutant of a transcript involved in antibiotic production. With use of a bacteriophage-based integrative vector, a promotorless xylE reporter gene was inserted into a previously undescribed gene for an early step in biosynthesis of the red antibiotic undecylprodigiosin, located in the red gene cluster. With this transcriptional fusion present at unit copy number in the chromosome, xylE expression in a bldA+ strain was maximal late in growth in a liquid production medium and was virtually absent in a bldA mutant. On plates of a different medium, the bldA mutant was able to produce undecylprodigiosin and to express the red::xylE fusion, but both abilities were repressed by increasing the concentration of phosphate in the medium. These experiments showed that the undecylprodigiosin deficiency of bldA mutants cannot be accounted for by the presence of TTA codons in the red structural genes, but rather that bldA influences red gene mRNA abundance. In low-phosphate conditions, an alternative regulatory pathway can lead to red gene expression.

DNA amplification in Streptomyces achromogenes subsp. rubradiris is accompanied by a deletion, and the amplified sequences are conditionally stable and can be eliminated by two pathways.

Southern blot analysis of BglII-digested DNA isolated from wild-type Streptomyces achromogenes, which harbors the 8.8-kilobase amplifiable unit of DNA, AUD-Sar 1, and of similarly digested DNA from 12 strains carrying an array of 200 to 300 tandem copies of a specific AUD-Sar 1-derived 8.0-kilobase DNA sequence, ADS-Sar 1, revealed the absence of the 12.4-kilobase BglII AUD-Sar 1-chromosome right junction band in the latter strains, whereas the corresponding 26.0-kilobase left junction band remained unaltered. Further Southern analyses indicated in all of the seven amplified strains tested the occurrence of a deletion of at least 10 kilobases of the DNA adjacent to the right side of the AUD. The deletion has one endpoint in the vicinity of the ADS array. Corroborating and expanding upon previously reported results, we found that the amplified DNA of strain C010 was stably maintained for at least 20 transfers when the transfers involved mycelia propagated in spectinomycin-free liquid medium. In contrast, when strain C010 was subjected separately to one cycle of protoplast formation and regeneration or to three cycles of spore germination, aerial mycelium formation, and sporulation on spectinomycin-free media, only approximately 20% of the protoplast regenerants and spores retained the reiterated DNA sequences and the ability subsequently to form colonies on media containing high levels of spectinomycin. Approximately 80% of these units completely deleted the reiterated DNA and left adjacent sequences and exhibited sensitivity to 25 micrograms of spectinomycin per ml. One among 24 protoplast-derived deletants apparently retained the left portion of the AUD-ADS left direct repeat plus left adjacent sequences.

Relationship between aeration, carbon source, and respiration yield and the ethylene production and differentiation of static cultures of Penicillium cyclopium and P. velutinum

Fully aerated surface cultures of Penicillium cyclopium and P. velutinum grown on liquid medium with 2-oxoglutarate or glutamate (34 mM) produced ethylene in association with the formation of aerial hyphae at about the 5th day (ethylene peak EP 1), its concentration then declined, and rose again during conidiation at about the 7th day (peak EP 2). In P. cyclopium glucose (22.2 mM) shortened differentiation, EP 1 and EP 2 merged, and culture pH dropped to 1.8. Methionine (34 mM) prevented the formation of aerial mycelium in P. cyclopium and penicilli initiation in P. velutinum; ethylene production was low. Under conditions of delayed aeration, P. cyclopium grown on glucose produced no ethylene and weak formation of immature conidia was observed. On other substrates both conidiation and ethylene production occurred after reaeration, but phase EP 1 was lacking. Penicillium velutinum grown on 2-oxoglutarate and glutamate with delayed aeration developed immature conidia within 36 h and traces of ethylene were produced. In the cultures with methionine and glucose, conidiation and ethylene production occurred after reaeration, but phase EP 1 was missing. In both strains, no ethylene was formed under anaerobic conditions. Intensively producing cultures were characterized by a narrow range of respiration yields (6.8–7.5 g dry weight per mole of consumed O2). A prerequisite for high ethylene production was a drop in the phosphate content of the medium to less than 0.45 mM.Key words: Penicillium cyclopium, Penicillium velutinum, ethylene, conidiation.

Calcium ion regulates aerial mycelium formation in actinomycetes.

Ca2+ induced the formation of aerial mycelia in Streptomyces ambofaciens. Its effect was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, a Ca2+ specific chelating agent. A survey of 36 strains of actinomycetes showed that Ca2+ regulated aerial mycelium formation in 21 (58%) of them. The Ca2+ concentration required for aerial mycelium formation in the culture medium ranged from 0.1 to 1.5 mM.

New products related to kinamycin from Streptomyces murayamaensis. I. Taxonomy, production, isolation and biological properties.

Six new products of Streptomyces murayamaensis sp. nov. Hata et Ohtani, the producer of the kinamycins, were isolated by silica gel column chromatography. The antibacterial activities of the new products, as well as that of dehydrorabelomycin and murayaquinone, previously isolated products of the same organism, were compared to the kinamycins. Three of the products had antibacterial activities similar to the kinamycins, while two others had activity only against Gram-positive bacteria. Dehydrorabelomycin and one other metabolite had no detectable antibacterial activity. The organism was found to be capable of aerial mycelium formation, with sporophores branched at regular intervals bearing square-ended spores with smooth surfaces. The culture contains L,L-diaminopimelic acid in the cell wall (Type I), is highly resistant to lysozyme, and lecithinase- and melanin-positive, suggesting a relationship with the genus Streptoverticillium and the lavendulae group of the genus Streptomyces.

Promoter determining the timing and spatial localization of transcription of a cloned Streptomyces coelicolor gene encoding a spore-associated polypeptide

Streptomyces coelicolor is a filamentous, gram-positive bacterium that exhibits a complex cycle of morphological differentiation involving the formation of an aerial mycelium of multinucleoid hyphae which undergo septation to form long chains of spores. We report the identification of two proteins of 13 and 3 kilodaltons, designated SapA and SapB, respectively, that are produced during formation of the aerial mycelium and are found in assocation with purified, mature spores. We cloned the structural gene (sapA) for one of these spore-associated proteins. Nucleotide sequence analysis suggests that the 13-kilodalton polypeptide is derived from a larger pre- or preproprotein containing a leader sequence of 37 amino acids. Nuclease protection-hybridization analysis and experiments using the Vibrio harveyi, luciferase-encoding luxAB operon as a gene tag demonstrated that expression of sapA is controlled from a promoter contained within a region of less than 110 base pairs in length, whose transcription start site is located approximately 50 base pairs upstream from the initiation codon for the sapA open reading frame. Transcription of sapA was induced at the time of appearance of the aerial mycelium, and the level of sapA transcripts was significantly reduced in certain mutants blocked in aerial mycelium (bld) and or spore (whi) formation. As further evidence of the association of sapA transcription with morphological differentiation, experiments in which we monitored sapA transcription topographically by use of a sapA-luxAB operon fusion demonstrated a close spatial correlation between colony regions undergoing aerial mycelium formation and zones of sapA-promoted light emission.

New loci required for Streptomyces coelicolor morphological and physiological differentiation.

Streptomyces coelicolor colonies differentiate both morphologically, producing aerial spore chains, and physiologically, producing antibiotics as secondary metabolites. Single mutations, which block both aspects of differentiation, define bld (bald colony) genes. To identify new bld genes, mutagenized colonies were screened for blocks in the earliest stage of sporulation, the formation of aerial mycelia, and blocks in antibiotic synthesis. The mutations in 12 mutants were mapped; in each strain, the pleiotropic phenotype was due to a single mutation. Seven of the strains contained mutations in known bld loci, bldA and bldB. Three strains contained mutations in a new locus, bldG, and two contained mutations in another new locus, bldH. Like the previously defined bldA mutants, the bldG and bldH mutants were developmentally blocked on glucose. On a variety of carbon sources whose utilization was subject to glucose repression, the developmental blocks were partially relieved for bldG (and bldA) mutants and fully relieved for bldH mutants. These results are compatible with an hypothesis which suggests that there are two alternative controls on S. coelicolor differentiation, one of which is glucose repressible.

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).

Isolation and characterization of Streptomyces lividans mutants deficient in intraplasmid recombination.

Plasmid pIF132 containing two direct repeats of the mel (melanin) sequence was used to monitor intraplasmid recombination. Five mutants of Streptomyces lividans TK64 deficient in intraplasmid recombination were isolated. Four contained additional defects in aerial mycelium formation, pigmentation, and nutrient requirements; among these two showed extensive amplification of chromosomal sequences. Mutant JT46 had no pleiotropic defects but had the most severe blockage in recombination. Only one of the mutants was slightly more sensitive to UV and two were slightly more sensitive to mitomycin C. Plasmid pWCL1 (containing pIJ702, pUC12, and HBVsAg sequences; Lee et al. 1986) could not stably replicate in TK64 without spontaneous deletions. In contrast the mutant JT46 maintained the integrity of pWCL1 much more stably.