Aerial Mycelium Formation Research Articles

Streptomyces Species as Boundary Microorganisms: Eucaryotic Regulatory Systems for Secondary Metabolism and Morphological Differentiation.

The genus Streptomyces is characterized by its ability to produce a wide variety of secondary metabolites and complex morphological differentiation resembling that of fungi. The regulation controlling these two characteristic aspects include “eucaryotic” systems, such as a hormonal control represented by the A-factor regulatory system and protein phosphorylation represented by the afsK/afsR system. A-factor is a chemical signaling molecule that triggers both secondary metabolite formation and aerial mycelium formation in Streptomyces griseus. Characterization of the A-factor-specific receptor gene suggests that the receptor acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early stage of growth and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The A-factor signal is then transferred, via one or more steps, to a transcriptional activator for the strR gene, a transcriptional activator for the whole streptomycin biosynthetic genes. Involvement of protein serine/threonine kinases in signal transduction leading to antibiotic production and morphogenesis, either as a member in the A-factor regulatory cascade or as a regulator in the pathway independent of the cascade, has also been demonstrated by the studies with afsK/afsR of Streptomyces coelicolor A3(2) and their homologs of S. griseus.

Emerging Concepts of Secondary Metabolism in Actinomycetes.

Secondary metabolism in actinomycetes is brought on by exhaustion of a nutrient and/or by a growth rate decrease. These events generate signals which effect a cascade of regulatory events resulting in chemical differentiation (secondary metabolism) and morphological differentiation (morphogenesis). The signal is often a low molecular weight butyrolactone inducer (autoregulatory factor) which acts by negative control, i.e. by binding to a regulatory protein (repressor protein / receptor protein) which prevents secondary metabolism and morphogenesis during rapid growth and nutrient sufficiency. Nutrient/growth rate signals presumably activate a “master gene” which either acts at the level of translation by encoding a rare tRNA, or by encoding a positive transcription factor. Such master genes control both secondary metabolism and morphogenesis. At the second level of regulatory hierarchy are genes which control one branch of the cascade, i.e. either secondary metabolism or morphogenesis but not both. In the secondary metabolism branch, genes at the third level control formation of particular groups of secondary metabolites. At the fourth level are genes which control smaller groups, and finally, fifth level genes control individual biosynthetic pathways; these are usually positively acting but at least one acts negatively. These are also several levels of hierarchy on the morphogenesis branch. The second level includes genes which control aerial mycelium formation plus all the conidial genes lower in the cascade. Each third level locus controls a particular stage of conidiation (e.g. coiling, septation, wall thickening, spore maturation or spore pigmentation). Many of the these conidiation loci are complex, containing a number of genes; at least two code for sigma factors. Feedback regulation also plays a role in secondary metabolite control.

Cloning and analysis of a gene cluster from Streptomyces coelicolor that causes accelerated aerial mycelium formation in Streptomyces lividans.

We describe the cloning and analysis of two overlapping DNA fragments from Streptomyces coelicolor that cause aerial mycelium to appear more rapidly than usual when introduced into Streptomyces lividans on a low-copy-number plasmid vector. Colonies of S. lividans TK64 harboring either clone produce visible aerial mycelia after only 48 h of growth, rather than the usual 72 to 96 h. From deletion and sequence analysis, this rapid aerial mycelium (Ram) phenotype appears to be due to a cluster of three genes that we have designated ramA, ramB, and ramR. Both ramA and ramB potentially encode 65-kDa proteins with homology to ATP-dependent membrane-translocating proteins. A chromosomal ramB disruption mutant of S. lividans was found to be severely defective in aerial mycelium formation. ramR could encode a 21-kDa protein with significant homology to the UhpA subset of bacterial two-component response regulator proteins. The overall organization and potential proteins encoded by the cloned DNA suggest that this is the S. coelicolor homolog of the amf gene cluster that has been shown to be important for aerial mycelium formation in Streptomyces griseus. However, despite the fact that the two regions probably have identical functions, there is relatively poor homology between the two gene clusters at the DNA sequence level.

The Streptomyces aureofaciens homologue of the whiG gene encoding a putative sigma factor essential for sporulation

A putative σ factor-encoding gene, rpoZ, was cloned from Streptomyces aureofaciens. Its deduced protein product showed high similarity to the putative σ factor encoded by the Streptomyces coelicolor whiG gene. Disruption of rpoZ blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores.

Induction of aerial mycelium formation in a bld mutant of Streptomyces tendae by borrelidin--a macrolide antibiotic.

In a screening programme for substances with morphogenic effects on the nikkomycin producer strain Streptomyces tendae Tü 901 we identified a metabolite, which induced aerial mycelium formation in the bld mutant Tü 901/S 2566-EM 1. By using a HPLC UV/Vis absorbance spectral library we could confirm that this compound was identical with the macrolide antibiotic borrelidin. 100 ng borrelidin/paperdisc were sufficient to show an evident morphological effect.

Induction of aerial mycelium formation in a bld mutant of Streptomyces tendae by Borrelidin—a macrolide antibiotic

In a screening programme for substances with morphogenic effects on the nikkomycin producer strain Streptomyces tendae Tü 901 we identified a metabolite, which induced aerial mycelium formation in the bld mutant Tü 901/S 2566-EM 1. By using a HPLC UV/Vis absorbance spectral library we could confirm that this compound was identical with the macrolide antibiotic borrelidin. 100 ng borrelidin/paperdisc were sufficient to show an evident morphological effect.

Stimulation of genetic instability in Streptomyces ambofaciens ATCC 23877 by antibiotics that interact with DNA gyrase

In wild-type Streptomyces ambofaciens ATCC 23877, pigment-defective (Pig-) mutants arise at a frequency of about 0.5%; this genetic instability is related to genomic rearrangements such as deletions and/or amplifications of DNA sequences. On media containing oxolinic acid and novobiocin, which interact with the A and B subunits of DNA gyrase, respectively, the frequency of variants increased dramatically. The Pig- mutant frequency was increased to almost 100% on a medium containing oxolinic acid at a concentration allowing 55% survival. On solid medium containing either oxolinic acid or novobiocin at subinhibitory concentrations, most colonies exhibited a 'patchwork' phenotype, characterized by the presence of numerous Pig- sectors. Similar phenomena were not observed on media containing the transcriptional inhibitor rifampicin or the translational inhibitor streptomycin. Many of the Pig- mutants exhibited a pleiotropic phenotype and were affected in aerial mycelium formation, colony growth and/or prototrophy. Moreover, the same kinds of rearrangements (deletions and/or amplifications of DNA sequences) were found in both induced and spontaneous Pig- mutants. The results suggest either that DNA gyrase is directly involved in genetic instability or that an SOS-like system is implicated.

ChemInform Abstract: Synthetic Microbial Chemistry. Part 25. Synthesis of the Enantiomers of Differolide (4‐(2′,5′‐Dihydro‐2′‐oxo‐3′‐furanyl)‐3a,4,5,6‐ tetrahydro‐1(3H)‐isobenzofuranone), a Microbial Bioregulator for the Formation of Aerial Mycelium and Spores of Streptomyces glaucescens.

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Catabolic activity on humic acids of Streptomyces viridosporus grown under oxygen

When Streptomyces viridosporus was grown in batch culture on mineral salts – glucose medium and under oxygen, aerial mycelium formation and sporulation were suppressed. Under these conditions, biodegradation of soil or synthetic humic acids (expressed as the rate of decolorization of growth medium and the loss of radioactivity of labeled melanoidin, respectively) increased. Concurrently, both the catabolic activity of cells (expressed as peroxidase production, measured by the oxidation of N,N,N′,N′-tetramethyl-1,4-phenylenediamine as substrate) and the excretion of siderophores were stimulated.Key words: humic acid biodegradation, melanoidin mineralization, Streptomyces viridosporus, peroxidase, siderophore.

Genetics of differentiation in Streptomyces.

The use of mutants and molecular genetics has begun to reveal how differentiation is brought about in multicellular, mycelial Streptomyces spp. Alternative pathways to aerial mycelium formation may be activated on different media. In some cases, extracellular signals are transmitted or exchanged. Extracellular proteins may act as morphogens in the erection of aerial hyphae. A rare codon, UUA, is apparently confined to mRNAs from a few genes important only for early stages of differentiation; it is absent from vegetative or developmentally late mRNAs. Sporulation of aerial hyphae involves at least four specific regulatory proteins, including a sigma factor and an unusual small protein. A complex interplay between the sporulation regulatory genes, rather than a simple linear dependence cascade, is emerging.

Synthetic Microbial Chemistry, XXV. Synthesis of the Enantiomers of Differolide [4‐(2′,5′‐Dihydro‐2′‐oxo‐3′‐furanyl)‐3a,4,5,6‐tetrahydro‐1(3H)‐isobenzofuranone], a Microbial Bioregulator for the Formation of Aerial Mycelium and Spores of Streptomyces glaucescens

AbstractBoth the enantiomers of differolide [4‐(2′,5′‐dihydro‐2′‐oxo‐3′‐furanyl)‐3a,4,5,6‐tetrahydro‐1(3H)‐isobenzofuranone (1)] were synthesized by starting from the known lactone rac‐2. The key step was the optical resolution of hemiacetal rac‐3 by acetal formation with (+)‐ or (−)‐menthol. A single crystal X‐ray analysis of acetal (−)‐4a established its absolute configuration. Since the natural product was known to be racemic, the present synthesis of both (+)‐ and (−)‐1 with known absolute configuration provided molecular probes to examine the enantioselectivity at the receptor site of Streptomyces glaucescens.

Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces coelicolor.

The formation of an aerial mycelium by the filamentous bacterium Streptomyces coelicolor is determined in part by a small morphogenetic protein called SapB. A collection of representative bald (bld) mutants, which are blocked in aerial mycelium formation, are all defective in the production of this protein and regain the capacity to undergo morphological differentiation when SapB is supplied exogenously. We now report that most of the bld mutants are rescued for SapB production and aerial mycelium formation when grown near certain other bld mutants. Extracellular complementation experiments of this kind indicate that morphological differentiation is governed by a hierarchical cascade of at least four kinds of intercellular signals. At least one such signal is present in conditioned medium. It is resistant to boiling and protease treatment, and it remains effective even when diluted up to eightfold in fresh medium.

Accumulation of bldA-specified tRNA is temporally regulated in Streptomyces coelicolor A3(2)

Deletion of the bldA gene of Streptomyces coelicolor A3(2), which encodes the only tRNA for the rare UUA codon, had no obvious effects on primary growth but interfered with aerial mycelium formation and antibiotic production. To investigate the possible regulatory role of bldA, its transcription start point was identified, and time courses were determined for the appearance of its primary transcript, the processing of the primary transcript to give a mature 5' end, and the apparent efficiency of translation of ampC mRNA, which contains multiple UUA codons. The bldA promoter was active at all times, but processing of the 5' end of the primary transcript was comparatively inefficient in young cultures. This may perhaps involve an antisense RNA, evidence of which was provided by promoter probing and in vitro transcription. The presence of low levels of the processed form of the tRNA in young cultures followed by increased abundance in older cultures contrasted with the pattern observed for accumulation of a different, presumably typical tRNA which was approximately equally abundant throughout growth. The increased accumulation of the 5' processed form of bldA tRNA coincided with more-efficient translation of ampC mRNA in older cultures, supporting the hypothesis that in at least some physiological conditions, bldA may have a regulatory influence on events late in growth, such as morphological differentiation and antibiotic production.

A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators.

Mutants of Streptomyces griseus deficient in A-factor production are sporulation negative, since A-factor is an essential hormonal regulator for the induction of morphological and physiological differentiation in this bacterium. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this mutant strain. Subcloning experiments and nucleotide sequencing showed that two open reading frames, ORF1 with 656 amino acids and ORF2 with 201 amino acids, were required in order to induce sporulation. The amino acid sequence of ORF1 significantly resembled that of the Escherichia coli HlyB protein, a member of a family of bacterial membrane proteins engaged in ATP-dependent secretion mechanisms. Conserved features of this surface translocator family, such as the transmembrane structure predicted by their hydropathy profiles and the amino acid sequence forming an ATP-binding fold, were also conserved in ORF1. The ORF1 gene appeared to constitute a transcriptional unit with an additional upstream gene encoding ORF3, which was greatly similar to ORF1 in size and amino acid sequence. The other protein, ORF2, showed significant end-to-end homology with the E. coli uhpA product, a regulatory protein for the uptake of sugar phosphates. Like UhpA as a response regulator of a bacterial two-component regulatory system, ORF2 contained a helix-turn-helix DNA-binding domain at its COOH-terminal portion and an Asp residue (Asp-54) probably to be phosphorylated at its NH2-terminal portion. An amino acid replacement from Asp-54 to Asn resulted in the loss of the ability of ORF2 to induce sporulation in strain HH1.

"Eucaryotic" Signal Transduction Systems in the Bacterial Genus Streptomyces.

A microbial hormone, A-factor, controls streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. A-factor exerts its regulatory role by binding to a specific receptor protein which, in the absence of A-factor, acts as a repressor-type regulator for these morphological and physiological differentiation. In the signal relay leading to streptomycin production, the A-factor signal is transmitted from the A-factor receptor to the upstream activation sequence of a regulatory gene, strR, in the streptomycin biosynthetic gene cluster via an A-factor-dependent protein that acts as a transcription factor for strR. The signal relay leading to aerial mycelium formation includes proteins similar to response regulators of the procaryotic two-component regulatory systems and to a family of membrane translocators engaged in ATP-dependent secretion mechanisms. Accumulating data show that a large family of γ-butyrolactones act as autoregulators in a wide variety of Streptomyces spp. In addition to the hormonal control by the autoregulators, secondary metabolism and morphogenesis are controlled by signal transduction systems via both procaryotic and eucaryotic protein kinases. A pair of the AfsQ1 and AfsQ2 proteins constituting a procaryotic-type histidine-aspartate phosphotransfer system globally controls secondary metabolite formation in Streptomyces coelicolor A3(2). Another pair of proteins, AfsK and AfsR, which also serves as a global regulator for secondary metabolism in S. coelicolor, constitutes an eucaryotic-type phosphotransfer system in which a protein serine/threonine/tyrosine kinase AfsK phosphorylates serine and threonine residues of AfsR. A wide distribution of DNA sequences homologous with the afsQ1/afsQ2 and afsK/afsR genes suggests that both procaryotic and eucaryotic protein kinases control secondary metabolism in general in Streptomyces. Inhibition by K-252a and staurosporine, known as eucaryotic protein kinase inhibitors, of antibiotic production and aerial mycelium formation of S. griseus and S. coelicolor supports this idea.

Differential expression of principal sigma factor homologues of Streptomyces aureofaciens correlates with the developmental stage.

In previous experiments, Streptomyces aureofaciens has been shown to contain four genes hrdA, hrdB, hrdD, and hrdE, encoding polypeptides very similar to principal sigma factors of RNA polymerase. Two apparent tandem promoters were identified for each of the hrdA, hrdB and hrdD genes by S1 nuclease mapping using RNA prepared of S. aureofaciens in various developmental stages. Under all the conditions studied, tandem promoters of each gene differed significantly in their respective strengths. Transcription from the hrd promoters depended on developmental stage. While hrdB is transcribed from both promoters in all developmental stages, both tandem promoters of the hrdD gene are active only in vegetative stage and transcription of the hrdA tandem promoters temporally correlates with the aerial mycelium formation. In addition to a promoter, hrdB-P2, which lies upstream of the open reading frame, the hrdB gene, proposed to encode functional principal factor, appeared to contain at least one internal promoter, hrdB-P1. Activity of all promoters was consistent with S1 mapping experiments after insertion of promoter-bearing DNA fragments to promoter-probe vectors pIJ486 and pARC1. The results implicate temporally different expression of the hrd genes during the differentiation of S. aureofaciens.

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

Sporulation-inducing factor inStreptomyces avermitilis

The excretion of a diffusible factor was detected in sporulating, avermectin-producing strain ofStreptomyces avermitilis. The factor induced the formation of aerial mycelium and spores by a nonsporulating mutant.

Autoregulatory factors and communication in actinomycetes.

The ability to produce a wide variety of secondary metabolites and a mycelial form of growth that develops into spores are two biological aspects characteristic of the gram-positive bacterial genus Streptomyces. Secondary metabolism and cell differentiation are controlled by diffusible low-molecular-weight chemical substances called autoregulators. A-factor, the representative of the autoregulators, triggers streptomycin production and aerial-mycelium formation in Streptomyces griseus. A-factor exerts its regulatory function with the aid of a receptor protein that itself acts as a repressor-type regulator. The A-factor signal via the A-factor-receptor protein is transferred to downstream genes, such as streptomycin-production genes and sporulation genes, through multiple regulatory genes in a complex regulatory cascade. Thus, A-factor can be termed a "microbial hormone." This review deals with the A-factor-regulatory cascade as a model system for other autoregulators. The biosynthesis of A-factor, the structures and characteristics of other autoregulators, and the importance of these autoregulators in the ecosystem are also included.

Two promoters for the whiB sporulation gene of Streptomyces coelicolor A3(2) and their activities in relation to development.

Two transcripts corresponding to the whiB sporulation gene of Streptomyces coelicolor A3(2) that differed in length at their 5' ends by 164 nucleotides were identified by S1 mapping. Their presumptive promoters differed from each other; the more downstream, P2, resembled typical prokaryotic promoters (i.e., those recognized by the major form of RNA polymerase) at five of six positions in its -10 and -35 regions; and the more upstream, P1, was comparably similar, instead, to the previously described hrdDp1 promoter (M. J. Buttner, K. F. Chater, and M. J. Bibb, J. Bacteriol. 172:3367-3378, 1990) around -40, -10, and +1. In surface cultures of the wild-type strain, the abundance of transcripts from the weak P1 promoter showed no obvious correlation with the developmental stage, whereas transcripts from P2 were barely detectable until aerial mycelium was present and then became relatively abundant, consistent with the developmental role of whiB. Both types of transcript were detected during and, to a lesser extent, after rapid growth in liquid culture. In addition, both promoters were utilized in vitro by RNA polymerase purified from a liquid culture of S. coelicolor. Transcription from P1 and P2 was observed during surface culture in strains carrying mutations blocking aerial mycelium formation (bldA and bldB) or the formation of spores in aerial mycelium (whiA, whiB, whiG, and whiH). Thus, whiB transcription is not severely dependent on any of these developmental genes, among which whiG is the determinant of a putative sigma factor specific for, and crucial to, sporulation.