Strict regulation of morphological differentiation and secondary metabolism by a positive feedback loop between two global regulators AdpA and BldA in Streptomyces griseus.

AdpA is a global transcriptional regulator that is induced by the microbial hormone A-factor and activates many genes required for morphological differentiation and secondary metabolism in Streptomyces griseus. We confirmed that the regulatory tRNA gene bldA was required for translation of TTA-containing adpA. We also demonstrated that AdpA bound two sites upstream of the bldA promoter and activated transcription of bldA. Thus, we revealed a unique positive feedback loop between AdpA and BldA in S. griseus. Forced expression of bldA in an A-factor-deficient mutant resulted in the partial restoration of aerial mycelium formation and streptomycin production, suggesting that the positive feedback loop could prevent premature transcriptional activation of the AdpA-target genes in the wild-type strain. We revealed that the morphological defect of the bldA mutant could be attributed mainly to the TTA codons of only two genes: adpA and amfR. amfR encodes a transcriptional activator essential for aerial mycelium formation and is a member of the AdpA regulon. Thus, amfR is regulated by a feedforward mechanism involving AdpA and BldA. We concluded that the central regulatory unit composed of AdpA and BldA plays important roles in the initiation of morphological differentiation and secondary metabolism triggered by A-factor.

Proteomic analysis of the Streptomyces griseus ribosomal fraction.

The Streptomyces griseus 70S ribosome fraction was analyzed by radical-free and highly reducing two-dimensional (RFHR 2D) gel electrophoresis and mass spectrometry. Among the 60 putative ribosomal proteins that are encoded by the S. griseus genome, 48 were identified in the 70S ribosome fraction prepared from mycelia grown in liquid culture for 12, 36, and 48 h. Ribosomal protein S3 was detected at two different positions on the 2D gel, and the distribution changed completely in the course of the growth, suggesting that it was modified or processed. The SGR3624 protein was also identified in the 70S ribosome fraction, but detailed cellular fractionation analysis indicated that it localizes mainly at the membrane rather than the ribosome. An SGR3624-deleted mutant showed slow growth on solid media, indicating that SGR3624 has an important role in the growth of the substrate mycelium in solid culture.

Chromate reduction by PVA-alginate immobilized Streptomyces griseus in a bioreactor.

Microbial reduction of toxic Cr6+ to the less toxic Cr3+ is potentially a useful bioremediation process. Among the matrices tested for whole cell immobilization of an efficient chromate-reducing Streptomyces griseus strain, PVA-alginate was the most effective and was used for reduction of Cr(VI) in a bioreactor. Cr6+ reduction efficiency decreased as Cr6+ was increased from 2 to 12 mg l(-1) but increased with an increase in biomass concentration. However, increasing the flow rate from 2 to 8 ml h(-1) did not significantly affect Cr(6+) reduction. The reduction was faster in simulated effluent than in synthetic medium and complete removal of 8 mg Cr6+ l(-1) from effluent and synthetic medium occurred in 2 and 12 h, respectively. Our results indicate that immobilized S. griseus cells could be applied for the large-scale bioremediation of chromate-containing effluents and wastewaters.

Biofiltration technology for the removal of toluene from polluted air using Streptomyces griseus.

Biofiltration technology has been recognized as a promising biotechnology for treating the volatile organic compounds (VOCs) present in polluted air. This study aims to investigate the performance of a biofiltration system of Streptomyces griseus sp. DSM-40759 immobilized on activated carbon (PICA S23) towards the adsorption and degradation of toluene vapour as well as to regenerate the activated carbon in situ. The batch studies were performed using nutrient agar medium and basal salt medium (BSM) for microbial growth. Initially the pre-cultures were incubated at a temperature of 28°C on a rotary shaker at 150 rpm. After two days, the strain S. griseus DSM-40759 was immobilized on a known weight of activated carbon (12 g). The results of biofilter performance showed three different stages with a quick adsorption phase with approximately 95% of toluene removal after 70 min, a slow biotransformation phase by immobilized cells. In the later, the removal efficiency decreased significantly with the extension of time and reached 60% during this stage. Moreover, a final quick removal phase by the immobilized cells had an average removal efficiency of toluene around 95% after 500 min. The toluene degradation was found to be more than 84% after the second cycle and the biofilter was still capable of removing additional toluene. Thus, the results demonstrated the feasibility and reusability of a new biofilter system for toluene removal as well as extending the activated carbon's capacity and this could be a potential solution to reuse the activated carbon in industrial application.

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2).

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network.

Characterization of heat-induced interaction of neutral liposome with lipid membrane of Streptomyces griseus cell.

The interaction between the neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes and cell membrane of Streptomyces griseus induced by the heat treatment at specific temperature was investigated, focusing on the internalization of the neutral POPC liposomes with S. griseus cells. In an attempt to clarify the modes of liposome internalization, various kinds of inhibitors of endocytotic pathways were used to treat S. griseus cells. The efficiency of the heat treatment on liposome-cell membrane interactions was finally characterized based on the hydrophobic, electrostatic interactions and hydration effect. In fact, the internalization of the neutral liposomes into these cells was found to show higher rate and greater amount at higher temperatures. The kinetic study showed that the maximum amount of the internalized liposomes was, respectively, 469 x 10(5) and 643 x 10(5) liposomes/cell at 37 and 41 degrees C. The internalization of the neutral liposomes induced by the heat treatment was characterized, implying that the endocytosis occurred. The interactions involving the internalization, adsorption, and fusion of these liposomes with S. griseus cells were mainly contributed by the hydrophobic interaction and the unstable hydrogen bonds caused by the loss of water of surface hydration of cell membrane rather than the electrostatic interaction under the specific heat condition.

Hexavalent chromate reduction by immobilized Streptomyces griseus.

Hexavalent chromium, which is a mutagen and carcinogen, was efficiently reduced by Streptomyces griseus. This activity was associated with the cell. Cr(6+) reduction by free as well as immobilized cells was studied: cells in PVA-alginate had the highest (100%) Cr(6+) removal efficiency in 24 h with reduction rates similar to free cells. Immobilized cells completely reduced 25 mg Cr(6+) l(-1) in 24 h. PVA-alginate immobilized cells could be reused four times to completely reduce 25 mg Cr(6+) l(-1) in 24 h each time. Chromate in a simulated effluent containing Cu(2+), Mg(2+), Mn(2+) and Zn(2+) was completely reduced by PVA-alginate immobilized cells within 9 h.

Dual transcriptional control of amfTSBA, which regulates the onset of cellular differentiation in Streptomyces griseus.

The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus. Here we examined the transcriptional control mechanism for the promoter preceding amfT (PamfT) directing the transcription of the amfTSBA operon. High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT. Low-resolution analysis showed that PamfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The -35 region of PamfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2). The cloned bldD locus of S. griseus showed high sequence similarity to the S. coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S. griseus, which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation assay revealed that purified BldD and AmfR recombinant proteins specifically bind PamfT. Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of PamfT activity. An amfT-depleted mutant also showed a bald phenotype but PamfT activity was not affected. Both the bldD-overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control PamfT activity via direct binding to determine the transcriptional level of the amf operon responsible for the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S. griseus.

Ultrasonic characterization of mycelial morphology as a fractal structure.

Preliminary measurements of scattering of ultrasound from filamentous microorganisms in aqueous-medium suspensions are reported. The data are used to characterize the structuring (morphology) between the organisms quantitatively by modelling the morphology in terms of fractal structures and obtaining the fractal dimension as the characteristic parameter. The fractal dimension is calculated from the slope of a log-log plot of scattered intensity versus the scattering vector magnitude. The intensity is measured over a range of magnitudes of the scattering vector by varying the scattering angle as well as the ultrasonic frequency. Results from microbial suspensions of different species and different morphologies indicate that the fractal dimension can be used to quantify the morphological state of a suspension. The fractal dimension ranges from around 1.5 for suspensions with pelletous morphology to around 2.5 for suspensions with filamentous morphology.

A-factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus.

A-factor, containing a gamma-butyrolactone in its structure, is an autoregulatory factor or a 'microbial hormone' controlling secondary metabolism and cellular differentiation 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 morphological and physiological differentiation. In the signal relay leading to streptomycin production in S. griseus, the A-factor signal is transferred 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 serves as a transcription factor for strR. The StrR protein thus induced appears to activate the transcription of other streptomycin-production genes. The presence of A-factor homologues in a wide variety of Streptomyces species and distantly related bacteria implies the generality of gamma-butyrolactones as chemical cellular signalling molecules in microorganisms.

A screening method for autoregulators of anthracycline-producing streptomycetes.

A novel method was proposed for screening of autoregulators by anthracycline-producing strains of Streptomyces griseus. By means of a modified cosynthesis procedure, an indicator strain was detected capable of producing both leukaemomycin and aerial mycelium only in the presence of specific inducing factors (autoregulators) which were produced not only by anthracycline-yielding organisms and their mutants, but also by a streptomycin-producing strain of Streptomyces griseus as well as by its non-differentiating derivative.

Intracellular cyclic adenosine 3',5'-monophosphate levels and streptomycin production in cultures of Streptomyces griseus.

Changes in the amount of cyclic 3',5'-adenosine monophosphate within the mycelium of Streptomyces griseus were measured as cultures progressed through trophophase and idiophase in a complex medium supporting growth and streptomycin synthesis. Concentrations were highest before the cultures entered stationary phase and had declined 90% by 5 h before the antibiotic was produced. This low conentration was maintained while the antibiotic accumulated during the idiophase. The results indicate that the onset of streptomycin synthesis is not directly mediated by an increase in intracellular cyclic 3',5'-adenosine monophosphate concentration, and thus that antibiotic production in S. griseus is not controlled by catabolite repression.

Genetic segregation in a high-yielding streptomycin-producing strain of Streptomyces griseus.

The streptomycin-producing Streptomyces griseus HP spontaneously segregated non-reverting derivatives with altered phenotypes. Clones characterized by increased spore formation and decreased streptomycin production were found. Two other types of derivatives were defective in aerial mycelium and streptomycin formation as well, but differed in the capacity to synthesize a yellow pigment. These derivatives were examined with respect to further properties. The stability of S. griseus HP was investigated in relation to conditions of continuous culture. Both at 26 and 30 degrees C, under glycerol and NH4Cl limitation a rapid segregation and enrichment of streptomycin-non-producing derivatives occurred. At 34 degrees C and glycerol limitation segregation began only after about 35 generations of continuous culture. In NH4Cl-limited chemostats the original strain was stable during 80 generations. In the course of the continuous culture experiments it was shown that the onset of genetic segregation within mycelia can be detected before it becomes obvious in colonies grown from the mycelia. This was achieved by fractionation of the mycelia by protoplast formation and subsequent plating on regeneration medium allowing colony growth and differentiation.

Expression analysis of the ssgA gene product, associated with sporulation and cell division in Streptomyces griseus.

The ssgA gene of Streptomyces griseus B2682, when present in high copy number, results in both suppression of sporulation and fragmented growth of mycelia. Western analysis with polyclonal antibodies against the gene product (SsgA) revealed a close correlation between SsgA accumulation and the onset of sporulation in wild-type cells. The protein was only detected in the cytoplasm. Certain developmental mutants of S. griseus (afs, reIC and brgA) which are defective in aerial mycelium formation in solid culture and submerged spore formation in liquid culture failed to accumulate SsgA. The SsgA protein appeared shortly (1 h) after nutritional shift-down of strain B2682 cells. afs mutant cells sporulated and expressed SsgA only when A-factor was present both before and after nutritional shift-down. Introduction of the ssgA gene in a low-copy-number vector into strain B2682 resulted in fivefold overexpression of SsgA, and was accompanied by fragmented growth of mycelia and suppression of submerged spore formation (in liquid culture) and aerial mycelium formation (in solid culture). Streptomycin production was not inhibited. In a control experiment, a nonfunctional ssgA gene possessing a frameshift mutation near its N-terminus had no effect on either growth or sporulation. It is proposed that the ssgA gene product plays a role in promoting the developmental process of S. griseus.

Analysis of a gene that suppresses the morphological defect of bald mutants of Streptomyces griseus.

When present in multiple copies, orf1590 restored sporulation to class IIIA bald mutants of Streptomyces griseus, which form sporulation septa and thick spore walls prematurely. The orf1590 alleles from class IIIA bald mutants restored sporulation upon introduction at a high copy number into those same mutants, and the nucleotide sequence of one of these alleles was identical to that of the wild-type strain. We conclude that overexpression of orf1590 suppresses the defect in class IIIA bald mutants. Previous nucleotide sequence and transcript analyses suggested that orf1590 could encode two related proteins, P56 and P49.5, from nested coding sequences. A mutation that prevented the synthesis of P56 without altering the coding sequence for P49.5 eliminated the function of orf1590, as did amino acid substitutions in the putative helix-turn-helix domain located at the N terminus of P56 and absent from P49.5. To determine the coding capacity of orf1590, we analyzed translational fusions between orf1590 and the neo gene from Tn5. Measurement of the expression of fusions to the wild-type and mutant alleles of orf1590 indicated that P56 was the sole product of orf1590 during vegetative growth. Attempts to generate a nonfunctional frameshift mutation in orf1590 were unsuccessful in the absence of a second-site bald mutation, suggesting that orf1590 may be required during vegetative growth by preventing early sporulation. Our results are consistent with the hypothesis that P56 at a high level delays the premature synthesis of sporulation septa and spore walls in class IIIA mutants.

Characterization of the gene for factor C, an extracellular signal protein involved in morphological differentiation of Streptomyces griseus.

The gene encoding factor C (facC), an extracellular signal protein involved in cellular differentiation, was cloned from Streptomyces griseus 45H, and the complete nucleotide sequence was determined. The deduced amino acid sequence was confirmed by HPLC/electrospray ionization-mass spectrometry analysis. The full-length protein consists of 324 amino acids and has a predicted molecular mass of 34,523 Da. The mature extracellular 286 amino acid protein (31,038 Da) is probably produced by cleaving off a 38 amino acid secretion signal sequence. Southern hybridization detected facC in several other Streptomyces strains, but database searches failed to identify a protein with significant homology to factor C. Expression of facC from a low-copy-number vector in S. griseus 52-1 resulted in a phenotypic effect similar to that given by exogenously added factor C protein.

ATP-binding cassette transport system involved in regulation of morphological differentiation in response to glucose in Streptomyces griseus.

Streptomyces griseus NP4, which was derived by UV mutagenesis from strain IFO13350, showed a bald and wrinkled colony morphology in response to glucose. Mutant NP4 formed ectopic septa at intervals along substrate hyphae, and each of the compartments developed into a spore which was indistinguishable from an aerial spore in size, shape, and thickness of the spore wall and in susceptibility to lysozyme and heat. The ectopic spores of NP4 formed in liquid medium differed from "submerged spores" in lysozyme sensitivity. Shotgun cloning experiments with a library of the chromosomal DNA of the parental strain and mutant NP4 as the host gave rise to DNA fragments giving two different phenotypes; one complementing the bald phenotype of the host, and the other causing much severe wrinkled morphology in the host. Subcloning identified a gene (dasR) encoding a transcriptional repressor belonging to the GntR family that was responsible for the reversal of the bald phenotype and a gene (dasA) encoding a lipoprotein probably serving as a substrate-binding protein in an ATP-binding cassette (ABC) transport system that was responsible for the severe wrinkled morphology. These genes were adjacent but divergently encoded. Two genes, named dasB and dasC, encoding a membrane-spanning protein were present downstream of dasA, which suggested that dasRABC comprises a gene cluster for an ABC transporter, probably for sugar import. dasR was transcribed actively during vegetative growth, and dasA was transcribed just after commencement of aerial hypha formation and during sporulation, indicating that both were developmentally regulated. Transcriptional analysis and direct sequencing of dasRA in mutant NP4 suggested a defect of this mutant in the regulatory system to control the expression of these genes. Introduction of multicopies of dasA into the wild-type strain caused ectopic septation in very young substrate hyphae after only 1 day of growth and subsequent sporulation in response to glucose. The ectopic spores of the wild type had a thinner wall than those of mutant NP4, in agreement with the observation that the former was sensitive to lysozyme and heat. Disruption of the chromosomal dasA or dasR in the wild-type strain resulted in growth as substrate mycelium, suggesting an additional role of these genes in aerial mycelium formation. The ectopic septation and sporulation in mutant NP4 and the wild-type strain carrying multicopies of dasA were independent of a microbial hormone, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), that acts as a master switch of aerial mycelium formation and secondary metabolism.

Carbon-source-dependent transcriptional control involved in the initiation of cellular differentiation in Streptomyces griseus.

Carbon source is one of the environmental factors that affects cellular differentiation of Streptomyces. We have identified the craA gene as a putative negative regulator involved in the carbon-source-dependent initiation of cellular differentiation in Streptomyces griseus. Carbon-source-dependent transcriptional repression of craA, which is caused by binding of a putative repressor protein to its promoter region, is proposed to result in the initiation of aerial mycelium formation. The presence of a craA homologue in the chromosome of Streptomyces coelicolor A3(2) implicates the existence of a similar regulatory mechanism in this organism. The repression of craA-promoter activity in glucose media could be alleviated not only by replacing glucose with maltose but also by supplying copper, which suggests that the stimulatory effect of copper on cellular differentiation in Streptomyces is excerted via abolishment of glucose-repression of craA.

Modification by genetic changes of the pleiotropic interference of butyrolactone-type autoregulators with differentiation of Streptomyces griseus.

Two series of aerial-mycelium-negative (Amy-), anthracycline-nonproducing (Ant-) mutants were obtained from ancestral Amy+Ant+ strains of S. griseus: a) derivatives represented by the met- strain 39 which could not differentiate although they were still producing both the butyrolactone-type autoregulator 1 and NADP-glycohydrolase, and b) mutants whose incapability to form spores and anthracycline pigments was apparently caused by the loss of autoregulator production. These latter mutants responded to the addition of 1 or the naturally occurring dihydro derivative 2 with complete or at least partial reconstitution of differentiation-associated functions. All of the b)-type mutant strains exhibited similar biochemical alterations in the presence of 1 or 2 regardless of the presence of additional genetic changes in the primary metabolism. Two mutants, however, displayed an altered pattern of secondary product formation. In submerged cultures the major biochemical changes observed in presence of 1 (or 2) were an increase of the lipid level in the mycelium, an alteration of the lipid composition, and a stimulation of neutral proteinase production. All of the blocked autoregulator-negative mutants were discernible from the ancestral strains and strain 39 by their lack of NADP-glycohydrolase production. This suggested the existance of a common genetic locus or a common pleiotropic regulator gene controling both gene functions. Present ideas concerning the role of butyrolactone-type autoregulator 1 as a pleiotropic effector molecule interacting with development of S. griseus are summarized in a hypothetical scheme.