Streptomyces umbrella toxin particles block hyphal growth of competing species.

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

Dihydropyriculol produced by Pyricularia oryzae inhibits the growth of Streptomyces griseus.

Dihydropyriculol is a major secondary metabolite of Pyricularia oryzae. However, the biological activity of dihydropyriculol has not been reported. Here, we showed that dihydropyriculol has inhibitory activity against Streptomyces griseus. Localization analysis of dihydropyriculol revealed that dihydropyriculol could reach to S. griseus under confrontation culture. These results suggest that dihydropyriculol can be used as a chemical weapon against S. griseus.

Mutation in afsR Leads to A-Factor Deficiency in Streptomyces griseus B2682.

BACKGROUND/AIMS: A-factor, a γ-butyrolactone autoregulator, in Streptomyces griseus is involved in the regulation of differentiation and antibiotic production. Here we studied the S. griseus B2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the afsR gene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. METHODS: Wild afsR was cloned to the pHJL401 shuttle vector and was transformed to the S. griseus AFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. RESULTS: Transformation of AFN by a wild afsR restored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of afsR to the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. CONCLUSIONS: AfsR has an effect on the regulation of A-factor production in S. griseus. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.

wblE2 transcription factor in Streptomyces griseus S4-7 plays an important role in plant protection.

Streptomyces griseus S4-7 was originally isolated from the strawberry rhizosphere as a microbial agent responsible for Fusarium wilt suppressive soils. S. griseus S4-7 shows specific and pronounced antifungal activity against Fusarium oxysporum f. sp. fragariae. In the Streptomyces genus, the whi transcription factors are regulators of sporulation, cell differentiation, septation, and secondary metabolites production. wblE2 function as a regulator has emerged as a new group in whi transcription factors. In this study, we reveal the involvement of the wblE2 transcription factor in the plant-protection by S. griseus S4-7. We generated ΔwblE, ΔwblE2, ΔwhiH, and ΔwhmD gene knock-out mutants, which showed less antifungal activity both in vitro and in planta. Among the mutants, wblE2 mutant failed to protect the strawberry against the Fusarium wilt pathogen. Transcriptome analyses revealed major differences in the regulation of phenylalanine metabolism, polyketide and siderophore biosynthesis between the S4-7 and the wblE2 mutant. The results contribute to our understanding of the role of streptomycetes wblE2 genes in a natural disease suppressing system.

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin.

Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ∼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.

Characteristics of Streptomyces griseus biofilms in continuous flow tubular reactors.

The purpose of this study was to investigate the feasibility of cultivating the biotechnologically important bacterium Streptomyces griseus in single-species and mixed-species biofilms using a tubular biofilm reactor (TBR). Streptomyces griseus biofilm development was found to be cyclical, starting with the initial adhesion and subsequent development of a visible biofilm after 24 h growth, followed by the complete detachment of the biofilm as a single mass, and ending with the re-colonisation of the tube. Fluorescence microscopy revealed that the filamentous structure of the biofilm was lost upon treatment with protease, but not DNase or metaperiodate, indicating that the extracellular polymeric substance is predominantly protein. When the biofilm was cultivated in conjunction with Bacillus amyloliquefaciens, no detachment was observed after 96 h, although once subjected to flow detachment. Electron microscopy confirmed the presence of both bacteria in the biofilm and revealed a network of fimbriae-like structures that were much less apparent in single-species biofilm and are likely to increase mechanical stability when developing in a TBR. This study presents the very first attempt in engineering S. griseus biofilms for continuous bioprocess applications.

[Influence of perfluorodecalin on growth of actinomycetes and intensification of Streptomycin and daunorubicin production by the genus Streptomyces kind bacteria in submerged culture].

Addition of perfluorodecalin with gas-transporting function to the liquid medium during submerged cultivation of actinomycetes of the genus Streptomyces resulted in higher intensity and level of the biomass synthesis and increased production of streptomycin and daunorubicin. Addition of perfluorodecalin to the medium provided a 2.0-2.3-fold surpass of the maximum antibiotic production (achieved by the 120th-144th hours of the culture growth) vs. the antibiotic accumulation peaks in the control.

An alternative sigma factor governs the principal sigma factor in Streptomyces griseus.

In bacteria, the RNA polymerase holoenzyme comprises a five-subunit core enzyme and a dissociable subunit, sigma factor, which is responsible for transcriptional initiation. The filamentous bacterium Streptomyces griseus has 52 sigma factors, including one essential 'principal' sigma factor (σ(HrdB) ) that is responsible for the transcription of housekeeping genes. Here we characterized an alternative sigma factor (σ(ShbA) ), which is highly conserved within the genus Streptomyces. A σ(ShbA) -deficient mutant showed a severe growth defect and transcriptome analysis indicated that many housekeeping genes were downregulated in response to insufficient σ(ShbA) production. Biochemical and genetic analyses proved that σ(ShbA) is a major determinant of transcription of the σ(HrdB) gene. This observation of a principal sigma factor being governed by another sigma factor throughout growth is unprecedented. We found that increasing σ(ShbA) production with mycelial growth maintained a high σ(HrdB) level late in growth. Furthermore, a hrdB-autoregulatable σ(ShbA) -deficient mutant, in which the principal sigma factor gene can be transcribed by RNA polymerase containing σ(HrdB) itself, showed several defects: rapid mycelial lysis in stationary phase in liquid culture and delayed morphological development and impaired streptomycin production in solid culture. From these observations, we discuss the biological significance of control of σ(HrdB) by σ(ShbA) in S. griseus.

Pleiotropic role of the Sco1/SenC family copper chaperone in the physiology of Streptomyces.

Antibiotic production and cell differentiation in Streptomyces is stimulated by micromolar levels of Cu(2+) . Here, we knocked out the Sco1/SenC family copper chaperone (ScoC) encoded in the conserved gene cluster 'sco' (the S treptomycescopper utilization) in Streptomyces coelicolor A3(2) and S. griseus. It is known that the Sco1/SenC family incorporates Cu(2+) into the active centre of cytochrome oxidase (cox). The knockout caused a marked delay in antibiotic production and aerial mycelium formation on solid medium, temporal pH decline in glucose-containing liquid medium, and significant reduction of cox activity in S. coelicolor. The scoC mutant produced two- to threefold higher cellular mass of the wild type exhibiting a marked cox activity in liquid medium supplied with 10 µM CuSO(4) , suggesting that ScoC is involved in not only the construction but also the deactivation of cox. The scoC mutant was defective in the monoamine oxidase activity responsible for cell aggregation and sedimentation. These features were similarly observed with regard to the scoC mutant of S. griseus. The scoC mutant of S. griseus was also defective in the extracellular activity oxidizing N,N'-dimethyl-p-phenylenediamine sulfate. Addition of 10 µM CuSO(4) repressed the activity of the conserved promoter preceding scoA and caused phenylalanine auxotrophy in some Streptomyces spp. probably because of the repression of pheA; pheA encodes prephenate dehydratase, which is located at the 3' terminus of the putative operon structure. Overall, the evidence indicates that Sco is crucial for the utilization of copper under a low-copper condition and for the activation of the multiple Cu(2+) -containing oxidases that play divergent roles in the complex physiology of Streptomyces.

[Features of biosynthesis of chitinolytic enzymes by Streptomyces griseus var. streptomycini].

The previously selected strain Streptomyces griseus var. streptomycini is able to hydrolyze colloid as well as crystal forms of chitin. During the submerged cultivation in the medium with crystal chitin, the chitinase activity achieved the maximal value after 46-50 h of culturing. Use of colloid chitin as an inductor allowed increasing the chitinolytic activity by 33%. Adding of mannose to the medium increased the chitinase activity of the producer by two times. It has been shown that the chitinase biosynthesis bears an inducible nature.

Pilot-plant cultivation of Streptomyces griseus producing homologues of nonactin by precursor-directed biosynthesis and their identification by LC/MS-ESI.

Precursor-directed biogenetic approach was used to produce a range of nonactin homologues in a 50 l fermentor cultivation of strain Streptomyces griseus 34/249 obtained by UV mutagenesis. The production medium contained sodium propionate, isobutyrate and isovalerate as individual precursors, and 10 g l(-1) Diaion HP20 styrene-divinylbenzene resin that maintains suitable precursor concentration by reversibly adsorbing and releasing it. The produced nonactin homologues were separated on two C18 reversed-phase liquid chromatography columns in series and analyzed by MS with ESI source in the positive ion mode. Formation of doubly charged ions was suppressed by an excess of Na(+) ions throughout the process. The production of the homologues increased up to day 5 and then it leveled off. Cultivations with individual precursors yielded a total of 18 nonactin homologues whose spectrum depended on the precursor used. The total production of the homologues was lowered but their spectrum was shifted to higher-molecular-weight compounds.

CebR as a master regulator for cellulose/cellooligosaccharide catabolism affects morphological development in Streptomyces griseus.

Streptomyces griseus mutants exhibiting deficient glucose repression of beta-galactosidase activity on lactose-containing minimal medium supplemented with a high concentration of glucose were isolated. One of these mutants had a 12-bp deletion in cebR, which encodes a LacI/GalR family regulator. Disruption of cebR in the wild-type strain caused the same phenotype as the mutant, indicating that CebR is required for glucose repression of beta-galactosidase activity. Recombinant CebR protein bound to a 14-bp inverted-repeat sequence (designated the CebR box) present in the promoter regions of cebR and the putative cellobiose utilization operon, cebEFG-bglC. The DNA-binding activity of CebR was impaired by cellooligosaccharides, including cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose. In agreement with this observation, transcription from the cebE and cebR promoters was greatly enhanced by the addition of cellobiose to the medium. Seven other genes containing one or two CebR boxes in their upstream regions were found in the S. griseus genome. Five of these genes encode putative secreted proteins: two cellulases, a cellulose-binding protein, a pectate lyase, and a protein of unknown function. These five genes and cebEFG-bglC were transcribed at levels 4 to 130 times higher in the DeltacebR mutant than in the wild-type strain, as determined by quantitative reverse transcription-PCR. These findings indicate that CebR is a master regulator of cellulose/cellooligosaccharide catabolism. Unexpectedly, the DeltacebR mutant formed very few aerial hyphae on lactose-containing medium, demonstrating a link between carbon source utilization and morphological development.

Identification and gene disruption of small noncoding RNAs in Streptomyces griseus.

Small noncoding RNAs (sRNAs) have been shown to control diverse cellular processes in prokaryotes. To identify and characterize novel bacterial sRNAs, a gram-positive, soil-inhabiting, filamentous bacterium, Streptomyces griseus, was examined, on the assumption that Streptomyces should express sRNAs as important regulators of morphological and physiological differentiation. By bioinformatics investigation, 54 sRNA candidates, which were encoded on intergenic regions of the S. griseus chromosome and were highly conserved in those of both Streptomyces coelicolor A3(2) and Streptomyces avermitilis, were selected. Of these 54 sRNA candidates, 17 transcripts were detected by Northern blot analysis of the total RNAs isolated from cells grown on solid medium. Then, the direction of transcription of each sRNA candidate gene was determined by S1 nuclease mapping, followed by exclusion of four sRNA candidates that were considered riboswitches of their downstream open reading frames (ORFs). Finally, a further sRNA candidate was excluded because it was cotranscribed with the upstream ORF determined by reverse transcription-PCR. Thus, 12 sRNAs ranging in size from 40 to 300 nucleotides were identified in S. griseus. Seven of them were apparently transcribed in a growth phase-dependent manner. Furthermore, of the 12 sRNAs, the expression profiles of 7 were significantly influenced by a mutation of adpA, which encodes the central transcriptional regulator of the A-factor regulatory cascade involved in both morphological differentiation and secondary metabolism in S. griseus. However, disruption of all 12 sRNA genes showed no detectable phenotypic changes; all the disruptants grew and formed aerial mycelium and spores with the same time course as the wild-type strain on various media and produced streptomycin similarly to the wild-type strain.

tmRNA of Streptomyces collinus and Streptomyces griseus during the growth and in the presence of antibiotics.

Streptomycetes are soil microorganisms with the potential to produce a broad spectrum of secondary metabolities. The production of antibiotics is accompanied by a decrease in protein synthesis, which raises the question of how these bacteria survived the transition from the primary to the secondary metabolism. Translating ribosomes incapable to properly elongate or terminate polypeptide chain activate bacterial trans-translation system. Abundance and stability of the tmRNA during growth of Streptomyces collinus and Streptomyces griseus producing kirromycin and streptomycin, respectively, was analysed. The level of tmRNA is mostly proportional to the activity of the translational system. We demonstrate that the addition of sub-inhibitory concentrations of produced antibiotics to the cultures from the beginning of the exponential phase of growth leads to an increase in tmRNA levels and to an incorporation of amino acids into the tag-peptides at trans-translation of stalled ribosomes. These findings suggest that produced antibiotics induce tmRNA that facilitate reactivation of stalled complex of ribosomes and maintain viability. The effect of antibiotics that inhibit the cell-wall turnover, DNA, RNA or protein synthesis on the level of tmRNA was examined. Antibiotics interfering with ribosomal target sites are more effective at stimulation of the tmRNA level in streptomycetes examined than those affecting the synthesis of DNA, RNA or the cell wall.

Toxic effects of Streptomyces griseus spores and exudate on gill pathology of freshwater fish.

Many unexplained fish-kills in British waters are considered microbial in origin and a large proportion of field sites contains elevated concentrations of filamentous actinobacteria. The present study has shown that a strain of Streptomyces griseus, isolated from field sites, elicits pathological changes to the gills of fish under laboratory conditions which mirror those found in situ. These changes include hyperplasia leading to fusion of the secondary lamellae and loss of microridging on the filamental epithelium of the primary lamellae. Juveniles of up to six fish species were exposed to spore suspensions or exudate of S. griseus in the range of 1 x 10(2)-1 x 10(6)spores ml(-1) for up to 96 h. The exudate was more potent than the spores and there was a positive correlation between exudate concentration and the rate and extent of fish gill pathology with bream and rainbow trout being more sensitive than carp, tench and roach. The results are discussed in the context of recognising and managing potential fish mortalities caused by microbial toxins.

Identification of sas, a conserved gene cluster involved in the regulation of aerial mycelium formation in Streptomyces griseus.

We cloned a DNA fragment that suppressed the aerial-mycelium-deficient phenotype in an amfS mutant of Streptomyces griseus when it was introduced into the cells via a high-copy-number plasmid. The sasABCDR gene cluster was identified as being responsible for this suppressive activity. The proteins encoded by sasABCD were of unknown function, but the operon structure was found to be conserved in all the strains of Streptomyces spp. and related organisms whose genomes have been sequenced. sasR, the flanking opposite coding sequence, encoded a putative DNA-binding protein. Subcloning revealed that the presence of all five coding sequences was essential for complete suppression. Scanning electron microscopy of Streptomyces griseus strains carrying the sas gene cluster at a high copy-number revealed that bundle-like structures consisting of several aerial hyphae were often formed. S1 nuclease protection analyses were performed to identify the transcriptional start site in the promoters preceding sasA and sasR. The promoter preceding sasA was highly active during vegetative growth. Null mutants for sasABCD among the S. griseus and S. coelicolor A3(2) cells exhibited bald phenotypes; this suggested a positive regulatory role of this gene cluster in the onset of morphogenesis in these two phylogenetically distinct Streptomyces species.

Streptomyces griseus enhances denitrification by Ralstonia pickettii K50, which is possibly mediated by histidine produced during co-culture.

Ralstonia pickettii K50 (strain K50) is a denitrifying bacterium that produces low levels of N(2)O under aerobic conditions. In this study, we found that co-culturing of strain K50 with Streptomyces griseus significantly enhanced the denitrification activity of strain K50 in an artificial wastewater (AWW) system. Most factors that enhance denitrification activity were in the high molecular weight fraction of the cell-free broth of S. griseus, and were suggested to be extracellular proteases. Further investigation revealed that the cultivation of strain K50 in protease-treated AWW medium fully enhanced denitrification, and that a shortage of amino acids in the medium limited it. Among the 20 standard amino acids tested, only histidine had a significant effect in inducing denitrification by strain K50. Our results indicate that histidine is a novel inducer of bacterial denitrification.

Process analysis of macrotetrolide biosynthesis during fermentation by means of direct infusion LC-MS.

The optimization of the biosynthetic pathways is highly attractive for the large-scale preparation of macrotetrolides, because overall yields in the chemical synthesis of compounds like nonactin have been very low. A key success factor determining the outcome of such optimizations is the adequate process analysis for the envisioned product. The analytical methods for process control involved in the past spectrophotometric and chromatographic measurements. LC-MS offers a modern approach to obtain more detailed data than the spectrophotometric and chromatographic measurements used in the past. In this work, a fast and versatile analytical LC-MS method has been set up, which has proven of much value for the in-process analysis of macrotetrolides during fermentation and which has allowed rapid large-scale bioprocess development.

Production of streptomycin from chitin using Streptomyces griseus in bioreactors of different configuration.

Streptomyces griseus was cultured in three different bioreactors in a medium containing chitin flakes. When a conventional bioreactor stirred by two sets of Rushton impellers and operated at high speed was used, the yield of streptomycin (3.1mg/l) was the highest observed and occurred at approximately 500 h. Cultivation of S. griseus in a bioreactor stirred at low speed by a U-shaped paddle resulted in a lower yield of streptomycin (1.8 mg/l) but this was achieved in a shorter period of time (400 h). Increasing the concentration of chitin from 5% to 10% w/v had no significant effect on either of these two parameters. The use of a novel vertical basket bioreactor in which the chitin flakes were contained within a wire mesh basket and were gently fluidised by air, enabled comparatively high yields of streptomycin (2.8 mg/l) in the relatively short time of 300 h.