Genome-wide screening antifungal genes in Streptomyces griseus S4-7, a Fusarium wilt disease suppressive microbial agent.

Streptomyces is a widely studied bacterial genus, particularly with regard to secondary metabolites and antibiotics production. Streptomyces griseus S4-7 was isolated from a strawberry Fusarium wilt disease suppressive soil, and its biological control ability has been well established. However, the antifungal mechanism of strain S4-7 is not yet fully understood at the molecular and biochemical level. Therefore, in this study we created a random mutant library for strain S4-7 with the Tn5 transposon element to investigate antifungal traits on a genome-wide scale. In total 4646 individual mutant strains were created and 13 mutants were selected based on loss of antifungal activity. The knockout genes were identified as electron transfer oxidoreductase (eto),sigma factor-70(sig70) and nrps by Inverse PCR (I-PCR). eto regulates the geranylgeranyl reductase gene, which is involved in terpenoid-quinone biosynthesis, an important factor in cell fitness. In the △eto strain, expression of wbl, a master regulator of the production of secondary metabolites, was significantly reduced. sig70 is responsible for the cell differentiation sensing mechanism in genus Streptomyces. △nrps showed decreased production of hybrid peptide-polyketide siderophores. These results suggest that S. griseus S4-7 may have various antifungal mechanisms, and each mechanism is essential to maximal antifungal activity.

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.

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.

Cross-interaction of anti-σH factor RshA with BldG, an anti-sigma factor antagonist in Streptomyces griseus.

Stress-response sigma factor σ(H) is negatively regulated by its cognate anti-sigma factor RshA in Streptomyces griseus. As the overexpression of RshA in the wild-type strain confers a distinctive bald phenotype (deficiency in aerial mycelium formation and streptomycin production), RshA is supposed to associate with not only σ(H) but also another regulatory element that plays a crucial role in the developmental control of S. griseus. Here, we show that an anti-sigma factor antagonist BldG associates with RshA and negatively regulates its activity. The bald phenotype conferred by the overexpression of rshA was restored to the wild-type phenotype by the coexpression with bldG. The in vivo and in vitro protein interaction analyses demonstrated the specific association between RshA and BldG. A bldG mutant exhibited a distinctive bald phenotype and was defective in the σ(H) -dependent transcription activities. The positive regulatory role of BldG regarding the σ(H) activity was verified by an in vitro transcriptional analysis, in which the inhibition of σ(H) -dependent transcription by RshA was abolished by the addition of BldG in a dose-responsive manner. Overall, evidence suggests that BldG serves as a master switch for both stress-response and developmental gene expression based on its association with multiple anti-sigma factors in S. griseus.

Control of aerial mycelium formation by the BldK oligopeptide ABC transporter in Streptomyces griseus.

An oligopeptide permease family ATP-binding cassette (ABC) transporter encoded by SGR2418-SGR2414 was shown to be essential for aerial mycelium formation on glucose-containing media in Streptomyces griseus. In spite of only weak sequence similarity, the operon was equivalent to the bldK operon of Streptomyces coelicolor A3(2) in terms of chromosomal location and function. Transcription of the operon appeared not to be directly regulated by AdpA, a global regulator of morphological and physiological development in S. griseus, although it was affected by adpA inactivation. This study revealed that an ABC transporter was essential for aerial mycelium formation not only in S. coelicolor A3(2) but also in S. griseus, indicating that extracellular signaling by certain peptides should be conserved among different Streptomyces species.

Dynamic changes in the extracellular proteome caused by absence of a pleiotropic regulator AdpA in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers morphological development and secondary metabolism by inducing a pleiotropic transcriptional regulator AdpA. Extracellular proteome analysis of the wild-type and DeltaadpA strains grown to the end of the exponential phase in liquid minimal medium revealed that 38 secreted proteins, including many catabolic enzymes, such as protease, glycosyl hydrolase and esterase, were produced in an AdpA-dependent manner. Transcriptome analysis showed that almost all of these AdpA-dependent secreted proteins were regulated at the transcriptional level. In vitro AdpA-binding assays and determination of transcriptional start sites led to identification of 11 promoters as novel targets of AdpA. Viability staining revealed that some hyphae lysed during the exponential growth phase, which could explain the detection of 3 and 23 cytoplasmic proteins in the culture media of the wild-type and DeltaadpA strains respectively. In the wild-type strain, due to high protease activity in the culture medium, cytoplasmic proteins that leaked from dead cells seemed to be degraded and reused for the further growth. The existence of many AdpA-dependent (i.e. A-factor-inducible) secreted catabolic enzymes, which are likely involved in the assimilation of material that leaked from dead cells, reemphasizes the importance of A-factor in the morphological differentiation of S. griseus.

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.

Taxonomic study of a chromomycin-producing strain and reclassification of Streptomyces cavourensis subsp. washingtonensis as a later synonym of Streptomyces griseus.

A chromomycin-producing actinomycete, strain AP19, was isolated from a sample of faeces collected from Foping national nature reserve in China. Chemotaxonomic and morphological properties indicated that the novel isolate was a member of the genus Streptomyces. Phylogenetic analyses based on an almost complete 16S rRNA gene sequence of the strain and on the 120-nt nucleotide variable gamma-region of this molecule revealed that it was closely related to Streptomyces griseus ISP 5236(T) and Streptomyces cavourensis subsp. washingtonensis ATCC 27732(T). DNA-DNA relatedness values among these strains were above 70 %. Streptomyces cavourensis subsp. washingtonensis could be readily distinguished from Streptomyces cavourensis ATCC 14889(T) by differing BOX-PCR fingerprinting patterns, relatively low 16S rRNA gene sequence similarity and a low DNA-DNA relatedness value. It is proposed, therefore, that Streptomyces cavourensis subsp. washingtonensis is a later synonym of Streptomyces griseus.

Identification and utility of FdmR1 as a Streptomyces antibiotic regulatory protein activator for fredericamycin production in Streptomyces griseus ATCC 49344 and heterologous hosts.

The fredericamycin (FDM) A biosynthetic gene cluster, cloned previously from Streptomyces griseus ATCC 49344, contains three putative regulatory genes, fdmR, fdmR1, and fdmR2. Their deduced gene products show high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family (FdmR1) or to MarR-like regulators (FdmR and FdmR2). Here we provide experimental data supporting FdmR1 as a SARP-type activator. Inactivation of fdmR1 abolished FDM biosynthesis, and FDM production could be restored to the fdmR1::aac(3)IV mutant by expressing fdmR1 in trans. Reverse transcription-PCR transcriptional analyses revealed that up to 26 of the 28 genes within the fdm gene cluster, with the exception of fdmR and fdmT2, were under the positive control of FdmR1, directly or indirectly. Overexpression of fdmR1 in S. griseus improved the FDM titer 5.6-fold (to about 1.36 g/liter) relative to that of wild-type S. griseus. Cloning of the complete fdm cluster into an integrative plasmid and subsequent expression in heterologous hosts revealed that considerable amounts of FDMs could be produced in Streptomyces albus but not in Streptomyces lividans. However, the S. lividans host could be engineered to produce FDMs via constitutive expression of fdmR1; FDM production in S. lividans could be enhanced further by overexpressing fdmC, encoding a putative ketoreductase, concomitantly with fdmR1. Taken together, these studies demonstrate the viability of engineering FDM biosynthesis and improving FDM titers in both the native producer S. griseus and heterologous hosts, such as S. albus and S. lividans. The approach taken capitalizes on FdmR1, a key activator of the FDM biosynthetic machinery.

Conditionally positive effect of the TetR-family transcriptional regulator AtrA on streptomycin production by Streptomyces griseus.

AtrA, a transcriptional activator for actII-ORF4, encoding the pathway-specific transcriptional activator of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2), has been shown to bind the region upstream from the promoter of strR, encoding the pathway-specific transcriptional activator of the streptomycin biosynthetic gene cluster in Streptomyces griseus [Uguru et al. (2005) Mol Microbiol 58, 131-150]. The atrA orthologue (atrA-g) in S. griseus was constitutively transcribed throughout growth from a promoter located about 250 nt upstream of the translational start codon, as determined by S1 nuclease mapping. DNase I footprinting showed that histidine-tagged AtrA-g bound an inverted repeat located upstream of strR at positions -117 to -142 relative to the transcriptional start point of strR as +1. This AtrA-g-binding site was between two AdpA-binding sites at approximately nucleotide positions -270 and -50. AdpA is a central transcriptional activator in the A-factor regulatory cascade and essential for the transcription of strR. AtrA-g and AdpA simultaneously bound the respective binding sites. In contrast to AdpA, AtrA-g was non-essential for strR transcription; an atrA-g-disrupted strain produced streptomycin on routine agar media to the same extent as the wild-type strain. However, the atrA-g-disrupted strain tended to produce a smaller amount of streptomycin than the wild-type strain under some conditions, for example, on Bennett agar containing 1 % maltose and on a minimal medium. Therefore, AtrA-g had a conditionally positive effect on streptomycin production, as a tuner, probably by enhancing the AdpA-dependent transcriptional activation of strR in a still unknown manner.

Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsin.

The sgtR1 and sgtR2 genes encoding putative regulators similar to the Aha1 and ArsR families, respectively, were identified downstream from the sprT gene. To investigate their function, expression vectors containing various combinations of sprT, sgtR1, and sgtR2 were transformed into Streptomyces lividans and Streptomyces griseus. The trypsin activity levels produced by S. lividans harboring pWHM3-TR2 (sprT and sgtR2) or pWHM3-TR1R2 (sprT, sgtR2, and sgtR2) were, respectively, 6.6 or 8.9 times that of S. lividans transformed with pWHM3-T (sprT). In the pWHM3-TR1R2 transformant, the transcription of sprT consistently occurred during the earlier stages of growth and was maintained at a higher level throughout the 6 days of cultivation. Streptomyces griseus IFO13350 harboring pWHM3-TR1R2 also produced trypsin activity 2.1 times that of the pWHM3-T transformant. However, all S. griseus Delta adpA transformants produced lower SGT activity than the wild-type strain, and none could overcome the deficiency in AdpA transcriptional activator, suggesting that AdpA is an absolute prerequisite for sprT expression. The sprT transcript was detected at a high level only in the wild-type strain, but the sgtR1 and sgtR2 transcript levels were very similar between the S. griseus IFO13350 and Delta adpA strains. This clearly demonstrates that the transcription of the sgtR1 and sgtR2 genes is not dependent on AdpA and that they are therefore not members of the AdpA regulon.

Role of sigmaH paralogs in intracellular melanin formation and spore development in Streptomyces griseus.

Streptomyces griseus possesses multiple stress-response sigma factors including sigma(H). Previously, we have suggested that sigma(H) and related sigma factors are involved in the developmental control of S. griseus. Herein, we studied the role of two sigma(H) paralogs--sigma(F) and sigma(N)--which are encoded in tandem coding sequences of sigF-sigN in S. griseus [sigma(N) has been described as sigma(L) previously (Gene 320:127, 2003)]. A sigF mutant produced decreased levels of intracellular melanin and formed irregular spores. A triple mutant for sigHNF exhibited defective melanin production. While sigN was transcribed by three tandem promoters during the early to late growth phases, sigF was transcribed in the late developmental phase by a single promoter. The activity of the promoter preceding the rpp operon (Prpp), which is responsible for the intracellular melanin biosynthesis, was decreased in the sigF mutant and abolished in the sigHNF, adpA and A-factor biosynthesis mutants. The in vitro transcription assay demonstrated that Esigma(F) transcribed the rpp promoter. Both Esigma(F) and Esigma(N) transcribed a sigma(H)-dependent promoter that preceded the sigH operon, and their activities were repressed by the addition of RshA, an anti-sigma(H) protein. Overall, the results suggest that the three sigma factors have similar functions and that they are required for spore development and pigmentation. The transcription of the rpp operon is regulated both by the stress-response sigma factors and the A-factor regulatory cascade.

The CarA/LitR-family transcriptional regulator: Its possible role as a photosensor and wide distribution in non-phototrophic bacteria.

LitR is a MerR-family regulator that controls light-induced carotenogenesis in Streptomyces coelicolor. The introduction of litR conferred photo-dependent transcription activities on Streptomyces griseus, which does not perform photo-responsive carotenogenesis. LitR discovers homologs encoded at illumination-related loci of various non-phototrophic bacterial genomes. Probably, LitR acts not only as a transcriptional regulator but also a photosensor.

Structure determination of germacrane-type sesquiterpene alcohols from an endophyte Streptomyces griseus subsp.

Three germacrane-type sesquiterpene alcohols were isolated from an endophyte of mangrove plant Kandelia candel. Their structures were characterized as 1(10)E,5E-germacradiene-11-ol (1), 1(10)E,5E-germacradiene-3,11-diol (2), 1(10)E,5E-germacradiene-2,11-diol (3) based on the extensive NMR studies. Among them, 2 and 3 are identified as new compounds.

Classification of Streptomyces griseus (Krainsky 1914) Waksman and Henrici 1948 and related species and the transfer of 'Microstreptospora cinerea' to the genus Streptomyces as Streptomyces yanii sp. nov.

A soil actinomycete, strain 80-133(T), with the non-validly published name 'Microstreptospora cinerea', was the subject of a polyphasic study designed to clarify its taxonomic status. Comparative 16S rRNA gene sequence studies indicated that the organism belonged to the genus Streptomyces, a result in line with previous chemotaxonomic and morphological data. The strain belonged to the Streptomyces griseus clade, but could be distinguished from representatives of species assigned to this taxon by using DNA-DNA relatedness and phenotypic data. In light of these findings, it is proposed that the organism should be recognized as a novel species of the genus Streptomyces. The name proposed for this taxon is Streptomyces yanii sp. nov., with isolate 80-133(T) (=AS 4.1146(T)=JCM 3331(T)) as the type strain. It was also shown that representative strains of Streptomyces argenteolus, Streptomyces caviscabies, S. griseus and Streptomyces setonii belong to the same genomic species and have key phenotypic properties in common. It is proposed that S. caviscabies and S. setonii should be considered as later heterotypic synonyms of S. griseus and that S. argenteolus AS 4.1693(T) should also be assigned to this taxon.

Structures of grixazone A and B, A-factor-dependent yellow pigments produced under phosphate depletion by Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) acts as a microbial hormone that induces morphological development and secondary metabolism in Streptomyces griseus. A diffusible yellow pigment is produced by S. griseus in an A-factor-dependent manner under phosphate depletion. Detailed analysis of the pigment production by S. griseus cultivated in minimal liquid medium containing different concentrations of phosphate showed that the pigment was actively produced in the presence of low concentrations of phosphate and the production of the pigment was completely repressed in the presence of 2.5 mM KH2PO4. HPLC analysis of the culture supernatant showed that the pigment consisted of two major, structurally related compounds and they were produced at different ratios depending on the concentration of phosphate in the medium. The structures of the two major compounds, designated as grixazone A and B, were determined by spectroscopic analyses as 1-[[2-(acetylamino)-2-carboxyethyl]thio]-2-amino-3-oxo-8-formyl-3H-phenoxiazine and 1-[[2-(acetylamino)-2-carboxyethyl]thio]-2-amino-3-oxo-8-carboxyl-3H-phenoxiazine, respectively. Grixazone A was a novel compound, although grixazone B was reported in a patent as a parasiticide produced by Streptomyces sp. DSM3813.

Transcriptional switch on of ssgA by A-factor, which is essential for spore septum formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers morphological development and secondary metabolism in Streptomyces griseus. A transcriptional activator (AdpA) in the A-factor regulatory cascade switches on a number of genes required for both processes. AdBS11 was identified in a library of the DNA fragments that are bound by AdpA and mapped upstream of ssgA, which is essential for septum formation in aerial hyphae. Gel mobility shift assays and DNase I footprinting revealed three AdpA-binding sites at nucleotide positions about -235 (site 1), -110 (site 2), and +60 (site 3) with respect to the transcriptional start point, p1, of ssgA. ssgA had two transcriptional start points, one starting at 124 nucleotides (p1) and the other starting at 79 nucleotides (p2) upstream of the start codon of ssgA. Of the three binding sites, only sites 1 and 2 were required for transcriptional activation of p1 and p2 by AdpA. The transcriptional switch on of ssgA required the extracytoplasmic function sigma factor, sigma(AdsA), in addition to AdpA. However, it was unlikely that sigma(AdsA) recognized the two ssgA promoters, since their -35 and -10 sequences were not similar to the promoter sequence motifs recognized by sigma(BldN), a sigma(AdsA) homologue of Streptomyces coelicolor A3(2). An ssgA disruptant formed aerial hyphae, but did not form spores, irrespective of the carbon source of the medium, which indicated that ssgA is a member of the whi genes. Transcriptional analysis of ssfR, located just upstream of ssgA and encoding an IclR-type transcriptional regulator, suggested that no read-through from ssfR into ssgA occurred, and ssgA was transcribed in the absence of ssfR. ssgA was thus found to be controlled by AdpA and not by SsfR to a detectable extent. SsfR appeared to regulate spore septum formation independently of SsgA or through interaction with SsgA in some unknown way, because an ssfR disruptant also showed a whi phenotype.

Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus.

To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors in Streptomyces, we examined total proteins of Streptomyces griseus by two-dimensional gel electrophoresis. Among five proteins that were present at high levels during sporulation but absent from vegetative cells, two of the proteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. The deduced amino acid sequence of the gene that encodes P3 (EshA) showed extensive similarity to proteins from mycobacteria and a cyanobacterium, Synechococcus, that are abundant during nutritional stress but whose functions are unknown. Uniquely among these proteins, EshA contains a cyclic nucleotide-binding domain, suggesting that the activity of EshA may be modulated by a cyclic nucleotide. The eshA gene was strongly expressed from a single transcription start site only during sporulation, and accumulation of the eshA transcript depended on a developmental gene, bldA. During submerged sporulation, a null mutant strain that produced no EshA could not extend sporogenic hyphae from new branch points but instead accelerated septation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA is required for the growth of sporogenic hyphae and localization of septation and spore maturation but not for spore viability.

The A-factor regulatory cascade and cAMP in the regulation of physiological and morphological development in Streptomyces griseus.

In the A-factor regulatory cascade leading to the onset of streptomycin biosynthesis and aerial mycelium formation in Streptomyces griseus, the A-factor receptor protein (ArpA) serves as a DNA-binding repressor and A-factor releases the repression by binding to ArpA and dissociating it from the DNA. Mutants defective in arpA therefore produce streptomycin and aerial hyphae in the absence of A-factor. A gene that inhibits streptomycin production and aerial hyphae formation in an arpA mutant was cloned on a high-copy-number plasmid and found to encode a eukaryotic-type adenylate cyclase (CyaA). Consistent with this, an exogenous supply of cAMP at high concentration almost abolished streptomycin production and aerial hyphae formation. On the other hand, cAMP at lower concentrations stimulated or accelerated these developmental processes. The effects of cAMP were detectable only in arpA mutants, and not in the wild -type strain; an exogenous supply of cAMP or cyaA disruption in the wild-type strain caused almost no effect on these phenotypes. Thus the effects of cAMP became apparent only in the arpA-defective background. cAMP at high concentrations inhibited stringent response factor ppGpp production, which is important for the onset of antibiotic biosynthesis. cAMP also influenced the timing of tyrosine phosphorylation of more than nine proteins. These findings show that a cAMP regulatory relay for physiological and morphological development functions in a concerted and interdependent way with other signal transduction pathways.

Cloning and characterization of the gene encoding penicillin-binding protein A of Streptomyces griseus.

An internal segment of the penicillin-binding protein gene, pbpA, of Streptomyces griseus was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. pbpA encodes a 485 amino acid sequence that conserves three motifs of PBPs, SXXK, SXN, and KTG. The pbpA gene was located downstream of a gene homologous to the Bacillus subtilis spoVE gene. The pbpA gene was disrupted by replacing an ApaI fragment of the pbpA gene in S. griseus chromosome with an apramycin resistance gene cassette or directly inserting this apramycin resistance gene cassette at the NcoI site of pbpA penicillin-binding domain. No obvious defects in growth, sporulation, or spore sonication resistance were observed in the constructed pbpA mutants, suggesting that PBPA is not essential for growth and sporulation under normal laboratory conditions in S. griseus.