Comparison of actinomycin peptide synthetase formation in Streptomyces chrysomallus and Streptomyces antibioticus.

Actinomycin peptide synthetase genes constitute two oppositely oriented transcriptional units, acmADR, and acmBC, separated by a non-coding intergenic region. Gene constructs of the intergenic region together with its adjoining gene acmA or acmB from the actinomycin biosynthetic gene cluster of Streptomyces chrysomallus were transferred into Streptomyces lividans TK64. Each construct expressed the respective synthetase indicating divergent promoters. Primer extension revealed for both directions -10 and -35 boxes similar to σ70 -dependent promoters from Streptomyces and E. coli. No conspicuous regulatory sequences were detected. Accordingly, S. chrysomallus-grown in glucose-containing medium-produced the peptide synthetases AcmA and AcmB/C as well as actinomycin during logarithmic growth phase. Alignments with the corresponding intergenic region of the actinomycin biosynthetic gene cluster in Streptomyces antibioticus identified analogous -10 and -35 boxes of σ70 consensus sequence. However, in S. antibioticus-cultivated in the same conditions-AcmA and AcmB/C were at maximum activity in late log phase and actinomycin formation peaked in stationary phase. The different patterns of formation of actinomycin and its peptide synthetases encoded by the highly homologous actinomycin biosynthetic gene clusters in S. chrysomallus and S. antibioticus suggest strain-specific control of biosynthesis in agreement with absence of pathway-specific regulatory genes.

Neoantimycins A and B, Two Unusual Benzamido Nine-Membered Dilactones from Marine-Derived Streptomyces antibioticus H12-15.

An actinomycete strain (H12-15) isolated from a sea sediment in a mangrove district was identified as Streptomycesantibioticus on the basis of 16S rDNA gene sequence analysis as well as the investigation of its morphological, physiological, and biochemical characteristics. Two novel benzamido nonacyclic dilactones, namely neoantimycins A (1) and B (2), together with the known antimycins A1ab (3a,b), A2a (4), and A9 (5), were isolated from the culture broth of this strain. Compounds 1 and 2 are the first natural modified ATNs with an unusual benzamide unit. The structures of these new compounds, including their absolute configuration, were established on the basis of HRMS, NMR spectroscopic data, and quantum chemical ECD calculations. Their cytotoxicities against human breast adenocarcinoma cell line MCF-7, the human glioblastoma cell line SF-268, and the human lung cancer cell line NCI-H460 were also tested. All compounds exhibited mild cytotoxic activity. However, Compounds 1 and 2 showed no activity against C. albicans at the test concentration of 1 mg/mL via paper disc diffusion, while the known antimycins showed obvious antifungal activity.

Nitrite formation from organic nitrogen by Streptomyces antibioticus supporting bacterial cell growth and possible involvement of nitric oxide as an intermediate.

The actinomycete Streptomyces antibioticus was shown to produce nitrite (NO-(2)) and ammonium (NH+(4)]) when aerobically incubated in an organic nitrogen-rich medium. The production of NO-(2) was synchronized with rapid cell growth, whereas most NH+(4)] was produced after cell proliferation had ceased. Intracellular formation of nitric oxide (NO) was also observed during the incubation. The production of these inorganic nitrogen compounds along with cell growth was prevented by several enzyme inhibitors (of nitric oxide synthase or nitrate reductase) or glucose. Distinct, membrane-bound nitrate reductase was induced in the NO-(2)-producing cells. Tungstate (a potent inhibitor of this enzyme) prevented the NO-(2) production and cell growth, whereas it did not prevent the NO formation. These results revealed the occurrence of novel nitrogen metabolic pathway in S. antibioticus forming NO-(2) from organic nitrogen by which rapid cell growth is possible. NO synthase, NO dioxygenase (flavohemoglobin), and dissimilatory nitrate reductase are possible enzymes responsible for the NO-(2) formation.

RNase III is required for actinomycin production in Streptomyces antibioticus.

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.

In vivo and in vitro patterns of the activity of simocyclinone D8, an angucyclinone antibiotic from Streptomyces antibioticus.

Simocyclinone D8 (SD8) exhibits antibiotic activity against gram-positive bacteria but not against gram-negative bacteria. The molecular basis of the cytotoxicity of SD8 is not fully understood, although SD8 has been shown to inhibit the supercoiling activity of Escherichia coli gyrase. To understand the mechanism of SD8, we have employed biochemical assays to directly measure the sensitivities of E. coli and Staphylococcus aureus type II topoisomerases to SD8 and microarray analysis to monitor the cellular responses to SD8 treatment. SD8 is a potent inhibitor of either E. coli or S. aureus gyrase. In contrast, SD8 exhibits only a moderate inhibitory effect on S. aureus topoisomerase IV, and E. coli topoisomerase IV is virtually insensitive to SD8. The antimicrobial effect of SD8 against E. coli has become evident in the absence of the AcrB multidrug efflux pump. As expected, SD8 treatment exhibits the signature responses to the loss of supercoiling activity in E. coli: upregulation of gyrase genes and downregulation of the topoisomerase I gene. Unlike quinolone treatment, however, SD8 treatment does not induce the SOS response. These results suggest that DNA gyrase is the target of SD8 in both gram-positive and gram-negative bacteria and that the lack of the antibacterial effect against gram-negative bacteria is due, in part, to the activity of the AcrB efflux pump.

A death round affecting a young compartmentalized mycelium precedes aerial mycelium dismantling in confluent surface cultures of Streptomyces antibioticus.

Development-associated cell-death processes were investigated in detail during the growth and differentiation of Streptomyces antibioticus ATCC 11891 on confluent surface cultures, by using fluorescent viability probes, membrane and activity fluorescence indicators, and electron microscopy analysis. A previously unsuspected complexity was revealed, namely the presence of a very young compartmentalized mycelium that dies following an orderly pattern, leaving alternating live and dead segments in the same hypha. This death round is followed by the growth of a second mycelium which develops rapidly from the live segments of the first mycelium and dies massively in a second death round, which extends over the phases of aerial mycelium formation and sporulation.

Mycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves.

BACKGROUND: The current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface. RESULTS: The work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 x 10(6) spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 x 10(3) spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases. CONCLUSION: An early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.

Cloning and characterization of a Streptomyces antibioticus ATCC11891 cyclophilin related to Gram negative bacteria cyclophilins.

Cyclophilins are folding helper enzymes and represent a family of the enzyme class of peptidyl-prolyl cis-trans isomerases. Here, we report the molecular cloning and biochemical characterization of SanCyp18, an 18-kDa cyclophilin from Streptomyces antibioticus ATCC11891 located in the cytoplasm and constitutively expressed during development. Amino acid sequence analysis revealed a much higher homology to cyclophilins from Gram negative bacteria than to known cyclophilins from Streptomyces or other Gram positive bacteria. SanCyp18 is inhibited weakly by CsA, with a K(i) value of 21 microM, similar to cyclophilins from Gram negative bacteria. However, this value is more than 20-fold higher than the K(i) values reported for cyclophilins from other Gram positive bacteria, which makes SanCyp18 unique within this group. The presence of SanCyp18 in Streptomyces is likely due to horizontal gene transmission from Gram-negative bacteria to Streptomyces.

Isolation of flavohemoglobin from the actinomycete Streptomyces antibioticus grown without external nitric oxide stress.

A flavocytochrome protein was isolated from the actinomycete Streptomyces antibioticus. The purified protein contained protoheme and FAD, and its M(r) was estimated to be 52000. The absorption spectra in its resting oxidized, dithionite-reduced, carbon monoxide-bound, and oxygenated (O(2)-bound) forms were characteristic of those of flavohemoglobin (Fhb). The N-terminal amino acid sequence showed high identities to those of other Fhb's. Furthermore, the actinomycete flavocytochrome scavenged nitric oxide in the presence of NADH. These results demonstrated that the flavocytochrome is the first Fhb purified from actinomycetes. The actinomycete Fhb was produced in S. antibioticus cells in large amounts without any external nitric oxide (NO) stress, which is indicative of a physiological function of Fhb other than detoxification of NO.

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels.

The pnp gene, encoding the enzyme polynucleotide phosphorylase (PNPase), was overexpressed in the actinomycin producer Streptomyces antibioticus. Integration of pIJ8600, bearing the thiostrepton-inducible tipA promoter, and its derivatives containing pnp into the S. antibioticus chromosome dramatically increased the growth rate of the resulting strains as compared with the parent strain. Thiostrepton induction of a strain containing pJSE340, bearing pnp with a 5'-flanking region containing an endogenous promoter, led to a 2.5-3 fold increase in PNPase activity levels, compared with controls. Induction of a strain containing pJSE343, with only the pnp ORF and some 3'-flanking sequence, led to lower levels of PNPase activity and a different pattern of pnp expression compared with pJSE340. Induction of pnp from pJSE340 resulted in a decrease in the chemical half-life of bulk mRNA and a decrease in poly(A) tail length as compared to RNAs from controls. Actinomycin production decreased in strains overexpressing pnp as compared with controls but it was not possible to attribute this decrease specifically to the increase in PNPase levels. Overexpression of pnp had no effect on ppGpp levels in the relevant strains. It was observed that the 3'-tails associated with RNAs from S. antibioticus are heteropolymeric. The authors argue that those tails are synthesized by PNPase rather than by a poly(A) polymerase similar to that found in Escherichia coli and that PNPase may be the sole RNA 3'-polynucleotide polymerase in streptomycetes.

Nuclease activities and cell death processes associated with the development of surface cultures of Streptomyces antibioticus ETH 7451.

The presence and significance of developmentally regulated nucleases in Streptomyces antibioticus ETH 7451 has been studied in relation to the lytic processes occurring during differentiation. The cell-death processes have been followed in surface cultures by a propidium iodide viability assay. This has allowed the visualization of dead (membrane-damaged, red fluorescent) and live (membrane-intact, green fluorescent) mycelium during development, and has facilitated the analysis of the role of nucleases in these processes. A parallel activity-gel analysis showed the appearance of 20-22 kDa, 34 kDa and 44 kDa nucleases, the latter appearing only when aerial mycelium is formed. The appearance of these nucleases shows a remarkable correlation with the death process of the mycelium during differentiation and with chromosomal DNA degradation. The 20-22 kDa enzymes are possibly related to the lytic phenomena taking place in the vegetative substrate mycelium before the emergence of the reproductive aerial mycelium, whereas the function of the 44 kDa nuclease seems to be related to the sporulation step. The 20-22 kDa nucleases require Ca2+ for activity and are inhibited by Zn2+. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. Conceivably, these enzymes work together and co-ordinate to achieve an efficient hydrolysis of DNA from dying cells. The results show that the biochemical reactions related with the lytic DNA degradation during the programmed cell death are notably conserved in Streptomyces. Some of the features of the process and the biochemical characteristics of the enzymes involved are analogous to those taking place during the DNA fragmentation processes in eukaryotic apoptotic cells.

Functional analysis of OleY L-oleandrosyl 3-O-methyltransferase of the oleandomycin biosynthetic pathway in Streptomyces antibioticus.

Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl- and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co(2+). The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.

Simocyclinones: diversity of metabolites is dependent on fermentation conditions.

Simocyclinones, a novel group of angucyclinone antibiotics, are produced by Streptomyces antibioticus Tü 6040. The compounds show antibacterial and antitumor properties. In submerged cultivation, the production of simocyclinones is strongly dependent on the carbon and nitrogen sources used in a chemically defined medium. Productivity of distinct components and diversity of simocyclinone compounds are influenced by the medium composition. Four series of simocyclinone compounds were detected by high-performance liquid chromatography (HPLC) diode array detector (DAD) and HPLC electrospray ionization (ESI) mass spectrometry (MS) analysis, isolated and the structures determined by nuclear magnetic resonance (NMR) techniques. Under optimized conditions, simocyclinone D8 was produced in an amount of 300 mg l(-1) and simocyclinone C4 in a concentration up to 50 mg l(-1).

Microbial growth and production kinetics of Streptomyces antibioticus Tü 6040.

Streptomyces antibioticus Tü 6040 is the producer of simocyclinones, which belong to a novel family of angucyclinone antibiotics some of which show antitumor activities. Growth and antibiotic production is dependent on the medium composition, especially on the carbon and nitrogen source, and on the fermentation conditions. The best results with respect to antibiotic productivity were achieved using a chemically defined medium with glycerol and L-lysine as carbon and nitrogen source, respectively, in an airlift fermenter with minimised shear stress at low gas flow rates withour oxygen limitation. These conditions led to a homogeneous formation of pellets of 1-2 mm in diameter and guaranteed reproducible product yields of the main compound, simocyclinone D8, in the range of 300 mg/l.

Interspecies complementation in Saccharopolyspora erythraea : elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives.

Two glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as L-rhamnose attached at position C-3 of an erythronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3, 4-isomerase.

relA is required for actinomycin production in Streptomyces antibioticus.

The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an Mr of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relA produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote.

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.

Effect of 5-azacytidine and sinefungin on Streptomyces development.

The effect of two DNA-methyltransferase inhibitors, 5-azacytidine (5azaC) and sinefungin (Sf), on the development of Streptomyces antibioticus ETH7451 (Sa) was studied. Pulse labeling experiments and SDS-PAGE analysis of proteins from cells grown in sporulation synthetic medium showed that both inhibitors affect a limited number of systems. Synthesis of the antibiotic rhodomycin was increased in the presence of 5azaC. 5azaC also stimulated the production of actinorhodin in cultures of S. coelicolor A3(2) grown in minimal medium. The analog did not affect the expression of whiB and whiG, two sporulation genes from S. coelicolor A3(2) whose homologues are present in Sa. Overall results indicated that 5azaC and Sf affect specific events associated with differentiation and secondary metabolism in Streptomyces.

Nutrient optimization for production of broad spectrum antibiotic by Streptomyces antibioticus SR15.4.

Antibiotic production by Streptomyces antibioticus Sr15.4 was studied under various cultural conditions. During nutrient optimization it was found that the strain utilized glycerol as the best source of carbon at 1.044 molar level, and 0.020 molar arginine as the best source of nitrogen. The strain exhibited significant enhancement in antibiotic production when grown at pH 6.8.