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Desferrioxamines (DF's) are siderophores produced by some groups of bacteria. Previously, we discovered that DFE, produced by Streptomyces griseus, induced divergent developmental phenotypes in various Streptomyces isolates. In this study, we isolated bacteria whose phenotype was affected by the presence of 0.1 mM DFB from soil samples, and studied their phylogenetic position via 16 S rRNA gene-based analysis. Isolates belonging to Microbacterium grew only in the presence of DFB in the medium. DFB promoted growth of some isolates, while significantly inhibiting that of other divergent bacteria. Different groups of isolates were affected, not because of growth-related changes, but because of changes in the colony morphology based on possible stimulation of motility. An isolate affiliated with Janthinobacterium was stimulated for violacein production as well as for pilus formation. The wide and divergent effects of DFB suggest that availability of siderophores significantly affect the structure of microbial community.

Today, the frequency of discovery of new antibiotics in microbial culture is significantly decreasing. The evidence from whole-genome surveys suggests that many genes involved in the synthesis of unknown metabolites do exist but are not expressed under conventional cultivation conditions. Therefore, it is urgently necessary to study the conditions that make otherwise silent genes active in microbes. Here we overview the knowledge on the antibiotic production promoted by cocultivation of multiple microbial strains. Accumulating evidence indicates that cocultivation can be an effective way to stimulate the production of substances that are not formed during pure cultivation. Characterization of the promotive factors produced by stimulator strains is expected to give clues to the development of effective cultivation conditions for drug discovery.

Widespread interspecific stimulation of antibiotic production occurs in strains of Streptomyces owing to the activity of diffusible substances, as previously determined in our investigations of the cross-feeding effect. In this study, we newly isolated a substance produced by a Streptomyces strain closely related to Streptomyces scabrisporus, based on the observation that this substance induced the production of an unknown antibiotic in another strain related to Streptomyces griseorubiginosus. This substance, named promomycin, is a polyether structurally related to lonomycin. Promomycin itself had an antibiotic activity, but it stimulated antibiotic production in multiple Streptomyces strains at sub-inhibitory concentrations. Evidence implies that this stimulation effect is widespread within this group of bacteria.

Leptolstatin, a new inhibitor of the progression of G1 and G2 phases of the mammalian cell cycle, was discovered through a unique screening system, in which effects of microbial metabolites on the cell cycle progression of the cultured rat fibroblasts were monitored by flow cytometry. The new inhibitor was extracted from the fermentation broth of Streptomyces sp. SAM1595 with ethyl acetate, and purified by silica gel column chromatography and HPLC. Leptolstatin showed a strong cytostatic effect on rat normal fibroblasts 3Y1 with an IC50 value of 0.4 ng/ml, but its antimicrobial activity was very weak. A 24-hour treatment of the fibroblast cells with 10 ng/ml of leptolstatin caused an arrest at G1 or G2 phase, as determined by flow cytometry. When the G2-arrested cells were freed from leptolstatin, those containing 4C DNA entered a new S phase without intervening M phase, resulting in the formation of proliferative tetraploid cells.

Astaxanthin dirhamnoside, a new astaxanthin derivative produced by a radio-tolerant bacterium, Sphingomonas astaxanthinifaciens

Biological activities of four chemically synthesized trichostatin-related compounds, (R)-trichostatin A, (S)-trichostatin A, (R)-trichostatic acid, and (S)-trichostatic acid, were investigated. Assays of differentiation-inducing activity in Friend leukemia cells and G2-arresting activity in the cell cycle of normal rat fibroblast cells were used as monitoring systems for comparing the bioactivities of these compounds. The results clearly showed that both of the enantiomers of trichostatic acid had no activity in both the assay systems. In the case of (S)-trichostatin A, the antipode of naturally occurring trichostatin A, 50% effective concentrations were determined to be 50-70-fold higher than those of (R)-trichostatin A. The relationship between this ratio and the value of enantiomeric excess strongly suggests that (S)-trichostatin A is also biologically inactive. These results indicate that the absolute configuration and the presence of the hydroxamate group of trichostatin A are essential for its biological activity.

Nitrosoguanidine-induced melanin-negative mutants of Streptomyces griseus were classified according to their ability to produce streptomycin and A-factor, and to form aerial mycelium. A large proportion of the mutants showed deficiency in either antibiotic production and morphological development or both, suggesting close regulatory correlation between melanogenesis and morphological and physiological differentiation. The tyrosinase-encoding mel operon of S. griseus was cloned and examined for its role in melanogenesis of this organism. As in other Streptomyces homologues, the operon consisted of two open reading frames, melC1 encoding the putative cofactor and melC2 encoding the tyrosinase. Regardless of the distinct sequence similarity, introduction of the operon on plasmids failed to confer melanin production in the melanin-negative mutants, and the disruption of melC2 barely affected the melanin productivity, which indicated the presence of another enzyme involved in the melanogenesis in S. griseus. On the other hand, mel on a high-copy-number plasmid caused precocious aerial mycelium formation in Streptomyces lividans TK21 suggesting a stimulatory role of tyrosinase in morphological development.

A 7.0-kb DNA fragment that conferred redbrown pigment production on Streptomyces griseus was shotgun-cloned with a multicopy vector pIJ486 from this microorganism. By restriction endonuclease mapping and subcloning, a 1.5-kb fragment which is essential for the production of redbrown pigment was determined. The nucleotide sequence of this region revealed the presence of two open reading frames, ORF1 with 109 amino acids (named RppA) and ORF2 with 262 amino acids (RppB), in addition to a truncated ORF3. The termination codon of rppA and the initiation codon of rppB overlapped, sharing one common nucleotide, which strongly suggests that these two genes are cotranscribed. Both rppA and rppB were essentially required for the pigmentation. The RppB protein showed great similarity in amino acid sequence to a chalcone synthase, a key enzyme of central importance in the biosynthetic pathway of all classes of flavonoids in plants. Part of RppA showed sequence similarity to the 33kDa phosphoprotein of adenovirus. Nucleotide sequences homologous to rppA and rppB were widely distributed in Streptomyces species, as determined by Southern hybridization. Further nucleotide sequencing of the entire orf-3 gene showed that ORF3 with 403 amino acids was a cytochrome P-450 (named P-450RPP). These data suggested that the cloned fragment contained part of a gene cluster for the biosynthesis of a certain metabolite. Introduction of the subcloned 1.5-kb fragment into Streptomyces lividans as well as Escherichia coli also caused production of redbrown pigment, suggesting that RppA and RppB are capable of synthesizing the redbrown pigment from metabolites commonly present in bacteria.

Leptolstatin, a product from Streptomyces sp. SAM1595, is a gap phase-specific inhibitor of the mammalian cell cycle. Physico-chemical properties and spectrometric analyses showed that the structure of leptolstatin is (2Z,6E,8Z,12E,14E,22E)-19,24-dihydro xy-8,10,14,16,18,20,22-heptamethyl-17-oxo-2 , 6,8,12,14,22-tetracosahexaen-5-olide.

A strain of Streptomyces was found to produce new antifungal antibiotics. The active compounds were purified and separated into two substances named leptomycin A and B by high performance liquid chromatography. The molecular formulae of leptomycins A and B are C32H46O6 and C33H48O6 respectively, and physicochemical and biological properties of them are very similar to each other. Leptomycins A and B exhibit strong inhibitory activity against Schizosaccharomyces and Mucor.

Guanidylfungin A, C58H103N3O18, and guanidylfungin B, C57H101N3O18, were isolated from the mycelia of Streptomyces hygroscopicus No. 662 by means of silica gel absorption and reversed phase liquid chromatographies. The guanidylfungins were active against fungi and Gram-negative bacteria.

Mode of action of leptomycin B (LMB), a new antifungal antibiotic, was studied with Schizosaccharomyces pombe. A low concentration of LMB caused inhibition of cell division, producing elongated cells with morphologically altered nuclei and several cell plates, while it inhibited nucleic acid synthesis in intact cells at 100-fold higher concentration. Addition of LMB during G2 phase in synchronous culture blocked following events in cell cycle. Analysis of the effect of LMB on cdc mutants suggested the antibiotic inhibited some specific step, possibly in M phase just prior to nuclear division.

B-Factor, 3'-(1-butylphosphoryl)adenosine, which was isolated from yeast extract, is an inducer of rifamycin production in a rifamycin non-producing Nocardia mutant. Feeding of B-factor to the mutant culture demonstrated that the induction process was triggered during early stationary phase. Rifamycin production in the mutant was also induced by an exogenous supply of 3-amino-5-hydroxybenzoic acid, an intermediate of the antibiotic pathway, suggesting that a step upstream from the intermediate is regulated by B-factor. B-Factor analogues, i.e., alkylesters of 3'-AMP with alkyl side chains of C(2) approximately C(12) and n-butyl esters of 3'-GMP and 2'-AMP all showed the B-factor activity. Among these n-octyl ester of 3'-AMP showed the lowest effective concentration of approximately 3 x 10(-10) M. An intrinsic substance of the Nocardia sp. with potent B-factor activity and a UV absorption maximum at 260 nm was isolated from the cells of the parental strain.

A novel, chlorine containing sulfamoyl nucleoside antibiotics, AT-265, was found from Streptomyces rishiriensis subspecies and its structure and biological activities were determined, The antibiotic is active in vitro against some Enterobacteriaceae and some Gram-positive bacteria.

"Curing" treatment of a rifamycin-producing Nocardia sp. resulted in a mutant deficient in the synthesis of antibiotics. This deficiency was reversed in a medium containing yeast extract. The active substance, named B-factor, which induced rifamycin production in the mutant was purified from yeast extract, and its structure, 3'-(1-butylphosphoryl) adenosine, was determined by structural analysis and chemical synthesis. An extremely low concentration of B-factor (10 ng/ml) caused recovery of rifamycin B synthesis in the mutant and stimulated synthesis of the antibiotic in the parental strain.

The effect of A-factor on streptomycin resistance and productivity in Streptomyces griseus and S. bikiniensis was studied using A-factor-negative mutants. Resistance of several of these mutants was markedly increased by adding A-factor to the growing medium, as also was their streptomycin productivity. The A-factor induced resistance was due to inactivation by streptomycin-6-phosphotransferase, and enzyme synthesis in these mutants was completely dependent on the presence of A-factor. In the case of S. griseus 2247 where streptomycin productivity was independent of A-factor, resistance and synthesis of the inactivating enzyme were also independent of A-factor. A-Factor-negative mutants of S. griseus showed a decreased level of NADP-glycohydrolase and an increased level of several NADP-linked dehydrogenases, but these enzymes did not return to parental levels in cultures supplemented with A-factor. A-Factor seems to regulate streptomycin biosynthesis, not through an indirect metabolic sequence involving these enzymes but, more likely, by directly stimulating synthesis of enzyme(s) in the biosynthetic pathway.

The structures of guanidylfungins A and B were elucidated from the physico-chemical properties of these compounds and the structures of the degradation products by ozonolysis and periodate oxidation. The guanidylfungins consist of a 36-membered polyhydroxyl lactone ring, a guanidine and a monoester of malonic acid. The labelling experiments with sodium [1-13C]acetate and sodium [1-13C]propionate revealed that twelve units of acetate and nine of propionate were incorporated into the molecule of guanidylfungin A.

By cleavage with restriction endonucleases and cloning the resultant fragments, the A-factor determinant cloned from streptomycin-producing Streptomyces bikiniensis (Horinouchi et al., J. Bacteriol. 158: 481 approximately 487, 1984) was narrowed down to a 1.1-kilobase (kb) fragment. A hybrid multicopy plasmid (pAFB15) carrying the 1.1-kb fragment conferred A-factor production in a large quantity to A-factor-deficient mutants of S. bikiniensis and Streptomyces griseus as well as afsA mutants of Streptomyces coelicolor A3(2). A transcriptional control signal in the 1.1-kb fragment was identified by using a promoter-probe vector pARC 1. Plasmid pAFB15 also caused A-factor production with a marked gene dosage effect in four different Streptomyces strains which originally had no ability to produce A-factor and no DNA sequence homologous to the S. bikiniensis A-factor determinant, suggesting that precursors of A-factor are common metabolites in streptomycetes and that acquisition of only a single key enzyme encoded by the 1.1-kb fragment is sufficient for Streptomyces strains to synthesize A-factor.

The structures of new antifungal antibiotics, leptomycins A and B produced by Streptomyces sp. ATS1287 were determined as described below (Fig. 1) on the basis of their spectral and chemical character. Leptomycins have unique structures which belong to the unsaturated, branched-chain fatty acids with delta-lactone rings at the end.

Guanidylfungin A was chemically modified by alkylation, reduction and/or demalonylation. Demalonylmethylguanidylfungin A became soluble in water and showed approximately eight-fold higher activity against fungi and Gram-positive bacteria than guanidylfungin A along with strongly fungicidal effect. Similarly, copiamycin was converted to demalonylmethylcopiamycin, which also showed higher antifungal activity than copiamycin itself.

Strain S15-1 which produces antiviral antibiotic S15-1 belonging to the streptothricin group of antibiotics was isolated from a soil sample. Cell analysis, colony morphology, the absence of sporangia-like vesicles, the formation of spores in chains of the Rectus Flexibilis type, and the ability to produce melanoid pigment, indicate that this strain belongs to the genus Streptomyces Waksman and Henrici. A comparison of the characteristics of strain S15-1 with related Streptomyces show that it should be identified as Streptomyces purpeofuscus Yamaguchi and Saburi. The investigation of cultural conditions show that soluble starch and meat extract are the most suitable carbon and organic nitrogen sources for the production of antibiotic S15-1. Strain S15-1 grew poorly on media with no organic nitrogen sources, and did not produce the antibiotic. Antiviral antibiotic S15-1 is accumulated at the highest level after 3 or 4 days growth of the producing organism.

Ninety-five streptomycin-nonproducing mutants derived from Streptomyces griseus FT-1 by UV-irradiation could be classified into major two classes by cosynthesis tests. Class I mutants (42 strains) were mutants blocked in the pathway of streptomycin biosynthesis while class II mutants (49 strains) required a factor for streptomycin biosynthesis which was excreted by the parental or class I mutant strains. The factor could be replaced by synthetic A-factor (2S-isocapryloyl-3-S-hydroxymethyl-gamma-butyrolactone) which restored both streptomycin biosynthesis and spore formation in the class II mutants. A-Factor deficient mutants were obtained from several strains of S. griseus and S. bikiniensis at high frequency by treatment with acridine orange or incubation at high temperature. A mutant whose streptomycin biosynthesis was independent of A-factor deficiency was found. The production of A-factor was distributed among various species of actinomycetes.

A covalently closed circular (ccc) DNA with a size of 5.9 kb was isolated from a strain producing streptothricins , Streptomyces lavendulae No. 1080, and its restriction map was determined. Colonies of the plasmid-carrying strain formed pocks on the lawn of the plasmid-free derivatives of the same organism. The pock-forming ability of pTA4001 was confirmed by transformation of S. lividans with the plasmid DNA. Insertion of the chromosomal streptothricin resistance gene of S. lavendulae No. 1080 into pTA4001 gave various composite plasmids with marked deletion in S. lividans as a host. By analyzing these derivatives, a 2.2 kb region essential for replication and a possible region for pock formation were determined in the map of pTA4001 . A cloning vector, pKST2 (4.3 kb) containing resistance genes to streptothricin and thiostrepton with several unique cloning sites was constructed from pTA4001 .

The mode of action of tomaymycin (TM) which is a new antibiotic with antitumor activity was studied. Inhibition of the incorporation of labeled precursors into the acid-insoluble fraction of Bacillus subtilis cells indicated that TM inhibits nucleic acids biosynthesis, while having relatively little effect on protein synthesis. TM displaced methyl green (MG) from a MG-DNA complex and a TM-DNA complex was isolated by gel filtration chromatography. The formation of the TM-DNA complex resulted in (1) shifts of UV absorption profiles of TM and the corresponding difference spectra, (2) increase in the melting temperature of DNA, (3) protection of native DNA from degradation by nuclease O. These findings are compatible with the proposition that TM exerts its action as a growth inhibitor by forming a complex with DNA which in turn prevents the DNA from participating as a template in the biosynthesis of nucleic acids.

In the screening for antiviral antibiotics produced by Streptomyces soil isolates, several strains were found to produce cytotoxic and/or antiviral antibiotics. One antiviral antibiotic designated S-15-1 was isolated from soil isolate Streptomyces S-15-1. The antibiotic S-15-1 shows noncytotoxic in vitro inhibitory activity against Newcastle disease virus, and it has also inhibitory activity against gram-positive, gram-negative bacteria as well as some yeasts.