The polyene antifungal candicidin is selectively packaged into membrane vesicles in Streptomyces S4.

In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of environments and niches. While the precise mechanism of biogenesis is unknown, it is focused on the modification of the Gram-negative cell wall to facilitate blebbing at sites of weakness in and around the characteristically thin peptidoglycan layer within the periplasm. Here, we investigate the biogenesis of membrane vesicles (MVs) in the Gram-positive organism Streptomyces albus S4 (Seipke et al. J Bacteriol 193:4270-4271, 2011 and Fazal et al. Antonie Van Leeuwenhoek 113:511-520, 2020). The S. albus S4 strain is an antifungal (candicidin and antimycin) producing organism that was isolated from attine ants (Barke et al. BMC Biol 8:109, 2010). The biogenesis and characterisation of S. albus S4 MVs is demonstrated using the wild-type (WT) and mutant strains ΔantC (no antimycin production) ΔfscC (no candicidin production) and ΔantC ΔfscC (produces neither antimycin nor candicidin). Here, we have shown that the S. albus S4 strain produces MVs and that these are comprised of both specific protein profiles and secondary metabolites, with a clear demonstration of the ability to selectively package one antifungal (candicidin) but not the other (antimycin).

Candicidin Isomer Production Is Essential for Biocontrol of Cucumber Rhizoctonia Rot by Streptomyces albidoflavus W68.

Diseases caused by soilborne fungal pathogens result in significant crop yield losses and quality reduction. Streptomyces albidoflavus strain W68 is effective in controlling several soilborne fungal diseases. To identify antifungal substances critical for biocontrol activity of W68, the genome of W68 was sequenced and a linear chromosome of 6.80 Mb was assembled. A total of 21 secondary metabolite biosynthesis gene clusters (BGCs), accounting for 12.27% of the genome, were identified. Core gene deletion mutants for each of all 8 BGCs for nonribosomal peptide synthetases and polyketide synthases were created. Among them, only the mutant lacking ctg1-5755 (the gene was renamed as fscDW68) in BGC 19, which shares 100% sequence similarity with the BGC for candicidin synthesis, showed obvious reduction in antifungal activity. A pot experiment revealed that biocontrol effects of the ΔfscDW68 mutant in Rhizoctonia rot of cucumber were also significantly compromised relative to W68. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that W68 but not the ΔfscDW68 mutant can produce candicidin isomers, indicating that the production of candicidin isomers is key for antifungal activity and biocontrol activity of S. albidoflavus W68.IMPORTANCE This study reports that candicidin-like secondary metabolites produced by microbial cells in natural soil environments can effectively control soilborne fungal diseases, revealing a novel mechanism of microbial biocontrol agents. We demonstrated that the main antifungal activity and biocontrol activity of Streptomyces albidoflavus strain W68 are attributable to the production of candicidin isomers, suggesting that gene clusters for candicidin-like compound biosynthesis might be used as molecular markers to screen and breed microbial strains for biocontrol agent development.

The pH shift and precursor feeding strategy in a low-toxicity FR-008/candicidin derivative CS103 fermentation bioprocess by a mutant of Streptomyces sp. FR-008.

CS103, the novel derivative of polyene macrolides antibiotic FR-008/candicidin with lower toxicity has been isolated from the culture mycelia of the mutant of Streptomyces sp. FR-008, with targeted deletions of the fscP cytochrome P450 gene from its chromosome. To enhance biosynthesis of CS103, pH shift and precursor feeding strategy for fermentation process by the mutant of Streptomyces sp. FR-008 in a stirred tank bioreactor was developed. According to the process parameters analysis, the effectiveness of the strategy was examined and confirmed by experiments. A maximal CS103 concentration of 139.98 microg/mL was obtained, 2.05-fold higher than that in the pH-uncontrolled fermentation. Compared to other three cases as pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch cultures, the proposed "pH shift and precursor feeding strategy" effectively avoided the scarcity of the antibiotic precursor, increased the CS103 yield from biomass (Y (P/X)) and substrate (Y (P/S)) by 110.61% and 48.52%, respectively, and at the time the fermentation time was shortened from 120 to 96 h. The highest CS103 production rate (1.46 microg mL(-1) h(-1)) of the pH shift and precursor feeding strategy was 284.21%, 97.30%, and 58.70% higher than that of pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch culture cases, respectively.

Organizational and mutational analysis of a complete FR-008/candicidin gene cluster encoding a structurally related polyene complex.

The complete gene cluster for biosynthesis of a polyene complex, FR-008, spans 137.2 kb of the genome of Streptomyces sp. FR-008 consisting of six genes for a modular PKS and 15 additional genes. The extensive similarity to the partially characterized candicidin gene cluster in Streptomyces griseus IMRU3570, especially for genes involved in mycosamine biosynthesis, prompted us to compare the compounds produced by Streptomyces sp. FR-008 and Streptomyces griseus IMRU3570, and we found that FR-008 and candicidin complex are identical. A model for biosynthesis of a set of four structurally related FR-008/candicidin compounds was proposed. Deletion of the putative regulatory genes abolished antibiotic production, while disruption of putative glycosyltransferase and GDP-ketosugar aminotransferase functionalities led to the productions of a set of nonmycosaminated aglycones and a novel polyene complex with attachment of altered sugar moiety, respectively.

Three genes hrdB, hrdD and hrdT of Streptomyces griseus IMRU 3570, encoding sigma factor-like proteins, are differentially expressed under specific nutritional conditions.

Three genes (hrd) homologous to the rpoD gene of Escherichia coli, that encode sigma factor-like proteins, have been cloned from DNA of the candicidin-producing strain Streptomyces griseus IMRU 3570. They are located in different regions of the chromosome. Sequence analysis showed that the first one is analogous to the hrdB gene of S. coelicolor. The second showed high similarity to the hrdD gene of S. coelicolor and S. aureofaciens and is linked, as in S. coelicolor, to a N-acetyltransferase-encoding gene (nat) distantly related to the pat and bar genes that encode resistance to bialafos. The third showed no close homology with other known hrd genes from actinomycetes and has been named hrdT. Functional domains in the three S. griseus Hrd proteins are highly conserved in relation to those of the sigma 70 protein family. Northern analysis showed that hrdB is expressed as a 1.9-kb transcript during active growth in phosphate-rich medium, but it is less efficiently transcribed under sporulation conditions (phosphate-starved) or after a heat-shock treatment. Two other shorter transcripts of 1.2 and 0.7 kb were also detected with the same probe. The hrdD gene is transcribed as a single 1.1-kb transcript under sporulation conditions following nutritional shiftdown and, to a lower extent, during growth conditions in phosphate-rich medium. The hrdT gene is weakly transcribed (1.5-kb RNA) under all conditions tested. The hrd-encoded sigma factors probably recognize actinomycetes promoters (SEP type) with E. coli-like consensus sequences.

[Oxidative destruction of the heptaene antibiotic levorin]. / Izuchenie okislitel'noi destruktsii geptaenovogo antibiotika levorina.

Levorin destruction in oxygen, argon or air at various temperature levels was studied. It was found that destruction of the antibiotic molecule was mainly due to its interaction with oxygen. As a result the heptaenic chromophore was oxidized and triene and tetraene formed. Oxidation of levorin was accompanied by splitting out of paraaminoacetophenone. Destruction of levorin in the absence of oxygen was retarded.

[Mechanism of polyene antibiotic inactivation. The effect of amino sugar oxidation on polyene chain destruction]. / Izuchenie mekhanizma inaktivatsii polienovykh antibiotikov. Vliianie okisleniia aminosakhara na destruktsiiu polienovoi tsepi.

Changes in the acid-base characteristics of levorin during its inactivation are discussed. It is shown that with a decrease in the biological activity a change in the basic characteristics of the levorin amino sugar moiety takes place. The kinetic studies with model compounds, i. e. octaenic alcohol and glucosamine showed that the processes of their oxidation were interrelated. Oxidation of the amino sugar played the role of an inducing factor in oxidation of the polyenic chromophore and in addition impaired correlation between the extinction specific coefficient of levorin and its biological activity.

The antihypercholesterolemic activity of candicdin as a function of dietary cholesterol in cockerels.

In the first of two experiments using White Leghorn cockerels, a multifactorial analysis of variance was employed to characterize the antihypercholesterolemic responses (AHC) to candicidin (a heptaene macrolide antifungal antibiotic)-containing diets and the manner in which hypercholesterolemic challenges of different magnitudes modify these responses. In hypercholesterolemic diets, oral candicidin reduced plasma cholesterol concentration in a dose-dependent manner. The relative AHC effects of the diet were dependent on and varied directly with the presence and magnitude of the hypercholesterolemic challenge. The AHC effects of oral candicidin appeared to be additive to the homeostatic mechanisms limiting the rise in plasma cholesterol concentration induced by hypercholesterolemic diets. In the second experiment the absorption of oral 14C-candicidin was studied. Radioactivity above background levels was not found in any of the sampled tissues 4 days after administration of the 14C-candicidin pulse. The total cumulative recovery of radioactivity in the excreta was approximately 95% of the administered dose after 4 days. Within the limits of detection, oral candicidin did not appear to be absorbed from the intestine and appeared to be rapidly and quantitatively eliminated from the body in the feces. Chronic provision of candicidin appeared to increase transit time through the gastrointestinal tract.

Mode of action of the polyene antibiotic candicidin: binding factors in the wall of Candida albicans.

The polyene antibiotic candicidin produces a rapid efflux of K(+) ions from a suspension of Candida albicans. Onset of K(+) leakage depends on the culture age, stationary-phase yeasts leaking K(+) more slowly than exponential-phase yeasts. The time taken for potassium leakage to begin represents the time taken by the antibiotic to cross the cell wall and produce membrane damage. It was shown that there were factors in the cell wall of C. albicans that increased their total binding capacity and their affinity for candicidin during growth. An attempt was made to relate changes in the lipid content of the yeast cell with the increased time taken to produce membrane damage.

Factors affecting the production of candicidin.

Factors affecting candicidin synthesis and mycelial growth of Streptomyces griseus IMRU 3570 were studied. Inorganic phosphate was found to inhibit candicidin synthesis but to stimulate mycelial growth. Zinc, iron, and magnesium ions stimulated candicidin synthesis at relatively high concentrations in a complex medium but not in a synthetic medium. No other factors studied, such as temperature, oxygen absorption rate, and sugar concentration, were found to differentially affect antibiotic synthesis and mycelial growth. Optimum concentration of inorganic phosphate for candicidin synthesis in a chemically defined medium was found to be between 5 x 10(-5) and 5 x 10(-4) M. The culture in idiophase stage can be reverted to typical trophophase growth by the addition of inorganic phosphate, suggesting the controlling role of inorganic phosphate in repression and derepression of secondary metabolic and primary metabolic activity of the culture. With a soya peptone-glucose medium, the maximum rate of candicidin production could be maintained and extended for a considerable length of time by controlling the culture pH at 8.0, using glucose to adjust the pH during the later stages of a batch fermentation. Carrying out fermentations in this way has given candicidin yields up to 4 g/liter.