Intriguing properties of the angucycline antibiotic auricin and complex regulation of its biosynthesis.

Streptomyces bacteria are major producers of bioactive natural products, including many antibiotics. We identified a gene cluster, aur1, in a large linear plasmid of Streptomyces aureofaciens CCM3239. The cluster is responsible for the production of a new angucycline polyketide antibiotic auricin. Several tailoring biosynthetic genes were scatted in rather distant aur1 flanking regions. Auricin was produced in a very narrow growth phase interval of several hours after entry into stationary phase, after which it was degraded to non-active metabolites because of its instability at the high pH values reached after the production stage. Strict transcriptional regulation of the auricin biosynthetic gene cluster has been demonstrated, including feed-forward and feedback control by auricin intermediates via several of the huge number of regulatory genes present in the aur1 cluster. The complex mechanism may ensure strict confinement of auricin production to a specific growth stage.

The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239.

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

Activity of translation system and abundance of tmRNA during development of Streptomyces aureofaciens producing tetracycline.

Transition from exponential phase of growth to stationary phase in Streptomyces aureofaciens is characterized by a decrease in the rate of translation and induction of tetracycline (Ttc) biosynthesis. In exponential phase, no significant changes were found in the activity of ribosomes at binding of ternary complex Phe-tRNA.EF-Tu.GTP to the A-site on ribosomes. Overexpression of Ttc in stationary phase is accompanied by a decrease in the binding of the ternary complex Phe-tRNA.EF-Tu.GTP to the A-site of ribosome and a formation of an aggregate with Ttc by part of the ribosomes. Antibiotics that cause ribosome to stall or pause could increase the requirement for tmRNA in the process called trans-translation. We found differences in the level of tmRNA during the development of S. aureofaciens. Subinhibitory concentrations of Ttc, streptomycin and chloramphenicol induced an increase in the tmRNA level in cells from the exponential phase of growth. In vitro trans-translation system of S. aureofaciens was sensitive to Ttc at a concentration of > 15 micromol/L; the trans-translation system can thus be considered to contribute to resistance against Ttc produced only at sublethal concentrations. These experiments suggest that the main role of the rising tmRNA level at the beginning of the Ttc production is connected with ribosome rescue.

Secondary metabolites from endophytic Streptomyces aureofaciens CMUAc130 and their antifungal activity.

Streptomyces aureofaciens CMUAc130 was isolated from the root tissue of Zingiber officinale Rosc. (Zingiberaceae). It was an antagonist of Colletotrichum musae and Fusarium oxysporum, the causative agents of anthracnose of banana and wilt of wheat, respectively. Evidence for the in vitro antibiosis of S. aureofaciens CMUAc130 was demonstrated by the zone of fungal-growth inhibition. Microscopic observations showed thickness and bulbous structures at the edges of the inhibited fungal hyphae. The culture filtrate and crude extract from this strain were all inhibitory to tested phytopathogenic fungi. The major active ingredients from the culture filtrate of S. aureofaciens CMUAc130 were purified by silica gel-column chromatography and identified to be (i) 5,7-dimethoxy-4-p-methoxylphenylcoumarin and (ii) 5,7-dimethoxy-4-phenylcoumarin by NMR and mass-spectral data, respectively. Bioassay studies showed that compounds (i) and (ii) had antifungal activities against tested fungi, and their MICs were found to be 120 and 150 microg ml(-1), respectively. This is the first report of compounds (i) and (ii) from micro-organisms as active ingredients for the control of phytopathogenic fungi.

Characterization of the polyketide spore pigment cluster whiESa in Streptomyces aureofaciens CCM3239.

A spore pigment polyketide gene cluster, whiESa, was cloned from Streptomyces aureofaciens CCM3239 using a probe from the S. coelicolor A3(2) whiE gene cluster. Sequence analysis of a 4,657-bp DNA fragment revealed five open reading frames with the highest similarity to the S. coelicolor A3(2) whiE locus responsible for spore pigment biosynthesis, with conservation of the size and position of the genes. The whiESa gene cluster was disrupted by a homologous recombination in S. aureofaciens CCM3239, replacing the most important whiESaIII gene encoding ketosynthase with a thiostrepton resistance gene. The mutation affected spore pigmentation. In contrast to wild-type grey-pink spore pigmentation, the mutant produced white spores, although overall spore morphology was not affected. Transcriptional analysis of whiESa revealed two divergently oriented promoters, whiESap1 and whiESap2, upstream of the whiESaI and whiESaVIII genes, respectively. Both promoters were developmentally regulated in S. aureofaciens CCM3239. They were induced at the late stages of differentiation, during sporulation of aerial hyphae and were dependent upon early sporulation-specific sigma factor sigma(RpoZ) and putative transcription factor WhiB. The level of the transcript originating from the whiESap2 promoter was substantially reduced in a sigF mutant of S. aureofaciens CCM3239, indicating its dependence upon the late sporulation sigma factor sigma(F). Comparison of the whiE promoters in three different spore pigment polyketide clusters revealed a highly conserved region upstream of the -35 promoter region that may bind a transcriptional regulator.

[Application of element and metabolism balancing for the cultivation process with Streptomyces aureofaciens].

On the base of element and metablism balancing, the mathematical model of the cultivation process with Streptomyces aureofaciens was developed, and the unknown parameters in the model were estimated with the method of nonlinear optimization. Firstly the energetic coefficient of CTC biosynthesis was gained, which was 1.8 - 2.8 mol-ATP x C-mol(-1). The macroscopic reaction rates were predicted in the process and compared with the experimental values. The results show that the model can preferably describe the relationships between several macroscopic reaction rates in the process and can supervise the optimization of CTC fermentation process theoretically.

Some features of DNA-binding proteins involved in the regulation of the Streptomyces aureofaciens gap gene, encoding glyceraldehyde-3-phosphate dehydrogenase.

A gapR gene, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators, was previously identified upstream of the gap gene, coding for glyceraldehyde-3-phosphate dehydrogenase in Streptomyces aureofaciens. The GapR protein overproduced in Escherichia coli was shown to bind to the gap-P promoter region. Using the gel mobility shift assay with cell-free protein extracts from different developmental stages of S. aureofaciens, we identified several other proteins, in addition to GapR, that specifically bound to the S. aureofaciens gap-P promoter region. When cell-free extracts from S. aureofaciens cultivated in liquid medium with glucose were analyzed, only one complex corresponding to GapR was detected. A new protein interacting with the gap-P promoter was detected in stationary culture of S. aureofaciens grown in the presence of mannitol as carbon sources. The GapR protein was partially purified from S. aureofaciens cultivated in liquid medium containing glucose and used for binding studies. DNA footprinting analysis revealed an identical protected region as previously identified for the GapR protein overproduced from Escherichia coli. The direct role of the GapR protein in the regulation of gap expression in S. aureofaciens in vivo was confirmed but regulation of gap expression seems to be more complex, possibly involving other regulatory protein(s), depending on the developmental stage of S. aureofaciens.

[Study on effect of Vitreoscilla hemoglobin gene expression on growth and metabolism of Streptomyces aureofaciens].

Vitreoscilla hemoglobin gene was expressed in S. aureofaciens through promoter of tetracycline resistance gene. Characteristics of S. aureofaciens growth and metabolism were studied in 1 m3 fermenter. In high dissolved oxygen concentrations, expression of hemoglobin gene had little effect on growth and metabolism of S. aureofaciens, and there were no obvious differences between engineering strain and control. The chlortetracycline of engineering strain was 22905 u/mL and of control was 22896 u/mL respectively. Under conditions of low dissolved oxygen, expression of hemoglobin enhanced growth, maintenance of energetic mycelium configuration and chlortetracycline yield of S. aureofaciens: the mycelium concentrations of engineering strain were more about 5%-10% and yield was more 11.4% than control.

Mechanism of the incidental production of a melanin-like pigment during 6-demethylchlortetracycline production in Streptomyces aureofaciens.

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.

Expression of the Csp protein family upon cold shock and production of tetracycline in Streptomyces aureofaciens.

A shift down in temperature causes in Streptomyces aureofaciens a transient repression of polypeptide synthesis. During the acclimation phase 32 proteins were synthesized. The addition of tetracycline (200 microg/ml) to cells from exponential phase of growth leads to induction of 27 novel proteins and 17 upregulated proteins migrated in 2-D gel as proteins expressed upon cold shock. Immunoblot analysis using antibodies raised against CspB, CspC, and CspD of Bacillus subtilis revealed five cross-reactive proteins of the Csp family. Proteins CspB and CspD are predominantly induced at low temperature or by the presence of tetracycline. Expression of Csp proteins during the acclimation phase is regulated on the transcription level. Proteins of the Csp family have been shown to be associated with ribosomes and can be removed by 1 M NH(4)Cl. As expression of Csp proteins differs during development or temperature shift down, these proteins can be considered as trans-acting factors to form contacts with the coding region of specific mRNAs.

Iron deficiency-induced tetracycline production in submerged cultures by Streptomyces aureofaciens.

Streptomyces aureofaciens ATCC 10762 grown in rotary-shaken submerged cultures produced substantial amounts of tetracycline only when the defined medium was deprived of iron. The biosynthesis of tetracycline was inhibited either by free iron at concentrations above 1-2 mumol l-1, or by chelated iron provided by the siderophores of this bacterial strain. Late static iron-containing cultures allowed cell differentiation and sporulation and led to tetracyclines synthesis. A nitrosoguanidine-induced mutant able to synthesize tetracycline in the presence of iron in shaken submerged cultures was isolated and compared to the wild-type strain. However, no constitutive siderophore-mediated iron transport occurred in the mutant. These results suggest the involvement of a putative iron-controlled repressor in the biosynthesis of these secondary metabolites during vegetative growth and primary metabolism of the bacterium.

The Streptomyces aureofaciens homologue of the whiB gene is essential for sporulation; its expression correlates with the developmental stage.

In previous experiments, a Streptomyces aureofaciens gene highly similar to the sporulation-specific whiB gene of Streptomyces coelicolor was identified. By integrative transformation, via double cross-over, a stable null mutant of the whiB-homologous gene of S. aureofaciens was obtained. The disruption blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores, producing white aerial hyphae without septation. Expression of the whiB gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared from S. aureofaciens in various developmental stages. Two putative promoters were identified upstream of the whiB coding region. The stronger promoter, whiB-P2, was induced at the beginning of aerial mycelium formation, and the weaker promoter, whiB-P1, was expressed fairly constantly during differentiation. No differences in the expression of the whiB promoters were detected in an rpoZ-disrupted S. aureofaciens strain. The promoter bearing DNA fragment was inserted into the promoter-probe vector pARC1 to produce an expression pattern consistent with the results of direct RNA analysis.

The use of protein synthesis elongation factor EF-Tu as internal calibration standard in two-dimensional electrophoretic studies of differentiation in Streptomyces.

Protein synthesis elongation factor EF-Tu is presented as an internal calibration standard for quantitative analysis of two-dimensional (2-D) protein electrophoresis gels. EF-Tu was selected on the basis of concentration measurements in cell-free extracts from Streptomyces aureofaciens, grown under conditions leading to production of tetracyclines, and separated on one-dimensional (1-D) and 2-D electrophoresis gels. The results demonstrated that the amount of EF-Tu synthesized in S. aureofaciens under conditions of slow growth during production of tetracyclines is constant in proportion to all other de novo synthesized proteins regardless of their total number. This makes EF-Tu an ideal internal protein standard for quantitation of protein spots on 2-D electrophoresis gels. For such quantitative analysis we developed a computer-aided image analysis system which provides preparation of a gel image for further analysis including calibration, background subtraction and cleaning for streaking in both directions. The system can locate any resolvable spot in the gel and measure the integrated density of the spot, even in the case of irregular spot shape in crowded and overlapping spot regions.

Differential expression of principal sigma factor homologues of Streptomyces aureofaciens correlates with the developmental stage.

In previous experiments, Streptomyces aureofaciens has been shown to contain four genes hrdA, hrdB, hrdD, and hrdE, encoding polypeptides very similar to principal sigma factors of RNA polymerase. Two apparent tandem promoters were identified for each of the hrdA, hrdB and hrdD genes by S1 nuclease mapping using RNA prepared of S. aureofaciens in various developmental stages. Under all the conditions studied, tandem promoters of each gene differed significantly in their respective strengths. Transcription from the hrd promoters depended on developmental stage. While hrdB is transcribed from both promoters in all developmental stages, both tandem promoters of the hrdD gene are active only in vegetative stage and transcription of the hrdA tandem promoters temporally correlates with the aerial mycelium formation. In addition to a promoter, hrdB-P2, which lies upstream of the open reading frame, the hrdB gene, proposed to encode functional principal factor, appeared to contain at least one internal promoter, hrdB-P1. Activity of all promoters was consistent with S1 mapping experiments after insertion of promoter-bearing DNA fragments to promoter-probe vectors pIJ486 and pARC1. The results implicate temporally different expression of the hrd genes during the differentiation of S. aureofaciens.

[Development of the system for protoplast generation in Streptomyces aureofaciens strains--chlortetracycline producers]. / Razrabotka sistemy protoplastirovaniia u shtammov Streptomyces aureofaciens--produtsentov khlortetratsiklina.

Conditions for preparation and regeneration of protoplasts in a commercial strain of the culture producing chlortetracycline and its derivatives were determined. The protoplasting level depended on the conditions of the mycelium cultivation and composition of a regeneration medium. Under the optimal conditions it amounted up to 10(6) protoplasts/ml. A mutant able to form regenerating protoplasts at a rate of 10(9) protoplasts/ml was isolated. An autoinhibition effect in regenerating protoplasts was observed. As a result ofprotoplast generationing, the morphological variation increased and the protoplast antibiotic activity changed within wide ranges. Variants with higher productivity in comparison to that of the initial strain were isolated. Stability of the inherited property of antibiotic production in the variants is being studied.

Biogenesis of some antibiotics in the presence of 2-chloroethylphosphonic acid.

2-Chloroethylphosphonic acid (CEPA) affected both the growth of and antibiotic production in Streptomyces aureofaciens, S. griseus, S. antibioticus, and Penicillium citrinum. Streptomyces strains seemed to be more sensitive to the presence of CEPA in the medium than did the fungus. A decrease in both growth and antibiotic production was observed with a concomitant increase in the concentration of the ethylene-releasing compound in the medium. Higher concentrations of CEPA completely inhibited the growth of the above microorganisms.

Dynamic of antibiotic accumulation by Streptomyces aureofaciens in a laboratory fermentor.

In a laboratory fermentor, the growth of Streptomyces aureofaciens on corn meal extract and its production of antibiotic was investigated. Weak biosynthesis of the antibiotic started after 12 hours of incubation, when phosphorus depletion in the medium occurred. During the third and fourth day of fermentation about 80.4% of the antibiotic was produced. The relationship between growth, antibiotic formation, and the uptake of both sugar and nitrogen was also studied. A chromatogram, showing the types of sugar present during fermentation period is given. Improvement of antibiotic accumulation either by adding sugar at the end of the logarithmic phase of growth or by determining the rate of aeration is also discussed.

[Possibility of the spectral analysis of heterogeneous biological systems. The determination of the mycelium concentration of Actinomyces aureofaciens, a producer of tetracycline, cultured on a medium with corn meal]. / O vozmozhnosti spektral'nogo analiza geterogennykh biologicheskikh sistem. Opredelenie kontsentratsii mitseliia Actinomyces aureofaciens--produtsenta tetratsiklina, kul'tiviruemogo na srede s kukuruznoi mukoi.

A possibility of using spectroscopy of attenuated total reflection in the IR region for analysis of the heterogenic system consisting of the microorganisms and plant cells is discussed. The method of spectroscopy is proposed for estimating the mycelium concentration of Act. aureofaciens producing tetracycline in the presence of corn meal in the medium. The experimental data confirming this possibility are presented. The peculiar properties of the spectral analysis under these particular conditions are discussed. It is supposed that the method may be used for analysis of heterogenous systems including other microorganisms.