Renata Novakova

Cloning and characterization of a polyketide synthase gene cluster involved in biosynthesis of a proposed angucycline-like polyketide auricin in Streptomyces aureofaciens CCM 3239.

A new polyketide gene cluster, aur1, was identified in Streptomyces aureofaciens CCM3239 by using genes for the spore-pigment polyketide synthase of the Streptomyces coelicolor whiE operon as a probe. Sequence analysis of three overlapping DNA fragments (encompassing 15,100 bp) revealed 15 open reading frames, the majority of which showed high similarity to the previously characterized type II polyketide synthase genes. The highest similarity was to three Streptomyces polyketide gene clusters involved in biosynthesis of angucycline antibiotics, jadomycin, urdamycin and landomycin. The proposed S. aureofaciens ketosynthase (Aur1D) was phylogenetically more related to all known ketosynthases for polyketide antibiotics in Streptomyces than to spore-pigment ketosynthases. Interestingly, the aur1 gene cluster contained a gene encoding a proposed malonyl-CoA:ACP transacylase that has not been identified in any of the previously characterized type II polyketide synthase cluster. Transcriptional analysis of aur1 revealed a single promoter upstream the first open reading frame (the aur1A gene) that was active in all stages of differentiation with increased activity at the time of aerial mycelium formation. The aur1 gene cluster was disrupted by a homologous recombination, replacing the three genes (aur1B,C,D) including ketosynthase, with antibiotic resistance marker gene in S. aureofaciens chromosome. Disruption did not affect growth and differentiation; disrupted strain produced spores with wild-type gray-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild-type and aur1-disrupted strains revealed an antibacterial compound that was missing in the mutant. The results indicated a role of the S. aureofaciens aur1 gene cluster in biosynthesis of a polyketide secondary metabolite (which we named auricin), and not in the spore pigment biosynthesis.

TheStreptomyces aureofaciens homologue of thewhiB gene is essential for sporulation; Its expression correlates with the developmental stage

In previous experiments, aStreptomyces aureofaciens gene highly similar to the sporulation-specificwhiB gene ofStreptomyces cœlicolor was identified. By intergrative transformationvia double cross-over, a stable null mutant of thewhiB-homologous gene ofS. 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 thewhiB gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared fromS. aureofaciens in various developmental stages. Two putative promoters were identified upstream of thewhiB 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 thewhiB promoters were detected in anrpoZ-disruptedS. 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 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.

The role of the TetR-family transcriptional regulator Aur1R in negative regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239.

Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.

Identification and characterization of an indigoidine-like gene for a blue pigment biosynthesis in Streptomyces aureofaciens CCM 3239

An incomplete oligoketide (PK; ‘polyketide’) gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.

A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ

We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, PREN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of PREN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (Mr 30006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).

The dpsA Gene of Streptomyces coelicolor: Induction of Expression from a Single Promoter in Response to Environmental Stress or during Development

The DpsA protein plays a dual role in Streptomyces coelicolor, both as part of the stress response and contributing to nucleoid condensation during sporulation. Promoter mapping experiments indicated that dpsA is transcribed from a single, sigB-like dependent promoter. Expression studies implicate SigH and SigB as the sigma factors responsible for dpsA expression while the contribution of other SigB-like factors is indirect by means of controlling sigH expression. The promoter is massively induced in response to osmotic stress, in part due to its sensitivity to changes in DNA supercoiling. In addition, we determined that WhiB is required for dpsA expression, particularly during development. Gel retardation experiments revealed direct interaction between apoWhiB and the dpsA promoter region, providing the first evidence for a direct WhiB target in S. coelicolor.

Differential expression of two sporulation specific σ factors of Streptomyces aureofaciens correlates with the developmental stage

In previous experiments, the Streptomyces aureofaciens (Sa) rpoZ, and sigF genes have been shown to encode putative sigma factors essential in differentiation. In an attempt to investigate expression of these genes during differentiation, we have performed S1 nuclease mapping using RNA prepared from Sa in various developmental stages. Two putative promoters were identified upstream of the rpoZ coding region. The promoters significantly differed in their strength, and were active in distinct developmental stages; the weaker, rpoZ-P1, was active only in substrate mycelium, and the stronger, rpoZ-P2, was induced at the beginning of aerial mycelium formation. Transcriptional analysis of sigF revealed two apparent transcription start points, both being detectable only in the late phase of aerial mycelium formation. No sigF transcription was detected in an rpoZ-disrupted Sa strain. Promoter-bearing DNA fragments from rpoZ and sigF were inserted into several promoter-probe vectors, to give expression patterns consistent with the results of direct RNA analysis. The results implicate temporally different expression of rpoZ and sigF during the differentiation of Sa, and direct or indirect dependence of sigF expression on the rpoZ-encoded putative sigma factor, thus indicating a cascade of sigma factors in Streptomyces development.

Multiple regulatory genes in the salinomycin biosynthetic gene cluster ofStreptomyces albus CCM 4719

A DNA fragment containing part of the salinomycin biosynthetic gene cluster from industrial strainStreptomyces albus CCM 4719 was cloned. Sequence analysis of the 25.809-kbp fragment revealed the presence of 8 open reading frames (ORFs), including two large ORFs encoding three modular sets of oligoketide synthase, followed by three genes (salRI, salRII, salRIII) encoding transcriptional regulators. The first two regulators, SalRI and SalRII, belonged to the novel LAL family of large transcriptional regulators. SalRIII was highly similar to the NysRIV, AmphRIV, and FscRI transcriptional regulators from the oligoene macrolides nystatin, amphotericin, and R008/candicidin clusters, respectively.

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.