Yuriy Rebets

SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors

Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed.

Production of landomycins in Streptomyces globisporus 1912 and S cyanogenus S136 is regulated by genes encoding putative transcriptional activators.

The regulatory genes lanI and lndI have been cloned from the landomycin A (LaA) producer Streptomyces cyanogenus S136 and from the landomycin E (LaE) producer Streptomyces globisporus 1912, respectively and both have been sequenced. A culture of S. globisporus I2-1 carrying a disrupted lndI gene did not produce LaE and other related intermediates. Complementation of S. globisporus I2-1 with either the lndI or lanI gene reconstituted LaE production indicating that LanI and LndI are involved in activation of structural genes in the respective clusters. Structural features of these regulatory genes are discussed.

Genetic factors that influence moenomycin production in streptomycetes

Moenomycin, a natural phosphoglycolipid product that has a long history of use in animal nutrition, is currently considered an attractive starting point for the development of novel antibiotics. We recently reconstituted the biosynthesis of this natural product in a heterologous host, Streptomyces lividans TK24, but production levels were too low to be useful. We have examined several other streptomycetes strains as hosts and have also explored the overexpression of two pleiotropic regulatory genes, afsS and relA, on moenomycin production. A moenomycin-resistant derivative of S. albus J1074 was found to give the highest titers of moenomycin, and production was improved by overexpressing relA. Partial duplication of the moe cluster 1 in S. ghanaensis also increased average moenomycin production. The results reported here suggest that rational manipulation of global regulators combined with increased moe gene dosage could be a useful technique for improvement of moenomycin biosynthesis.

Actinomycetes biosynthetic potential: how to bridge in silico and in vivo?

Actinomycetes genome sequencing and bioinformatic analyses revealed a large number of “cryptic” gene clusters coding for secondary metabolism. These gene clusters have the potential to increase the chemical diversity of natural products. Indeed, reexamination of well-characterized actinomycetes strains revealed a variety of hidden treasures. Growing information about this metabolic diversity has promoted further development of strategies to discover novel biologically active compounds produced by actinomycetes. This new task for actinomycetes genetics requires the development and use of new approaches and tools. Application of synthetic biology approaches led to the development of a set of strategies and tools to satisfy these new requirements. In this review, we discuss strategies and methods to discover small molecules produced by these fascinating bacteria and also discuss a variety of genetic instruments and regulatory elements used to activate secondary metabolism cryptic genes for the overproduction of these metabolites.

Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNALeuUAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.

Primer preactivation of peptidoglycan polymerases.

Peptidoglycan glycosyltransferases are highly conserved bacterial enzymes that catalyze glycan strand polymerization to build the cell wall. Because the cell wall is essential for bacterial cell survival, these glycosyltransferases are potential antibiotic targets, but a detailed understanding of their mechanisms is lacking. Here we show that a synthetic peptidoglycan fragment that mimics the elongating polymer chain activates peptidoglycan glycosyltransferases by bypassing the rate-limiting initiation step.

Function of lanI in regulation of landomycin A biosynthesis in Streptomyces cyanogenus S136 and cross-complementation studies with Streptomyces antibiotic regulatory proteins encoding genes.

The transcriptional regulator of landomycin A biosynthesis encoded by lanI gene has been inactivated within the chromosome of Streptomyces cyanogenus S136. The obtained mutant strain did not produce landomycin A and its known intermediates. Loss of landomycin A production caused significant changes in morphology of the lanI deficient strain. RT-PCR analysis confirmed complete cessation of transcription of certain lan genes, including lanJ (encoding putative proton dependent transporter) and lanK (presumably involved in lanJ expression regulation). Introduction of either lanI or lndI [lanI homologue controlling landomycin E biosynthesis in Streptomyces globisporus 1912, both encoding Streptomyces antibiotic regulatory proteins (SARPs)] restored landomycin A production in the mutant strain. Chimeric constructs ladI and ladR were generated by exchanging the DNA sequences corresponding to N- and C-terminal parts of LndI and LanI. None of these genes were able to activate the production of landomycins in regulatory mutants of S. cyanogenus and S. globisporus. Nevertheless, the production of novel unidentified compound was observed in the case of S. cyanogenus harboring ladI gene. Various genes encoding SARPs have been expressed in S. globisporus and S. cyanogenus regulatory mutants and the results of these complementation experiments are discussed.

Moenomycin Resistance Mutations in Staphylococcus aureus Reduce Peptidoglycan Chain Length and Cause Aberrant Cell Division

Staphylococcus aureus is a Gram-positive pathogen with an unusual mode of cell division in that it divides in orthogonal rather than parallel planes. Through selection using moenomycin, an antibiotic proposed to target peptidoglycan glycosyltransferases (PGTs), we have generated resistant mutants containing a single point mutation in the active site of the PGT domain of an essential peptidoglycan (PG) biosynthetic enzyme, PBP2. Using cell free polymerization assays, we show that this mutation alters PGT activity so that much shorter PG chains are made. The same mutation in another S. aureus PGT, SgtB, has a similar effect on glycan chain length. Moenomycin-resistant S. aureus strains containing mutated PGTs that make only short glycan polymers display major cell division defects, implicating PG chain length in determining bacterial cell morphology and division site placement.

Targeted disruption of Streptomyces globisporus lndF and lndL cyclase genes involved in landomycin E biosynthesis

Streptomyces globisporus strains with knockouts inlndF andlndL genes, previously identified as possibly encoding cyclases governing cyclization of the nascent oligoketide (‘polyketide’) chain during the biosynthesis of the antitumor angucycline landomycin E, were prepared. On combining the results of sequence analysis and HPLC of extracts from mutant strains,lndL was suggested to control the first cyclization-aromatization event andlndF to be responsible for the 3rd–4th ring formation.

Actinobacteria Isolated from an Underground Lake and Moonmilk Speleothem from the Biggest Conglomeratic Karstic Cave in Siberia as Sources of Novel Biologically Active Compounds

Actinobacteria isolated from unstudied ecosystems are one of the most interesting and promising sources of novel biologically active compounds. Cave ecosystems are unusual and rarely studied. Here, we report the isolation and characterization of ten new actinobacteria strains isolated from an ancient underground lake and moonmilk speleothem from the biggest conglomeratic karstic cave in Siberia with a focus on the biological activity of the obtained strains and the metabolite dereplication of one active strain. Streptomyces genera isolates from moonmilk speleothem demonstrated antibacterial and antifungal activities. Some of the strains were able to inhibit the growth of pathogenic Candida albicans.