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

Makitrynskyy R, Ostash B, Tsypik O, Rebets Y, Doud E, Meredith T, Luzhetskyy A, Bechthold A, Walker S, Fedorenko V - Open Biol (2013)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Biotechnology, Ivan Franko National University of Lviv, Hrushevskoho st. 4, Lviv 79005, Ukraine.

ABSTRACT
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 tRNA(Leu)UAA 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.

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The bldAgh gene directly affects translation of moeE5. (a) RT-PCR analysis of moeE5, moeO5 and moeGT4 transcription in S. ghanaensis wild-type (WT) and bldA-deficient (ΔbldAgh) strains. Lane C-, negative control (rrnA amplification from RNA sample in absence of RT). (b) Western blot analysis of cell-free lysates from WT and ΔbldAgh strains. The lysates were obtained from mycelium harvested in moenomycin production phase (TSB, 72 h) and probed with anti-MoeE5 rabbit serum (raised as described in §5).
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RSOB130121F4: The bldAgh gene directly affects translation of moeE5. (a) RT-PCR analysis of moeE5, moeO5 and moeGT4 transcription in S. ghanaensis wild-type (WT) and bldA-deficient (ΔbldAgh) strains. Lane C-, negative control (rrnA amplification from RNA sample in absence of RT). (b) Western blot analysis of cell-free lysates from WT and ΔbldAgh strains. The lysates were obtained from mycelium harvested in moenomycin production phase (TSB, 72 h) and probed with anti-MoeE5 rabbit serum (raised as described in §5).

Mentions: The transcription and translation of several moe genes was analysed in further detail to determine whether the bldA mutation affected moenomycin production directly (by blocking the translation of UUA-containing moeO5 and moeE5 mRNAs) or indirectly (by arresting adpAgh expression). Semiquantitative RT-PCR analysis of moeO5, moeE5 and moeGT4 showed that their transcription was not decreased in ΔbldAgh; in fact, it appeared to be increased (figure 4). Western blots revealed that MoeE5 protein is present in the cell-free lysate of the wild-type strain, but not in that of ΔbldAgh (figure 4), indicating a direct regulatory influence on the expression of TTA-containing moe genes by tRNALeuUAA.Figure 4.


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

Makitrynskyy R, Ostash B, Tsypik O, Rebets Y, Doud E, Meredith T, Luzhetskyy A, Bechthold A, Walker S, Fedorenko V - Open Biol (2013)

The bldAgh gene directly affects translation of moeE5. (a) RT-PCR analysis of moeE5, moeO5 and moeGT4 transcription in S. ghanaensis wild-type (WT) and bldA-deficient (ΔbldAgh) strains. Lane C-, negative control (rrnA amplification from RNA sample in absence of RT). (b) Western blot analysis of cell-free lysates from WT and ΔbldAgh strains. The lysates were obtained from mycelium harvested in moenomycin production phase (TSB, 72 h) and probed with anti-MoeE5 rabbit serum (raised as described in §5).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814723&req=5

RSOB130121F4: The bldAgh gene directly affects translation of moeE5. (a) RT-PCR analysis of moeE5, moeO5 and moeGT4 transcription in S. ghanaensis wild-type (WT) and bldA-deficient (ΔbldAgh) strains. Lane C-, negative control (rrnA amplification from RNA sample in absence of RT). (b) Western blot analysis of cell-free lysates from WT and ΔbldAgh strains. The lysates were obtained from mycelium harvested in moenomycin production phase (TSB, 72 h) and probed with anti-MoeE5 rabbit serum (raised as described in §5).
Mentions: The transcription and translation of several moe genes was analysed in further detail to determine whether the bldA mutation affected moenomycin production directly (by blocking the translation of UUA-containing moeO5 and moeE5 mRNAs) or indirectly (by arresting adpAgh expression). Semiquantitative RT-PCR analysis of moeO5, moeE5 and moeGT4 showed that their transcription was not decreased in ΔbldAgh; in fact, it appeared to be increased (figure 4). Western blots revealed that MoeE5 protein is present in the cell-free lysate of the wild-type strain, but not in that of ΔbldAgh (figure 4), indicating a direct regulatory influence on the expression of TTA-containing moe genes by tRNALeuUAA.Figure 4.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Biotechnology, Ivan Franko National University of Lviv, Hrushevskoho st. 4, Lviv 79005, Ukraine.

ABSTRACT
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 tRNA(Leu)UAA 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.

Show MeSH
Related in: MedlinePlus