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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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Translation of AdpAgh and MoeE5 is strongly affected on bldAgh-minus background. WT, ΔabsB and ΔbldA correspond to wild-type, absBgh and bldAgh  mutant strains, respectively, of S. ghanaensis expressing gusA fused to tested genes along with their promoters. adpA and moeE5 correspond to genes adpAgh and moeE5, respectively. As a negative control, promoterless versions of the above genes were fused to gusA and introduced into respective strains; these constructs had marginal or no GusA activity.
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RSOB130121F6: Translation of AdpAgh and MoeE5 is strongly affected on bldAgh-minus background. WT, ΔabsB and ΔbldA correspond to wild-type, absBgh and bldAgh mutant strains, respectively, of S. ghanaensis expressing gusA fused to tested genes along with their promoters. adpA and moeE5 correspond to genes adpAgh and moeE5, respectively. As a negative control, promoterless versions of the above genes were fused to gusA and introduced into respective strains; these constructs had marginal or no GusA activity.

Mentions: The recently described β-glucuronidase (GusA) reporter system [36] was applied to investigate the functional connection between the aforementioned pleiotropic regulators and moe genes. First, we measured transcription from the promoter of key structural gene moeE5 (moeE5p) in all of the S. ghanaensis mutants. The wild-type strain had relatively high levels of transcription from moeE5p (see, for comparison, the activity of other SM gene promoters [36]), but we failed to detect transcription in the ΔadpAgh strain (figure 5). The moeE5 transcription was increased more than twofold and threefold from wild-type levels in S. ghanaensisΔabsBgh and ΔbldAgh strains, respectively (figure 5), in agreement with RT-PCR data (figure 4). While the pattern of moeEp activity in ΔadpAgh and ΔabsBgh is as anticipated [25], increased levels of moeE5p transcript in the ΔbldAgh are somewhat unexpected. A plausible explanation is that moeE5p might be a target of an as-yet-unknown repressor(s) positively regulated by BldA, in which case the deletion of bldA would remove the repressive signal. To further delineate the involvement of bldAgh in the translational regulation of moenomycin production, we analysed GusA activity of translational fusions of gusA to moeE5 (plasmid pmoeE5transl) and adpAgh in a ΔbldAgh background. We found no GusA activity in ΔbldAgh carrying moeE5–gusA fusion (figure 6), underscoring the essentiality of bldAgh for translation of the two UUA codons in moeE5 mRNA. Surprisingly, GusA activity was detected in the ΔbldAgh strain carrying adpAgh–gusA fusion, although it was much weaker (15-fold) than that in wild-type strain (figure 6). This observation can be attributed to mistranslation of adpAgh UUA codon in the absence of tRNALeuUAA [37,38]. As the expression of AdpA in other cases has been shown to be strictly dependent on BldA [23,32,39], our data set a precedent for this important group of pleiotropic activators.Figure 5.


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)

Translation of AdpAgh and MoeE5 is strongly affected on bldAgh-minus background. WT, ΔabsB and ΔbldA correspond to wild-type, absBgh and bldAgh  mutant strains, respectively, of S. ghanaensis expressing gusA fused to tested genes along with their promoters. adpA and moeE5 correspond to genes adpAgh and moeE5, respectively. As a negative control, promoterless versions of the above genes were fused to gusA and introduced into respective strains; these constructs had marginal or no GusA activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB130121F6: Translation of AdpAgh and MoeE5 is strongly affected on bldAgh-minus background. WT, ΔabsB and ΔbldA correspond to wild-type, absBgh and bldAgh mutant strains, respectively, of S. ghanaensis expressing gusA fused to tested genes along with their promoters. adpA and moeE5 correspond to genes adpAgh and moeE5, respectively. As a negative control, promoterless versions of the above genes were fused to gusA and introduced into respective strains; these constructs had marginal or no GusA activity.
Mentions: The recently described β-glucuronidase (GusA) reporter system [36] was applied to investigate the functional connection between the aforementioned pleiotropic regulators and moe genes. First, we measured transcription from the promoter of key structural gene moeE5 (moeE5p) in all of the S. ghanaensis mutants. The wild-type strain had relatively high levels of transcription from moeE5p (see, for comparison, the activity of other SM gene promoters [36]), but we failed to detect transcription in the ΔadpAgh strain (figure 5). The moeE5 transcription was increased more than twofold and threefold from wild-type levels in S. ghanaensisΔabsBgh and ΔbldAgh strains, respectively (figure 5), in agreement with RT-PCR data (figure 4). While the pattern of moeEp activity in ΔadpAgh and ΔabsBgh is as anticipated [25], increased levels of moeE5p transcript in the ΔbldAgh are somewhat unexpected. A plausible explanation is that moeE5p might be a target of an as-yet-unknown repressor(s) positively regulated by BldA, in which case the deletion of bldA would remove the repressive signal. To further delineate the involvement of bldAgh in the translational regulation of moenomycin production, we analysed GusA activity of translational fusions of gusA to moeE5 (plasmid pmoeE5transl) and adpAgh in a ΔbldAgh background. We found no GusA activity in ΔbldAgh carrying moeE5–gusA fusion (figure 6), underscoring the essentiality of bldAgh for translation of the two UUA codons in moeE5 mRNA. Surprisingly, GusA activity was detected in the ΔbldAgh strain carrying adpAgh–gusA fusion, although it was much weaker (15-fold) than that in wild-type strain (figure 6). This observation can be attributed to mistranslation of adpAgh UUA codon in the absence of tRNALeuUAA [37,38]. As the expression of AdpA in other cases has been shown to be strictly dependent on BldA [23,32,39], our data set a precedent for this important group of pleiotropic activators.Figure 5.

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