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Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production.

Carata E, Peano C, Tredici SM, Ferrari F, Talà A, Corti G, Bicciato S, De Bellis G, Alifano P - Microb. Cell Fact. (2009)

Bottom Line: In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected.In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants.At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy. alifano@ilenic.unile.it.

ABSTRACT

Background: There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.

Results: Spontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.

Conclusion: Rifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

No MeSH data available.


Related in: MedlinePlus

Microarray analysis of the most relevant DEGs. Visualization by dChip of the most relevant genes belonging to each of the four clusters formed by phase a-DEGs. Red = up-regulation; Green = down-regulation.
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Figure 5: Microarray analysis of the most relevant DEGs. Visualization by dChip of the most relevant genes belonging to each of the four clusters formed by phase a-DEGs. Red = up-regulation; Green = down-regulation.

Mentions: This cluster is the largest one and comprises 122 genes that were up-regulated in rif6 and not affected in the rif1 compared to the wild type (Figure 5, left panel). This cluster includes genes involved in amino acid biosynthesis (metH, hisC2, lat corresponding to SACE 3898, SACE 0217 and SACE 0784, respectively) and uptake (SACE 2830) and in fatty acid biosynthesis (SACE 1694 coding for putative long-chain fatty acid ligase). Cluster 1 includes also genes coding for putative stress proteins (smpB [SACE 1108], SACE 0034, uspA3 [SACE 2443], SACE 1331 and SACE 1340), transcriptional factors (SACE 2101 coding for the omega subunit of RNAP) and global transcriptional regulators (SACE 3299, SACE 4349, SACE 6128), and genes involved in amino acid (dapD [SACE 1013], hisF [SACE 5756], SACE 5263), vitamin (pdx1 [SACE 2009], folK [SACE 0400]) and nucleotide metabolism (purF [SACE 7125], pyrE [SACE 7189], adk [SACE 6812]). Other very relevant genes belonging to this cluster are: rpsA (SACE 5431) coding for S1, the largest ribosomal protein, and genes encoding proteins involved in carbon metabolism (eno [SACE 0838] coding for the phosphopyruvate hydratase, SACE 5675 coding for the pyruvate dehydrogenase complex, E1 component, beta subunit, and SACE 7048 encoding the 2,5-diketo-D-gluconic acid reductase) and energy re-generation (ctaE [SACE 1684] coding for the cytochrome C oxidase subunit III, qcrC [SACE 1685] coding for the cytochrome C mono- and di-heme variants, atpD [SACE 6280] and atpF [SACE 6284] coding for the ATP synthase beta and B chains, respectively).


Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production.

Carata E, Peano C, Tredici SM, Ferrari F, Talà A, Corti G, Bicciato S, De Bellis G, Alifano P - Microb. Cell Fact. (2009)

Microarray analysis of the most relevant DEGs. Visualization by dChip of the most relevant genes belonging to each of the four clusters formed by phase a-DEGs. Red = up-regulation; Green = down-regulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667423&req=5

Figure 5: Microarray analysis of the most relevant DEGs. Visualization by dChip of the most relevant genes belonging to each of the four clusters formed by phase a-DEGs. Red = up-regulation; Green = down-regulation.
Mentions: This cluster is the largest one and comprises 122 genes that were up-regulated in rif6 and not affected in the rif1 compared to the wild type (Figure 5, left panel). This cluster includes genes involved in amino acid biosynthesis (metH, hisC2, lat corresponding to SACE 3898, SACE 0217 and SACE 0784, respectively) and uptake (SACE 2830) and in fatty acid biosynthesis (SACE 1694 coding for putative long-chain fatty acid ligase). Cluster 1 includes also genes coding for putative stress proteins (smpB [SACE 1108], SACE 0034, uspA3 [SACE 2443], SACE 1331 and SACE 1340), transcriptional factors (SACE 2101 coding for the omega subunit of RNAP) and global transcriptional regulators (SACE 3299, SACE 4349, SACE 6128), and genes involved in amino acid (dapD [SACE 1013], hisF [SACE 5756], SACE 5263), vitamin (pdx1 [SACE 2009], folK [SACE 0400]) and nucleotide metabolism (purF [SACE 7125], pyrE [SACE 7189], adk [SACE 6812]). Other very relevant genes belonging to this cluster are: rpsA (SACE 5431) coding for S1, the largest ribosomal protein, and genes encoding proteins involved in carbon metabolism (eno [SACE 0838] coding for the phosphopyruvate hydratase, SACE 5675 coding for the pyruvate dehydrogenase complex, E1 component, beta subunit, and SACE 7048 encoding the 2,5-diketo-D-gluconic acid reductase) and energy re-generation (ctaE [SACE 1684] coding for the cytochrome C oxidase subunit III, qcrC [SACE 1685] coding for the cytochrome C mono- and di-heme variants, atpD [SACE 6280] and atpF [SACE 6284] coding for the ATP synthase beta and B chains, respectively).

Bottom Line: In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected.In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants.At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy. alifano@ilenic.unile.it.

ABSTRACT

Background: There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.

Results: Spontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.

Conclusion: Rifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

No MeSH data available.


Related in: MedlinePlus