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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

Erythromycin production by S. erythraea NRRL2338 and derivative rif mutants on R3/1 or YS agar media. (A-B) Strains were grown for 7 days on R3/1 (A) or YS (B) agar media and antibiotic production was evaluated by microbiological assays. Data are shown as mean ± standard deviation of triplicate samples in representative experiments. Similar results were obtained in three independent experiments. (C) Pictures of S. erythraea NRRL2338 and rif derivatives rif1 and rif6 after 7 days growth on R3/1 solid medium. Note in rif6 the severe defect in aerial mycelium and spore formation.
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Figure 1: Erythromycin production by S. erythraea NRRL2338 and derivative rif mutants on R3/1 or YS agar media. (A-B) Strains were grown for 7 days on R3/1 (A) or YS (B) agar media and antibiotic production was evaluated by microbiological assays. Data are shown as mean ± standard deviation of triplicate samples in representative experiments. Similar results were obtained in three independent experiments. (C) Pictures of S. erythraea NRRL2338 and rif derivatives rif1 and rif6 after 7 days growth on R3/1 solid medium. Note in rif6 the severe defect in aerial mycelium and spore formation.

Mentions: Twenty spontaneous S. erythraea rif mutants were isolated on YS agar containing 36 μg ml-1 of rifampicin. By rapid screening on YS and R3/1 agar media, two of these mutants exhibited a clear higher-producing phenotype (rif1 and rif9), two mutants were severely impaired in the ability to produce erythromycin (rif6 and rif12), while the remaining others showed antibiotic yields slightly higher or lower with respect to that of the wild type strain (data not shown). Eight rif mutants, rif1, rif9, rif6, rif12, rif2, rif3, rif4 and rif5 were further analyzed (Figure 1). After a 7-day (168 h) incubation at 30°C on complex R3/1 solid medium, rif1 and rif9 produced an amount of antibiotic more than 4-fold higher than that of the parental strain (Figure 1A). In contrast, antibiotic production by rif6 and rif12 was barely detectable by microbiological assay. Antibiotic yields by rif2, rif3, rif4 and rif5 were almost identical and slightly lower than that of the wild type.


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)

Erythromycin production by S. erythraea NRRL2338 and derivative rif mutants on R3/1 or YS agar media. (A-B) Strains were grown for 7 days on R3/1 (A) or YS (B) agar media and antibiotic production was evaluated by microbiological assays. Data are shown as mean ± standard deviation of triplicate samples in representative experiments. Similar results were obtained in three independent experiments. (C) Pictures of S. erythraea NRRL2338 and rif derivatives rif1 and rif6 after 7 days growth on R3/1 solid medium. Note in rif6 the severe defect in aerial mycelium and spore formation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Erythromycin production by S. erythraea NRRL2338 and derivative rif mutants on R3/1 or YS agar media. (A-B) Strains were grown for 7 days on R3/1 (A) or YS (B) agar media and antibiotic production was evaluated by microbiological assays. Data are shown as mean ± standard deviation of triplicate samples in representative experiments. Similar results were obtained in three independent experiments. (C) Pictures of S. erythraea NRRL2338 and rif derivatives rif1 and rif6 after 7 days growth on R3/1 solid medium. Note in rif6 the severe defect in aerial mycelium and spore formation.
Mentions: Twenty spontaneous S. erythraea rif mutants were isolated on YS agar containing 36 μg ml-1 of rifampicin. By rapid screening on YS and R3/1 agar media, two of these mutants exhibited a clear higher-producing phenotype (rif1 and rif9), two mutants were severely impaired in the ability to produce erythromycin (rif6 and rif12), while the remaining others showed antibiotic yields slightly higher or lower with respect to that of the wild type strain (data not shown). Eight rif mutants, rif1, rif9, rif6, rif12, rif2, rif3, rif4 and rif5 were further analyzed (Figure 1). After a 7-day (168 h) incubation at 30°C on complex R3/1 solid medium, rif1 and rif9 produced an amount of antibiotic more than 4-fold higher than that of the parental strain (Figure 1A). In contrast, antibiotic production by rif6 and rif12 was barely detectable by microbiological assay. Antibiotic yields by rif2, rif3, rif4 and rif5 were almost identical and slightly lower than that of the wild type.

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