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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.


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Transcript analysis of the ery cluster and regulatory genes. (A) Microarray analysis. Visualization by dChip of the expression of the ery cluster (upper panels) and regulatory genes (lower panels) during the time course of the wild type strain (right panels) and during phase a in the wild type and the rif1 and rif6 mutants (left panels). Red = up-regulation; Green = down-regulation. (B). Semi-quantitative analysis of eryCII- and bldD-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.
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Figure 4: Transcript analysis of the ery cluster and regulatory genes. (A) Microarray analysis. Visualization by dChip of the expression of the ery cluster (upper panels) and regulatory genes (lower panels) during the time course of the wild type strain (right panels) and during phase a in the wild type and the rif1 and rif6 mutants (left panels). Red = up-regulation; Green = down-regulation. (B). Semi-quantitative analysis of eryCII- and bldD-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.

Mentions: For further analysis and discussion, we focused our attention on the 198 DEG of the growth phase a (see Additional file 1, Tables S2, S3, S4 and S5) when expression of erythromycin biosynthetic genes was maximal in the wild type strain (Figure 4A, right panel). Indeed, any comparison among the two mutant strains and the wild type during the phase b (see Additional file 1, Tables S6, S7, S8, S9 and S10) was considered uninformative because of the severe growth phenotype of rif6. The 198 DEG were grouped into four clusters (Cluster 1 to 4).


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)

Transcript analysis of the ery cluster and regulatory genes. (A) Microarray analysis. Visualization by dChip of the expression of the ery cluster (upper panels) and regulatory genes (lower panels) during the time course of the wild type strain (right panels) and during phase a in the wild type and the rif1 and rif6 mutants (left panels). Red = up-regulation; Green = down-regulation. (B). Semi-quantitative analysis of eryCII- and bldD-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Transcript analysis of the ery cluster and regulatory genes. (A) Microarray analysis. Visualization by dChip of the expression of the ery cluster (upper panels) and regulatory genes (lower panels) during the time course of the wild type strain (right panels) and during phase a in the wild type and the rif1 and rif6 mutants (left panels). Red = up-regulation; Green = down-regulation. (B). Semi-quantitative analysis of eryCII- and bldD-specific transcripts by RT real-time PCR. The RNAs were extracted from S. erythraea NRRL2338 and rif derivatives rif1 and rif6 grown in R3/1 medium up to phase a. Results were normalized to 16S rRNA levels. Transcript levels of S. erythraea NRRL2338 were arbitrarily given a value of 1. Data are shown as mean ± standard deviation from three independent experiments, each with triplicate samples, using distinct cDNA preparations for each RNA sample. The Student's t-test was used for statistical analysis. Statistically significant differences between values from S. erythraea NRRL2338 and rif mutants (asterisks) are declared at a p value < 0.05.
Mentions: For further analysis and discussion, we focused our attention on the 198 DEG of the growth phase a (see Additional file 1, Tables S2, S3, S4 and S5) when expression of erythromycin biosynthetic genes was maximal in the wild type strain (Figure 4A, right panel). Indeed, any comparison among the two mutant strains and the wild type during the phase b (see Additional file 1, Tables S6, S7, S8, S9 and S10) was considered uninformative because of the severe growth phenotype of rif6. The 198 DEG were grouped into four clusters (Cluster 1 to 4).

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