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In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure.

McArthur M, Bibb M - Nucleic Acids Res. (2006)

Bottom Line: DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA.The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome.This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK.

ABSTRACT
For a bacterium, Streptomyces coelicolor A3(2) contains a relatively large genome (8.7 Mb) with a complex and adaptive pattern of gene regulation. We discovered a correlation between the physical structure of the S.coelicolor genome and the transcriptional activity of the genes therein. Twelve genes were surveyed throughout 72 h of growth for both in vivo sensitivity to DNase I digestion and levels of transcription. DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA. The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome. This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

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Related in: MedlinePlus

Gene expression during growth of S.coelicolor strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.
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fig1: Gene expression during growth of S.coelicolor strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.

Mentions: We performed a comparative study of in vivo DNase I-sensitivity and transcriptional activity with a panel of 12 genes. Expression data were collected at three time points during growth of S.coelicolor in liquid culture (Figure 1) corresponding to early rapid growth (T1), transition phase (T2) and stationary phase (T3) (identified by determining the wet mass of the culture per ml). The physiology of S.coelicolor changes in each of these phases, with the onset of secondary metabolism generally occurring as growth slows. Three pathway-specific regulatory genes were included in the panel that control the production of three distinct antibiotics and whose expression was known to increase as the culture reaches stationary phase: actII-orf4, the pathway-specific regulatory gene for the blue-pigmented polyketide actinorhodin (Act); redD, encoding the activator for production of the red-pigmented undecylprodiginine complex (Red); and cdaR, which controls production of the calcium-dependent antibiotic [CDA; see (3) for references]. The remaining genes were chosen for their different patterns of expression as determined in (9) using the same medium as in this study (R5). afsK, which also influences secondary metabolism, is expressed throughout growth. Genes expressed predominantly early in growth were pgk (phosphoglycerate kinase) and fba (fructose 1,6-bisphosphate aldolase), both involved in primary metabolism, while the major vegetative sigma factor gene, hrdB, commonly used as a reference for transcriptional analysis, and the highly expressed sti1, encoding a protease inhibitor, represented genes expressed at high levels throughout growth. Finally, rlpA and rpmG3, both encoding ribosomal proteins, the ribonuclease gene rbnH, and the histidine kinase gene afsQ2 were chosen as genes expressed at relatively low levels.


In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure.

McArthur M, Bibb M - Nucleic Acids Res. (2006)

Gene expression during growth of S.coelicolor strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636467&req=5

fig1: Gene expression during growth of S.coelicolor strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.
Mentions: We performed a comparative study of in vivo DNase I-sensitivity and transcriptional activity with a panel of 12 genes. Expression data were collected at three time points during growth of S.coelicolor in liquid culture (Figure 1) corresponding to early rapid growth (T1), transition phase (T2) and stationary phase (T3) (identified by determining the wet mass of the culture per ml). The physiology of S.coelicolor changes in each of these phases, with the onset of secondary metabolism generally occurring as growth slows. Three pathway-specific regulatory genes were included in the panel that control the production of three distinct antibiotics and whose expression was known to increase as the culture reaches stationary phase: actII-orf4, the pathway-specific regulatory gene for the blue-pigmented polyketide actinorhodin (Act); redD, encoding the activator for production of the red-pigmented undecylprodiginine complex (Red); and cdaR, which controls production of the calcium-dependent antibiotic [CDA; see (3) for references]. The remaining genes were chosen for their different patterns of expression as determined in (9) using the same medium as in this study (R5). afsK, which also influences secondary metabolism, is expressed throughout growth. Genes expressed predominantly early in growth were pgk (phosphoglycerate kinase) and fba (fructose 1,6-bisphosphate aldolase), both involved in primary metabolism, while the major vegetative sigma factor gene, hrdB, commonly used as a reference for transcriptional analysis, and the highly expressed sti1, encoding a protease inhibitor, represented genes expressed at high levels throughout growth. Finally, rlpA and rpmG3, both encoding ribosomal proteins, the ribonuclease gene rbnH, and the histidine kinase gene afsQ2 were chosen as genes expressed at relatively low levels.

Bottom Line: DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA.The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome.This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK.

ABSTRACT
For a bacterium, Streptomyces coelicolor A3(2) contains a relatively large genome (8.7 Mb) with a complex and adaptive pattern of gene regulation. We discovered a correlation between the physical structure of the S.coelicolor genome and the transcriptional activity of the genes therein. Twelve genes were surveyed throughout 72 h of growth for both in vivo sensitivity to DNase I digestion and levels of transcription. DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA. The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome. This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

Show MeSH
Related in: MedlinePlus