This preprint is under consideration at Microbial Cell Factories. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Martyna Gongerowska-Jac
University of Wroclaw: Uniwersytet Wroclawski
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3395-6610
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Identifying the regulatory factors that control transcriptional activity is a major challenge of gene expression studies. Here, we describe the application of a novel approach for in vivo identification of regulatory proteins that may directly or indirectly control the transcription of a promoter of interest.
Results
A method based on the combination of Tn5 minitransposon-driven random mutagenesis and lux reporter genes was applied for the first time for the Streptomyces genus. As a proof of concept, we studied the topA supercoiling-sensitive promoter, whose activity is dependent on unknown regulatory factors. We found that the sco4804 gene product positively influences topA transcription in S. coelicolor, demonstrating SCO4804 as a novel player in the control of chromosome topology in these bacteria.
Conclusions
Our approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.
Keywords
Streptomyces, transcription regulation, lux reporter gene, transposon mutagenesis, TopA
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
- GongerowskaJacFig.S1.pdf
PCR confirming the presence of the pFLUXH integrated vector in clones from the WT-lux-tn library. PCR was performed on S. coelicolor colonies using topA_p1_fw and luxC_rv oligonucleotides. The amplicon (499 bp) is marked with a black arrow. M - DNA molecular mass marker.
- GongerowskaJacFig.S2.pdf
Structural analysis of the SCO4804 protein using PredictProtein software. The image shows the localization of the predicted secondary structures, DNA-binding domains (RI - reliability index reflecting the strength of a prediction, high value means high confidence for binding) and the predicted disordered regions.
- GongerowskaJacFig.S3.pdf
DNA supercoiling in the SCO4804 overproducing strain. DNA supercoiling density of the reporter plasmid pWHM3Hyg isolated from the SCO4804 overproducing MG66 strain derivative (MG66_RP), induced with 10 µg/ml thiostrepton for 45 minutes or cultured for 24 hours in the presence of inducer compared to the non-induced control, the wild-type strain derivative (MS10) and the TopA-depleted strain derivative (MS11) (representative images of two independent replicates are shown). The figure shows topoisomers detected in agarose gel as well as band intensity measurements performed using ImageJ software.
- GongerowskaJacFig.S4.pdf
Purification of 6His-SCO4804 recombinant protein and DNA binding analysis. A. SDS-PAGE analysis of the collected fractions obtained during 6His-SCO4804 purification from E. coli BL21 (DE3) groEL-groES. M - Molecular mass marker, 1 - non-induced E. coli cell extract, 2 - induced E. coli cell extract in sarcosyl buffer, 3 - induced E. coli cell extract in binding buffer, 4 - induced E. coli cell extract in binding buffer, soluble fraction, 5 - flow-through, 6 - proteins bound to Ni-NTA agarose, 7 - wash with binding buffer with 40 mM imidazole, 8 - proteins eluted by 200 mM imidazole, 9 - the resin after elution. B. Electrophoretic mobility shift assay (EMSA) performed with 30 ng of 461 bp dsDNA fragment of the topA promoter and two negative controls as follows: negative control 1, a 415 bp DNA fragment encompassing the non-coding region between sco4696 and sco4697 genes, and negative control 2, a 654 bp fragment of the sco3928 gene. Binding was performed in PBS containing 5 mg/ml BSA, 5% (v/v) glycerol and, optionally, 2 ng/µl poly(dI-dC). The samples were resolved on a 5% polyacrylamide gel run at 4°C in 0.25× Tris-borate-EDTA (TBE) buffer (22.5 mM Tris, 22.5 mM boric acid, 0.5 mM EDTA) at 100 V for 3-4 hours. The bands were visualized with ethidium bromide solution that was incubated for 30 min at room temperature and with a ChemiDoc XRS+ system (Bio-Rad).
- GongerowskaJacFig.S5.pdf
Gel electrophoresis demonstrating TopA activity in the presence of 6His-SCO4804 recombinant protein. The assay was performed using 120 ng of TopA and 100 ng of pUC19 plasmid and increasing concentrations of 6His-SCO4804 recombinant protein. The reaction was incubated at 37°C for 15 minutes and subsequently stopped by the addition of 2 μl of 0.5 M EDTA. The samples were subsequently resolved on a 0.8% agarose gel in TAE buffer for 14–16 hours at low voltage (2 V/cm).
- GongerowskaJacFig.S6.pdf
Pull-down experiment using 6His-SCO4804 recombinant protein bound to Ni-NTA agarose resin. The resin was subsequently incubated with lysate of the TopA-induced PS04 strain (TopA+ lysate). The negative controls served as lysates of the TopA-depleted PS04 (TopA-lysate) strain loaded on 6His-SCO4804 – Ni-NTA agarose and Ni-NTA resin lacking immobilized 6His-SCO4804 recombinant protein. The incubation was performed in TN buffer with 40 mM imidazole. Elution of specifically bound protein was performed using 200 mM imidazole in TN buffer. The samples for the Western blot analysis were prepared using 15 µl of lysate fractions and unbound fraction, 10 µl of eluted proteins and 5 µl of resin as a bound fraction. Samples were resolved using SDS-PAGE and visualized using Western blot and anti-TopA polyclonal antibodies as well as 6xHis tag monoclonal antibody (MA1-135, Thermo Fisher Scientific) to confirm efficient 6His-SCO4804 binding to the resin and its subsequent elution.
- Additionalfile1.docx
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On 13 Mar, 2021
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Subject Areas
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This preprint is under consideration at Microbial Cell Factories. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Martyna Gongerowska-Jac
University of Wroclaw: Uniwersytet Wroclawski
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3395-6610
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 18 Mar, 2021
No community comments so far
Editorial decision: Major Revision
On 05 Apr, 2021
Review #2 received
Received 04 Apr, 2021
Review #1 received
Received 28 Mar, 2021
Reviewer #2 agreed
On 16 Mar, 2021
Reviewer #1 agreed
On 13 Mar, 2021
Reviewers invited
Invitations sent on 13 Mar, 2021
Reviews received
Received 13 Mar, 2021
Editor assigned
On 11 Mar, 2021
Editor invited
On 11 Mar, 2021
Submission checks complete
On 11 Mar, 2021
First submitted
On 11 Mar, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Applied & Industrial Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Identifying the regulatory factors that control transcriptional activity is a major challenge of gene expression studies. Here, we describe the application of a novel approach for in vivo identification of regulatory proteins that may directly or indirectly control the transcription of a promoter of interest.
Results
A method based on the combination of Tn5 minitransposon-driven random mutagenesis and lux reporter genes was applied for the first time for the Streptomyces genus. As a proof of concept, we studied the topA supercoiling-sensitive promoter, whose activity is dependent on unknown regulatory factors. We found that the sco4804 gene product positively influences topA transcription in S. coelicolor, demonstrating SCO4804 as a novel player in the control of chromosome topology in these bacteria.
Conclusions
Our approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
- GongerowskaJacFig.S1.pdf
PCR confirming the presence of the pFLUXH integrated vector in clones from the WT-lux-tn library. PCR was performed on S. coelicolor colonies using topA_p1_fw and luxC_rv oligonucleotides. The amplicon (499 bp) is marked with a black arrow. M - DNA molecular mass marker.
- GongerowskaJacFig.S2.pdf
Structural analysis of the SCO4804 protein using PredictProtein software. The image shows the localization of the predicted secondary structures, DNA-binding domains (RI - reliability index reflecting the strength of a prediction, high value means high confidence for binding) and the predicted disordered regions.
- GongerowskaJacFig.S3.pdf
DNA supercoiling in the SCO4804 overproducing strain. DNA supercoiling density of the reporter plasmid pWHM3Hyg isolated from the SCO4804 overproducing MG66 strain derivative (MG66_RP), induced with 10 µg/ml thiostrepton for 45 minutes or cultured for 24 hours in the presence of inducer compared to the non-induced control, the wild-type strain derivative (MS10) and the TopA-depleted strain derivative (MS11) (representative images of two independent replicates are shown). The figure shows topoisomers detected in agarose gel as well as band intensity measurements performed using ImageJ software.
- GongerowskaJacFig.S4.pdf
Purification of 6His-SCO4804 recombinant protein and DNA binding analysis. A. SDS-PAGE analysis of the collected fractions obtained during 6His-SCO4804 purification from E. coli BL21 (DE3) groEL-groES. M - Molecular mass marker, 1 - non-induced E. coli cell extract, 2 - induced E. coli cell extract in sarcosyl buffer, 3 - induced E. coli cell extract in binding buffer, 4 - induced E. coli cell extract in binding buffer, soluble fraction, 5 - flow-through, 6 - proteins bound to Ni-NTA agarose, 7 - wash with binding buffer with 40 mM imidazole, 8 - proteins eluted by 200 mM imidazole, 9 - the resin after elution. B. Electrophoretic mobility shift assay (EMSA) performed with 30 ng of 461 bp dsDNA fragment of the topA promoter and two negative controls as follows: negative control 1, a 415 bp DNA fragment encompassing the non-coding region between sco4696 and sco4697 genes, and negative control 2, a 654 bp fragment of the sco3928 gene. Binding was performed in PBS containing 5 mg/ml BSA, 5% (v/v) glycerol and, optionally, 2 ng/µl poly(dI-dC). The samples were resolved on a 5% polyacrylamide gel run at 4°C in 0.25× Tris-borate-EDTA (TBE) buffer (22.5 mM Tris, 22.5 mM boric acid, 0.5 mM EDTA) at 100 V for 3-4 hours. The bands were visualized with ethidium bromide solution that was incubated for 30 min at room temperature and with a ChemiDoc XRS+ system (Bio-Rad).
- GongerowskaJacFig.S5.pdf
Gel electrophoresis demonstrating TopA activity in the presence of 6His-SCO4804 recombinant protein. The assay was performed using 120 ng of TopA and 100 ng of pUC19 plasmid and increasing concentrations of 6His-SCO4804 recombinant protein. The reaction was incubated at 37°C for 15 minutes and subsequently stopped by the addition of 2 μl of 0.5 M EDTA. The samples were subsequently resolved on a 0.8% agarose gel in TAE buffer for 14–16 hours at low voltage (2 V/cm).
- GongerowskaJacFig.S6.pdf
Pull-down experiment using 6His-SCO4804 recombinant protein bound to Ni-NTA agarose resin. The resin was subsequently incubated with lysate of the TopA-induced PS04 strain (TopA+ lysate). The negative controls served as lysates of the TopA-depleted PS04 (TopA-lysate) strain loaded on 6His-SCO4804 – Ni-NTA agarose and Ni-NTA resin lacking immobilized 6His-SCO4804 recombinant protein. The incubation was performed in TN buffer with 40 mM imidazole. Elution of specifically bound protein was performed using 200 mM imidazole in TN buffer. The samples for the Western blot analysis were prepared using 15 µl of lysate fractions and unbound fraction, 10 µl of eluted proteins and 5 µl of resin as a bound fraction. Samples were resolved using SDS-PAGE and visualized using Western blot and anti-TopA polyclonal antibodies as well as 6xHis tag monoclonal antibody (MA1-135, Thermo Fisher Scientific) to confirm efficient 6His-SCO4804 binding to the resin and its subsequent elution.
- Additionalfile1.docx
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