DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

Summary

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

Abstract

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

Introduction

The ability of bacteria to survive and thrive is critically dependent on how well they are able to perceive and respond to perturbations in their environments, and this in turn is dependent on their signal transduction systems. The number of signaling systems a bacterium encodes has been called its “microbial IQ” and can be an indication of both variability of its environment and its ability to sense multiple signals and fine tune its response¹. Two component signal transduction systems (TCS) are the most prevalent signaling systems used by bacteria, and they consist of a histidine kinase (HK) that senses the external signal and transmits via ph....

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Protocol

Note: The protocol below is tailored for determination of gene targets of the RR DVU3023 from the bacterium Desulfovibrio vulgaris Hildenborough. It can be adapted to any other transcriptional regulator of interest.

1. Clone and Purify RR

1. Clone the RR gene, specifically DVU3023, from D. vulgaris Hildenborough into an Escherichia coli expression vector such that the gene is C-terminally His-tagged and expression is under the control of a T7 promoter.
   Note: Several cloning methods may be used and is determined by the researcher. Alternate affinity tags may also be used.
2. Transform the exp....

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Results

The above method was applied to determine the global gene targets of the RRs in the model sulfate reducing bacterium Desulfovibrio vulgaris Hildenborough⁷. This organism has a large number of TCSs represented by over 70 RRs, indicating the wide variety of possible signals that it senses and responds to. In vivo analyses on the functions of these signaling systems are hard to perform since their signals and thus their activating conditions are unknown. Here the DAP-chip method was used to dete.......

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Discussion

The DAP-chip method described here was successfully used to determine the gene targets for several RRs in Desulfovibrio vulgaris Hildenborough⁷ of which one is shown here as a representative result. For RR DVU3023, choosing a candidate gene target was straightforward. DVU3025 is located immediately downstream of the RR gene, and the RR and target genes are conserved in several Desulfovibrio species, and additionally DVU3025 has a predicted sigma54-dependent promoter. The EMSA provides a simpl.......

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Materials

Name	Company	Catalog Number	Comments
HisTrapFF column (Ni-Sepharose column)	GE Lfe Sciences, Pittsburgh, PA, USA	17-5255-01
Akta explorer (FPLC instrument)	GE Lfe Sciences, Pittsburgh, PA, USA
HiPrep 26/10 Desalting column	GE Lfe Sciences, Pittsburgh, PA, USA	17-5087-01
Qiaquick Gel extraction kit	Qiagen Inc, Valencia, CA, USA	28704
Biotin-labeled oligonucleotides	Integrated DNA Technologies	N/A
6% polyacrylamide-0.5x TBE precast mini DNA retardation gel	Life Technologies, Grand Island, NY, USA	EC63652BOX	Alternately, you can pour your own gel.
Nylon membrane	EMD Millipore, Billerica, MA, USA	INYC00010
Trans-Blot SD Semi-dry electrophoretic transfer cell	Biorad, Hercules, CA, USA	170-3940
Extra thick blot paper, 8 x 13.5 cm	Biorad, Hercules, CA, USA	170-3967
UV crosslinker Model XL-1000	Fisher Scientific	11-992-89
Nucleic Acid chemiluminescent detection kit (Pierce)	Thermo fisher Scientific, Rockford, IL, USA	89880
Ni-NTA agarose resin	Qiagen Inc, Valencia, CA, USA	30210
GenomePlex Whole genome amplification kit (Fragmentation buffer, library preparation buffer, library stabilization solution, library preparation enzyme, 10x amplification master mix, WGA polymerase)	Sigma-Aldrich, St. Louis, MO, USA	WGA2-50RXN
Nanodrop ND-1000	Thermo Scientific, Wilmington, DE, USA	For quantitation of DNA
Perfecta Sybr Green SuperMix, with ROX	Quanta biosciences	95055-500	Any Sybr Green PCR mix may be used
PlateMax Ultra clear heat sealing film for qPCR	Axygen
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96-well clear low profile PCR microplate	Life Technologies, Grand Island, NY, USA	PCR-96-LP-AB-C
Applied Biosystems StepOne Plus Real time PCR system	Life Technologies, Grand Island, NY, USA	4376600	Any real time PCR system may be used
Qiaquick PCR purification kit	Qiagen Inc, Valencia, CA, USA	28104	Any PCR clean up kit may be used
Cy3/Cy5-labeled nonamers	Trilink biotechnologies, San Diego, CA, USA	N46-0001, N46-0002
Klenow polymerase 50,000 U/ml, 3'-5' exo-	New England Biolabs, Ipswich, MA	M0212M
Hybridization system	Roche-Nimblegen, Madison, WI, USA	N/A	This company no longer makes arrays or related items, so alternate sources such as Agilent or Affymetrix will need to be used.
Custom printed microarrays and mixers	Roche-Nimblegen, Madison, WI, USA	N/A
Hybridization kit (2x Hybridization buffer, Hybridization component A, Alignment oligo)	Roche-Nimblegen, Madison, WI, USA	N/A
Wash buffer kit (10x Wash buffer I, II, III, 1 M DTT)	Roche-Nimblegen, Madison, WI, USA	N/A
GenePix 4200A microarray scanner	Molecular Devices, Sunnyvale CA, USA	This model has been replaced by superior ones
GenePix Pro microarray software	Molecular Devices, Sunnyvale CA, USA
Nimblescan v.2.4, ChIP-chip analysis software	Roche-Nimblegen, Madison, WI, USA	N/A