Dataset Information

Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

ABSTRACT: Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track single LacI molecules in live cells by stochastically photoactivating and observing fluorescent proteins individually, there are no limitations on the copy number of the protein under study; as a result, we were able to study the behavior of LacI in bacterial strains containing the natural copy numbers (?40 monomers), as well as in strains with much higher copy numbers due to LacI overexpression. Our results allowed us to determine the relative abundance of specific, near-specific, and non-specific DNA binding modes of LacI in vivo, showing that all these modes are operational inside living cells. Further, we examined the spatial distribution of LacI in live cells, confirming its specific binding to lac operator regions on the chromosome; we also showed that mobile LacI molecules explore the bacterial nucleoid in a way similar to exploration by other DNA-binding proteins. Our work also provides an example of applying tracking photoactivated localization microscopy to studies of low-copy-number proteins in living bacteria.

SUBMITTER: Garza de Leon F

PROVIDER: S-EPMC5390046 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

Garza de Leon Federico F Sellars Laura L Stracy Mathew M Busby Stephen J W SJW Kapanidis Achillefs N AN

Biophysical journal 20170401 7

Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track s ...[more]

PMID: 28402875

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Project description:Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonellaâs close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique. Overall design The total RNA was isolated from Salmonella using âRNeasy Mini Kitâ (QIAGEN). Organism used: Salmonella enterica serovar Typhimurium LT2 * Slides: Agilentâs arrays (8x15k) AMADID: NO: 17809 * Starting material: Salmonella RNA in nuclease-free water * RNA Samples used: Wild type (WT), WT + pTrc99A+LacI (PTRC), WT + pTrc99A-LacI(Delta lac) * Labeling kit: MessageAmpâ¢ II-Bacteria RNA Amplification Kit from Ambion Cat # AM1790 * Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA * Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 * Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 * RNA quality was checked using Bioanalyzer. The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with mutant and rescue experiments and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.

Dataset Information

Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

Publications

Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

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