Abstract
BackgroundIn bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome.ResultsWe developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication.ConclusionRegions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.
Highlights
In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome
MalI as a Fluorescent Reporter-Operator System (FROS) reporter system Several methodologies have been employed to examine nucleoid structure, one of which is the use of Fluorescent Reporter Operator Systems (FROS) [8,9,10,11,12]
20 DNA sites for MalI were incorporated into the MalI operator array (MalO), which was targeted to specific positions in the chromosome of E. coli strain MG1655 using the gene doctoring recombineering method [18]
Summary
Many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. We have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. We developed a FROS probe based on the E. coli MalI DNA binding protein [13, 14], fused to mCherry fluorescent protein, and its cognate DNA target site. In combination with a modified LacI:GFP FROS probe, we have tagged the chromosome of E. coli strain MG1655, adjacent to AraC regulated genes, and determined the relative cellular locations by fluorescence microscopy. This was evident in dividing cells, where it was observed that induction of transcription facilitated separation of newly-replicated sister chromatids
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