Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12
Abstract Background In 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 reporter...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2017-08-01
|
| Series: | BMC Microbiology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12866-017-1079-2 |
| _version_ | 1819053505798733824 |
|---|---|
| author | Laura E. Sellars Jack A. Bryant María-Antonia Sánchez-Romero Eugenio Sánchez-Morán Stephen J. W. Busby David J. Lee |
| author_facet | Laura E. Sellars Jack A. Bryant María-Antonia Sánchez-Romero Eugenio Sánchez-Morán Stephen J. W. Busby David J. Lee |
| author_sort | Laura E. Sellars |
| collection | DOAJ |
| description | Abstract Background In 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. Results We 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. Conclusion Regions 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. |
| first_indexed | 2024-12-21T12:36:48Z |
| format | Article |
| id | doaj.art-cd26fb8c69544403a0d59e804d2bd85f |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-12-21T12:36:48Z |
| publishDate | 2017-08-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-cd26fb8c69544403a0d59e804d2bd85f2022-12-21T19:03:53ZengBMCBMC Microbiology1471-21802017-08-0117111010.1186/s12866-017-1079-2Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12Laura E. Sellars0Jack A. Bryant1María-Antonia Sánchez-Romero2Eugenio Sánchez-Morán3Stephen J. W. Busby4David J. Lee5Institute of Microbiology and Infection, School of Biosciences, University of BirminghamInstitute of Microbiology and Infection, School of Biosciences, University of BirminghamDepartamento de Genética, Facultad de Biología, Universidad de SevillaSchool of Biosciences, University of BirminghamInstitute of Microbiology and Infection, School of Biosciences, University of BirminghamInstitute of Microbiology and Infection, School of Biosciences, University of BirminghamAbstract Background In 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. Results We 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. Conclusion Regions 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.http://link.springer.com/article/10.1186/s12866-017-1079-2FROSGFPFluorescent microscopyChromosomeNucleoidEscherichia coli |
| spellingShingle | Laura E. Sellars Jack A. Bryant María-Antonia Sánchez-Romero Eugenio Sánchez-Morán Stephen J. W. Busby David J. Lee Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12 BMC Microbiology FROS GFP Fluorescent microscopy Chromosome Nucleoid Escherichia coli |
| title | Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12 |
| title_full | Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12 |
| title_fullStr | Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12 |
| title_full_unstemmed | Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12 |
| title_short | Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12 |
| title_sort | development of a new fluorescent reporter operator system location of arac regulated genes in escherichia coli k 12 |
| topic | FROS GFP Fluorescent microscopy Chromosome Nucleoid Escherichia coli |
| url | http://link.springer.com/article/10.1186/s12866-017-1079-2 |
| work_keys_str_mv | AT lauraesellars developmentofanewfluorescentreporteroperatorsystemlocationofaracregulatedgenesinescherichiacolik12 AT jackabryant developmentofanewfluorescentreporteroperatorsystemlocationofaracregulatedgenesinescherichiacolik12 AT mariaantoniasanchezromero developmentofanewfluorescentreporteroperatorsystemlocationofaracregulatedgenesinescherichiacolik12 AT eugeniosanchezmoran developmentofanewfluorescentreporteroperatorsystemlocationofaracregulatedgenesinescherichiacolik12 AT stephenjwbusby developmentofanewfluorescentreporteroperatorsystemlocationofaracregulatedgenesinescherichiacolik12 AT davidjlee developmentofanewfluorescentreporteroperatorsystemlocationofaracregulatedgenesinescherichiacolik12 |