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In search of a biosensor for DNT detection: Studies of inducer response and specificity of DntR
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Peter Brzezinski)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The primary aim of the work presented in this thesis was to change the inducer specificity of the DntR protein in order to improve the response to DNT. The long-term goal is to use this protein in a biosensor for DNT, a signature compound for detection of the explosive TNT. Another aspect of this work was to understand the mechanisms of inducer binding and how the binding of an inducer molecule changes the DntR structure into a state that triggers transcriptional activation.

In the papers included in this thesis the inducer specificity of wt DntR has been investigated under different conditions. The functional effects of specific mutations have also been investigated, in some cases in combination with structure determination using X-ray crystallography. In addition, structural data offering insights into the details of inducer binding and conformational changes upon inducer binding are presented and discussed in terms of mechanisms for transcriptional activation by DntR. Furthermore, a directed evolution strategy was employed in order to find variants of DntR with improved response to DNT. A variant with a large improvement in the DNT response was isolated and characterized. In optimized growth conditions, this DntR variant had a nearly 10-fold increase in fluorescence in response to DNT compared to wt DntR. Specific substitutions found in this DntR variant are suggested to be important for changing the inducer response.

Abstract [sv]

Syftet med denna avhandling har varit att förbättra förmågan hos proteinet DntR att upptäcka DNT. Det långsiktiga målet har varit att använda DntR i en biosensor för att upptäcka sprängämnet TNT, som avger DNT som en ”signaturmolekyl”. En annan aspekt har varit att bättre förstå den detaljerade mekanismen för hur DntR fungerar.

DntR är ett protein som binder till en viss DNA sekvens (promotor) och reglerar hur gener intill denna promotorsekvens läses av. När en inducerande molekyl som t.ex. DNT binder till DntR förändras proteinets struktur på ett sådant sätt att DntR kan aktivera transkription av de gener som finns intill promotor-sekvensen. För att mäta hur DntR reagerar på olika inducerande molekyler har DntR uttryckts i bakterien Escherichia coli, som också innehållit promotorn som DntR binder till. Intill promotorn sitter en gen som kodar för proteinet GFP. När en inducerande molekyl binder till DntR, slås avläses gfp-genen, och det fluorescerande proteinet GFP produceras. Ju mer GFP som produceras i cellerna, desto högre fluorescens kan uppmätas när cellerna analyseras.  

I de artiklar som presenteras i avhandlingen har vi undersökt hur olika substitutioner i DntR proteinet påverkar specificiten och sensitiviteten och hur dessa egenskaper kan påverkas av olika experimentella faktorer. Effekten av substitutioner har relaterats till strukturdata, där bilder av hur proteinet ser ut på molekylär nivå har tagits fram. Dessutom presenteras även en bild av hur DntR förändras beroende på om inducerande molekyler är bundna eller inte. En sådan strukturbild ökar förståelsen för de mekanismer som gör att bindning av en inducerande molekyl orsakar en förändring av formen hos DntR på så sätt att avläsning av gener kan aktiveras.

Vi har också använt en metod där evolutionära processer härmats för att få fram varianter av DntR med förbättrad respons till DNT. En variant med en drastisk ökning av DNT-responsen har isolerats, och dess egenskaper har karaktäriserats.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2011. , 68 p.
Keyword [en]
DntR, transcriptional regulation, gfp, nitro-aromatic compounds, LysR family, LTTR, TNT, DNT, FACS, directed evolution, random mutagenesis, recombination, structure determination
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-64129ISBN: 978-91-7447-404-6 (print)OAI: oai:DiVA.org:su-64129DiVA: diva2:455496
Public defence
2011-12-15, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: ManuscriptAvailable from: 2011-11-24 Created: 2011-11-09 Last updated: 2011-11-23Bibliographically approved
List of papers
1. In vivo and in vitro investigation of transcriptional regulation by DntR.
Open this publication in new window or tab >>In vivo and in vitro investigation of transcriptional regulation by DntR.
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2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, no 3, 571-82 p.Article in journal (Refereed) Published
Abstract [en]

DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-19210 (URN)10.1016/j.jmb.2007.06.076 | (DOI)000249494800002 ()17681542 (PubMedID)
Available from: 2007-10-23 Created: 2007-10-23 Last updated: 2017-12-13Bibliographically approved
2. Factors that influence the response of the LysR type transcriptional regulators to aromatic compounds
Open this publication in new window or tab >>Factors that influence the response of the LysR type transcriptional regulators to aromatic compounds
2011 (English)In: BMC Biochemistry, ISSN 1471-2091, E-ISSN 1471-2091, Vol. 12, 49- p.Article in journal (Refereed) Published
Abstract [en]

Background: The transcriptional regulators DntR, NagR and NtdR have a high sequence identity and belong to the large family of LysR type transcriptional regulators (LTTRs). These three regulators are all involved in regulation of genes identified in pathways for degradation of aromatic compounds. They activate the transcription of these genes in the presence of an inducer, but the inducer specificity profiles are different. Results: The results from this study show that NtdR has the broadest inducer specificity, responding to several nitro-aromatic compounds. Mutational studies of residues that differ between DntR, NagR and NtdR suggest that a number of specific residues are involved in the broader inducer specificity of NtdR when compared to DntR and NagR. The inducer response was also investigated as a function of the experimental conditions and a number of parameters such as the growth media, plasmid arrangement of the LTTR-encoding genes, promoter and gfp reporter gene, and the presence of a His(6) tag were shown to affect the inducer response in E. coli DH5 alpha. Furthermore, the response upon addition of both salicylate and 4-nitrobenzoate to the growth media was larger than the sum of responses upon addition of each of the compounds, which suggests the presence of a secondary binding site, as previously reported for other LTTRs. Conclusions: Optimization of the growth conditions and gene arrangement resulted in improved responses to nitro-aromatic inducers. The data also suggests the presence of a previously unknown secondary binding site in DntR, analogous to that of BenM.

Keyword
transcriptional regulator, LysR family, inducer specificity, gfp
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-64152 (URN)10.1186/1471-2091-12-49 (DOI)000295222700001 ()
Available from: 2011-11-11 Created: 2011-11-11 Last updated: 2017-12-08Bibliographically approved
3. Directed evolution of the transcriptional regulator DntR.: Isolation of mutants with improved DNT response
Open this publication in new window or tab >>Directed evolution of the transcriptional regulator DntR.: Isolation of mutants with improved DNT response
(English)Manuscript (preprint) (Other academic)
Keyword
LysR, LTTR, gfp, recombination, random mutagenesis, StEP, epPCR, DNT, salicylate
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-64155 (URN)
Available from: 2011-11-11 Created: 2011-11-11 Last updated: 2011-11-21Bibliographically approved
4. Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR-type transcriptional regulators
Open this publication in new window or tab >>Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR-type transcriptional regulators
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2011 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 81, no 2, 354-367 p.Article in journal (Refereed) Published
Abstract [en]

Activation of LysR-type transcription factors (LTTRs) is thought to result from conformational changes that occur when inducer molecules bind to their Inducer Binding Domains (IBDs). However, the exact nature of these changes remains to be fully elucidated. We present the crystal structures of two truncated constructs of the LTTR DntR in their apo- forms and in complex with its natural inducer molecule, salicylate. These provide a fuller picture of the conformational changes that can occur in LTTR IBDs and offer insights that may be relevant when considering the mechanism of activation of LTTRs. Two of the crystal structures show that DntR IBDs can bind up to two inducer molecules. The full extent of conformational changes observed is achieved only when inducer molecules are bound in both binding sites identified. Point mutations disrupting the putative secondary binding site produce DntR variants with a reduced response to salicylate in a whole cell system, suggesting that this site is functionally relevant.

Keyword
DntR, structure determination, inducer, gfp, LTTR
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-64153 (URN)10.1111/j.1365-2958.2011.07673.x (DOI)000292567200008 ()
Available from: 2011-11-11 Created: 2011-11-11 Last updated: 2017-12-08Bibliographically approved

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