Asunción Contreras

University of Alicante

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Paloma Salinas

University of Alicante

Javier Espinosa

University of Alicante

Raquel Cantos

University of Alicante

Rafaël Maldonado

Pompeu Fabra University

Karl Forchhammer

University of Tübingen

Vicente Rubio

University of Chile

Inmaculada Fuentes

University of Alicante

Antonio Llop

University of Alicante

José Ignacio Vega Labella

University of Alicante

Isabel Martínez Argudo

Cooperative Institutions

Consejo Superior de Investigaciones Científicas

University of Alicante

Centro de Investigación Biomédica en Red

Centre for Biomedical Network Research on Rare Diseases

Instituto de Biomedicina de Valencia

Instituto de Salud Carlos III

University of Tübingen

University of Sussex

Universidad de Sevilla

John Innes Centre

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In vivo features of signal transduction by the essential response regulator RpaB from Synechococcus elongatus PCC 7942

Microbiology (2012)

Félix Moronta-Barrios Javier Espinosa Asunción Contreras

The NblS-RpaB signalling pathway, the most conserved two-component system in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions and is involved in negative regulation of high-light-induced genes. However, relevant regulatory details of the NblS-RpaB signalling pathway remain to be elucidated. We recently showed that the response regulator RpaB is regulated by specific (de)phosphorylation from the histidine kinase NblS and that RpaB and its phosphorylatable residue Asp56 are both required for viability of Synechococcus elongatus PCC 7942. We show here that the phosphorylated form of RpaB is present in cells growing under standard laboratory conditions and that high light stress affected the ratio of phosphorylated to non-phosphorylated RpaB. It also decreased the amount of rpaB transcripts without appreciably changing the total levels of RpaB. Quantitative Western blotting and confocal microscopy analyses were consistent with RpaB being a very abundant regulator, with nucleoid localization. A genetically engineered RpaB-GFP (green fluorescent protein) fusion protein rescued lethality of the rpaB null mutant, indicating that it was functional. This is, to our knowledge, the first study demonstrating in a cyanobacterium, and for a two-component response regulator, that the in vivo ratio of phosphorylated to non-phosphorylated protein changes in response to environmental conditions.

Response regulator

Histidine kinase

Two-component regulatory system

10.1099/mic.0.057679-0

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Citations (32)

Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics

Applied and Environmental Microbiology (1991)

Asunción Contreras Søren Molin Juan L. Ramos

A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the Pseudomonas putida TOL plasmid-encoded meta -cleavage pathway operon (P m ) and the lacI gene encoding Lac repressor plus xylS , coding for the positive regulator of P m . The other element carries a fusion between the P tac promoter and the gef gene, which encodes a killing function. In the presence of XylS effectors, LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the P tac :: gef cassette is no longer prevented and a high rate of cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant XylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl-, and methoxybenzoates; salicylate; and methyl- and chlorosalicylates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown.

Pseudomonas putida

Lac repressor

TetR

10.1128/aem.57.5.1504-1508.1991

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Citations (94)

Effect of truncations and constitutive NtrB mutations in molecular interactions within the Klebsiella pneumoniae nitrogen-signal transduction pathway

CABI eBooks (2002)

Isabel Martínez Argudo Paloma Salinas Rafaël Maldonado Asunción Contreras

Transduction (biophysics)

Source

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Structure and dynamics of the cyanobacterial regulator SipA

Archives of Biochemistry and Biophysics (2024)

José L. Neira Maria Lopez-Redondo A. Cámara-Artigas Alberto Marina Asunción Contreras

The small, 78-residue long, regulator SipA interacts with the non-bleaching sensor histidine kinase (NblS). We have solved the solution structure of SipA on the basis of 990 nuclear Overhauser effect- (NOE-) derived distance constraints. The average pairwise root-mean-square deviation (RMSD) for the twenty best structures for the backbone residues, obtained by CYANA, was 1.35 ± 0.21 Å, and 1.90 ± 0.16 Å when all heavy atoms were considered (the target function of CYANA was 0.540 ± 0.08). The structure is that of a β-II class protein, basically formed by a five-stranded β-sheet composed of antiparallel strands following the arrangement: Gly6-Leu11 (β-strand 1), which packs against Leu66-Val69 (β-strand 5) on one side, and against Gly36-Thr42 (β-strand 2) on the other side; Trp50-Phe54 (β-strand 3); and Gly57-Leu60 (β-strand 4). The protein is highly mobile, as shown by measurements of R

Antiparallel (mathematics)

10.1016/j.abb.2024.109943

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The effect on the function of the transcriptional activator NtrC fromKlebsiella pneumoniaeof mutations in the DNA-recognition helix

Nucleic Acids Research (1988)

Asunción Contreras Martin Drummond

We have constructed mutations in what we predict to be the DNA-recognition helix of Klebsiellapneumoniae NtrC, which regulates transcription from promoters under global nitrogen control. Mutations which disrupt the helix lead to complete loss of function. All point mutants tested were able to activate transcription from the σ 54 -dependent glnA promoter, but only those retaining some ability to recognise NtrC binding sites, as evidenced by their ability to repress the ntrB promoter and the upstream glnA promoter, were able to activate the nifL promoter. One mutant, which contained an amino acid substitution in the turn of the DNA-binding motif as well as in the recognition helix, suppressed mutations in the NtrC binding sites upstream from the nifL promoter, but only if both sites bore equivalent transitions. This confirms that the DNA-binding motif for this class of transcriptional activator has been correctly identified and suggests that binding of NtrC can be cooperative.

Transcription

Helix-turn-helix

Upstream activating sequence

Recognition sequence

10.1093/nar/16.9.4025

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Citations (60)

Analysing the Cyanobacterial PipX Interaction Network Using NanoBiT Complementation in Synechococcus elongatus PCC7942

International Journal of Molecular Sciences (2024)

Carmen Jerez Antonio Llop Paloma Salinas Sirine Bibak Karl Forchhammer

The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding to them, in vivo studies are required to understand the physiological significance and contribution of environmental factors to the regulation of PipX complexes. Here, we have used the NanoBiT complementation system to analyse the regulation of complex formation in

10.3390/ijms25094702

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Citations (2)

PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

Frontiers in Microbiology (2017)

José Ignacio Vega Labella Raquel Cantos Javier Espinosa Alicia Forcada‐Nadal Vicente Rubio

Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essential PLP-holoenzymes or synthetic lethality with cysK. In addition, the related phenotypes of pipY and pipX mutants are consistent with genetic interactions in the contexts of survival to PLP-targeting antibiotics and transcriptional regulation. We also showed that PipY overexpression increased the length of S. elongatus cells. Taken together, our results support a universal regulatory role for COG0325 proteins, paving the way to a better understanding of these proteins and of their connections with other biological processes.

Regulon

10.3389/fmicb.2017.01244

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Citations (34)

The default cyanobacterial linked genome: an interactive platform based on cyanobacterial linkage networks to assist functional genomics

FEBS Letters (2020)

José Ignacio Vega Labella Antonio Llop Asunción Contreras

A database of cyanobacterial linked genomes that can be accessed through an interactive platform (https://dfgm.ua.es/genetica/investigacion/cyanobacterial_genetics/Resources.html) was generated on the bases of conservation of gene neighborhood across 124 cyanobacterial species. It allows flexible generation of gene networks at different threshold values. The default cyanobacterial linked genome, whose global properties are analyzed here, connects most of the cyanobacterial core genes. The potential of the web tool is discussed in relation to other bioinformatics approaches based on guilty-by-association principles, with selected examples of networks illustrating its usefulness for genes found exclusively in cyanobacteria or in cyanobacteria and chloroplasts. We believe that this tool will provide useful predictions that are readily testable in Synechococcus elongatus PCC7942 and other model organisms performing oxygenic photosynthesis.

Linkage (software)

Functional Genomics

10.1002/1873-3468.13775

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Citations (19)

Distinctive Features of PipX, a Unique Signaling Protein of Cyanobacteria

Life (2020)

José Ignacio Vega Labella Raquel Cantos Paloma Salinas Javier Espinosa Asunción Contreras

PipX is a unique cyanobacterial protein identified by its ability to bind to PII and NtcA, two key regulators involved in the integration of signals of the nitrogen/carbon and energy status, with a tremendous impact on nitrogen assimilation and gene expression in cyanobacteria. PipX provides a mechanistic link between PII, the most widely distributed signaling protein, and NtcA, a global transcriptional regulator of cyanobacteria. PII, required for cell survival unless PipX is inactivated or down-regulated, functions by protein–protein interactions with transcriptional regulators, transporters, and enzymes. In addition, PipX appears to be involved in a wider signaling network, supported by the following observations: (i) PII–PipX complexes interact with PlmA, an as yet poorly characterized transcriptional regulator also restricted to cyanobacteria; (ii) the pipX gene is functionally connected with pipY, a gene encoding a universally conserved pyridoxal phosphate binding protein (PLPBP) involved in vitamin B6 and amino acid homeostasis, whose loss-of-function mutations cause B6-dependent epilepsy in humans, and (iii) pipX is part of a relatively robust, six-node synteny network that includes pipY and four additional genes that might also be functionally connected with pipX. In this overview, we propose that the study of the protein–protein interaction and synteny networks involving PipX would contribute to understanding the peculiarities and idiosyncrasy of signaling pathways that are conserved in cyanobacteria.

10.3390/life10060079

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Citations (18)

The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of Klebsiella pneumoniae

Molecular Microbiology (1990)

Martin Drummond Asunción Contreras Lesley A. Mitchenall

A model for the domain structure of sigma 54-dependent transcriptional activators, based on sequence data, has been tested by examining the function of truncated and chimaeric proteins. Removal of the N-terminal domain of NtrC abolishes transcriptional activation, indicating that this domain is positively required for activator function. Over-expression of this domain as a separate peptide appears to titrate out the phosphorylating activity of NtrB. Removal of the N-terminal domain of NifA reduces activation 3-4-fold. The residual activity is particularly sensitive to inhibition by NifL, suggesting that the role of the N-terminal domain is to block the action of NifL in derepressing conditions. The C-terminal domain of NtrC showed repressor activity when expressed as a separate peptide. This domain is necessary for activator function even when NtrC binding sites are deleted from promoters. A point mutation in the ATP-binding motif of the NtrC central domain, Ser169 to Ala, also abolished activator function. Exchanging the N-terminal domains of Klebsiella pneumoniae NtrC, NifA and Escherichia coli OmpR, did not produce any hybrid activity, suggesting that N-terminal domains in the native proteins specifically recognize the rest of the molecule.

HAMP domain

PAS domain

10.1111/j.1365-2958.1990.tb02012.x

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Citations (110)