Study on the expression of recombinant resistance proteins domains

Towards a R protein crystal structure Alba de San Eustaquio Campillo Resistance (R) proteins are a key component of plant innate immunity. R proteins are cytoplasmic immune receptors in plants that recognize specific microbe-associated molecular patterns (MAMPs). This recognition activates the second layer of the immune system in plants, called effector-triggered immunity (ETI). Most R proteins are multi-domain proteins with a C-terminal leucine-rich repeat (LRR) domain, a central nucleotide-binding (NB)-ARC domain and a variable N-terminal domain. The N-terminal domain can be a coiled-coil (CC) region or a homologue of the Drosophila Toll and mammalian Interleukin-1 Receptors (TIR) structure. R proteins are members of the NB-ARC family of proteins, together with the human Apaf-1 and Caenorhabditis elegans CED-4, apoptosis receptors. The proposed activity of the NB-ARC domain is that of an ATPase. Studies conducted on R proteins have proved them to be ATPases. Nevertheless, a study with a subset of three R proteins showed their main activity was nucleotide phosphatases, not strict ATPases. In this project, expression, purification, and biochemistry experiments were conducted on Rx, R protein from potato that confers resistance against the potato virus X. Activity assays showed Rx-NBARC constructs to act as nucleotide phosphatases, not strict ATPases, supporting this newly found activity in R proteins

The Potato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor Rx1 is a Pathogen Dependent DNA-Deforming Protein

Plant NLR proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus, however, conserved nuclear targets that support their role in immunity are unknown. Previously we noted a structural homology between the NB domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA-binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger a Rx1-DNA interaction. DNA-binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signalling and defines DNA as a molecular target of an activated NLR

Développement d'un crible fonctionnel de mutants de MreB chez Bacillus subtilis et caractérisation d'un effecteur putatif de MreB

Acquisition and maintenance of the bacterial shape has been conscientiously studied for a long time. Nevertheless, there are still many unanswered questions. Gram-positive bacteria present a rigid external coating (cell wall) that allows them to preserve internal osmotic pressure and cell morphology. The cell wall (CW) is mainly formed by the peptidoglycan meshwork (PG), that confers its structure to the CW, to which are connected teichoic acids. The absence of this essential barrier causes the loss of shape and, ultimately, lysis of the cells. Integrity of the CW is, therefore, a matter of vital importance for bacteria. Proper CW synthesis and structure depends on the so-called peptidoglycan elongation machineries (PGEM) in charge of building the PG meshwork. The precise composition and functioning of the PGEM is not completely understood but they rely on a key player: MreB, a conserved prokaryotic actin-like protein. MreB is suspected to control PGEM activity and/or assembly but its precise function and mode of regulation are currently unknown. I used Bacillus subtilis, the model for Gram-positive bacteria, to gain a better understanding of MreB functions via i- the development and use of a genetic screen for loss-of-function mutants of mreB and ii- the study of a potential effector of MreB.(i) MreB has been studied for almost two decades now and still, little is known about its function(s). Since biochemical approaches proved to be difficult so far, most of the studies have focused on cellular localization and dynamics of the protein. Here, I have designed a genetic screen by means of which I have obtained a collection of functionally impaired mreB mutants in B. subtilis. Characterization of these mutants revealed numerous key residues for the functioning of the protein. Interestingly, my results indicate that some mutants have kept their dynamic properties (suggesting functional association to the PGEM) together with a wild type shape, while being strongly affected for growth. Preliminary results indicate an impaired ability to use certain carbon sources linking MreB to cellular metabolism. This suggests the existence of either a checkpoint or a coupling between carbon metabolism and CW expansion in B. subtilis.(ii) Unpublished results from our group revealed the existence of an uncharacterized operon (ydcFGH), whose expression is highly induced in the absence of MreB by comparison to the wild type. I have 1- deciphered the cause of ydcFGH induction in the absence of MreB, revealing the existence of multiple mutations in the MreB strain and 2- realized a thorough characterization of each gene of the ydcFGH operon. Although the exact link between MreB and ydcFGH is yet unknown, my results suggest a potential role of YdcH in the control of carbon metabolism and adaptation to stationary phase. In light of my mutagenesis screen data (i), these results are pointing towards a strong link between MreB and carbon metabolism.L'acquisition et le maintien de la forme bactérienne ont été consciencieusement étudiés pendant une très longue période. Néanmoins, il reste encore beaucoup de questions sans réponse. Les bactéries Gram-positives présentent une couche externe rigide (la paroi cellulaire) qui permet de préserver la pression osmotique interne et la morphologie cellulaire. La paroi cellulaire (CW) est principalement formée par un maillage de polymères de sucres, le peptidoglycane (PG), sur lequel sont accrochés des acides téichoïques. L'absence de cette barrière essentielle provoque la perte de forme et, finalement, la lyse de la cellule. L’intégrité du CW est par conséquent d'une importance vitale pour les bactéries. La structure ainsi que la synthèse correcte du CW dépendent de supposées machineries d'élongation du peptidoglycane (PGEM) chargées d’assembler le réseau du PG. Le fonctionnement et la composition des PGEMs restent incertains, mais on sait qu’ils dépendent d’une protéine essentielle : MreB, une protéine procaryote similaire à l'actine. MreB est suspectée de contrôler l’activité et/ou l’assemblage des PGEMs, mais sa fonction exacte comme son mode de régulation sont actuellement inconnus. J’utilise Bacillus subtilis, le modèle des bactéries Gram-positives, pour mieux comprendre les fonctions de MreB via i- le développement et l’utilisation d’un criblage génétique pour l’identification de mutants de mreB non fonctionnels et ii- l'étude d'un effecteur potentiel de MreB.(i) MreB a été étudié pendant près de deux décennies et pourtant, sa (ses) fonction(s) reste(nt) mal comprise(s). Comme les approches biochimiques se sont révélées particulièrement difficiles jusqu'à présent, la plupart des études se sont concentrées sur la localisation cellulaire et la dynamique de la protéine. Au cours de mes travaux, j’ai conçu un criblage génétique au moyen duquel j’ai obtenu une collection de mutants de mreB fonctionnellement déficients, chez B. subtilis. La caractérisation de ces mutants a révélé de nombreux résidus importants pour le fonctionnement de la protéine. De façon intéressante, mes résultats indiquent que certains mutants ont conservé leurs propriétés dynamiques (suggérant une association fonctionnelle aux PGEMs) en plus d'une morphologie de type sauvage, tout en étant fortement affectés pour la croissance. Des résultats préliminaires indiquent que ces mutants sont compromis dans leur capacité à utiliser certaines sources de carbone, reliant MreB au métabolisme cellulaire. Ceci suggère l'existence soit d'un point de contrôle, soit d'un couplage entre le métabolisme du carbone et l'expansion du CW chez B. subtilis.(ii) Des résultats non publiés de notre groupe ont révélé l'existence d'un opéron non caractérisé (ydcFGH) dont l'expression est fortement induite en absence de MreB, par comparaison à la souche sauvage. J’ai 1- mis en évidence la cause probable de l’induction de cet opéron en l’absence de MreB, révélant ainsi l’existence de nombreuses mutations dans la souche MreB et 2- réalisé une caractérisation poussée de chaque gène de l'opéron ydcFGH. Bien que le lien exact entre MreB et ydcFGH soit encore inconnu, nos résultats suggèrent un rôle potentiel d’YdcH dans le contrôle du métabolisme du carbone et l'adaptation à la phase stationnaire. À la lumière de mes données issues du criblage génétique (i), ces résultats indiquent un lien fort entre MreB et le métabolisme du carbone

A Tat ménage à trois — The role of Bacillus subtilis TatAc in twin-arginine protein translocation

AbstractThe twin-arginine translocation system (Tat) is a protein transport system that moves fully folded and cofactor-containing proteins across membranes of bacteria, archaea and thylakoids. The minimal Tat pathway is composed of two subunits, TatA and TatC. In some organisms TatA has been duplicated and evolved to form a third specialized subunit, TatB. The Bacillus subtilis genome encodes two TatC subunits (TatCd and TatCy) and three TatA subunits (TatAd, TatAy and TatAc). These subunits combine to form two parallel minimal pathways, TatAy-TatCy and TatAd-TatCd. The purpose and role of the third TatA component, TatAc, has remained ambiguous. In this study we examined the translocation of two natively expressed TatAy-TatCy-dependent substrates, EfeB and QcrA, in various Tat-deficient genetic backgrounds. More specifically, we examined the ability of different mutated TatAy subunits to complement for the absence of wild-type TatAy. We further detailed a graded growth phenotype associated with the functional translocation of EfeB. We found that in various instances where specific amino acid substitutions were made in TatAy, a definite TatAc-associated growth phenotype occurred in genetic backgrounds lacking TatAc. Altogether, our findings show that TatAy and TatAc interact and that this TatAy–TatAc interaction, although not essential, supports the translocation of the Tat substrate EfeB when TatAy function is compromised. This implies that the third TatA-like protein in B. subtilis could represent an intermediate evolutionary step in TatA-TatB specialization

PamR, a new MarR-like regulator affecting prophages and metabolic genes expression in <i>Bacillus subtilis</i>

<div><p><i>B</i>. <i>subtilis</i> adapts to changing environments by reprogramming its genetic expression through a variety of transcriptional regulators from the global transition state regulators that allow a complete resetting of the cell genetic expression, to stress specific regulators controlling only a limited number of key genes required for optimal adaptation. Among them, MarR-type transcriptional regulators are known to respond to a variety of stresses including antibiotics or oxidative stress, and to control catabolic or virulence gene expression. Here we report the characterization of the <i>ydcFGH</i> operon of <i>B</i>. <i>subtilis</i>, containing a putative MarR-type transcriptional regulator. Using a combination of molecular genetics and high-throughput approaches, we show that this regulator, renamed PamR, controls directly its own expression and influence the expression of large sets of prophage-related and metabolic genes. The extent of the regulon impacted by PamR suggests that this regulator reprograms the metabolic landscape of <i>B</i>. <i>subtilis</i> in response to a yet unknown signal.</p></div

The YdcH regulon.

<p>Pie charts summarizing genome-wide transcriptional profiling by RNAseq comparing gene expressions in a WT (ABS2005) and a Δ<i>ydcH</i> strain (ASEC56). The 363 genes retained (left chart) were reproducibly and statistically induced (182, right up) or repressed (181, right down) in the mutant compared to the wt by at least a two-fold factor. Genes were sorted by functional categories (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189694#pone.0189694.s007" target="_blank">S5 Table</a> for complete results), then regrouped into families of functions: Metabolism (carbon sources, amino acids, lipids, nucleotides and other metabolic pathways; electron transport & ATP synthesis; transport of sugars and other metabolites), stress response, information processing (DNA replication, segmentation, modification, recombination and repair; RNA and protein synthesis, modification and degradation), cellular processes (cell division; cell envelope synthesis, modification and degradation; ion homeostasis), lifestyles (motility & chemotaxis; biofilms formation; competence; sporulation), prophages & mobile genetic elements, and unknown. Numbers indicate the number of gene for each category.</p

<i>ydcFGH</i>, an operon of unknown function induced in a Δ<i>mreB</i> strain.

<p>A. Schematic representation of the genetic organization of the <i>B</i>. <i>subtilis ydcFGH</i> locus. Gene size of the orfs and putative functions are indicated above each gene. “P_<i>ydc</i> <i>lacZ</i>” shows the approximate size and localization on the locus of the DNA fragment amplified to construct the transcriptional reporter fusion to <i>lacZ</i> (strain ABS1761). B. A P_<i>ydc</i> <i>lacZ</i> fusion is induced in a strain lacking <i>mreB</i> (3725 “Δ<i>mreB”</i>; ABS1762) but not <i>mbl</i> (Δ<i>mbl</i>; ABS1769) nor in its wild type parent (Wt; ABS1761). C. Transformation of chromosomal DNA from strain ABS1761 (<i>amyE</i>::P_<i>ydc</i> <i>lacZ-spc</i>) into the recipient 3725 (neo- Δ<i>mreB</i>) leads to 100% of the spectinomycin/kanamycin resistant colonies expressing the <i>lacZ</i> reporter fusion (left) while the reverse transformation (right) leads to a limited number of blue colonies, indicating the absence of genetic link between Δ<i>mreB</i> and the factor inducing the reporter.</p

Sequence variations detected in strain 3725.

<p>Sequence variations detected in strain 3725.</p

The expression of <i>ydcFGH</i> is driven by two promoters.

<p>A. Schematic representation of the DNA fragments of the <i>ydcFGH</i> locus used for generating <i>lacZ</i> reporter fusions. The two putative promoters are indicated by arrows and the names of the resulting transcriptional fusion to <i>lacZ</i> are indicated below. On the right is displayed a picture of an X-Gal-LB plate to visualize LacZ activity of colonies harboring <i>lacZ</i> transcriptional fusions to P_<i>ydc1</i>, P_<i>ydc2</i>, P_{<i>ydc1-2</i>} or P_<i>ydc0</i> placed in either WT (ABS1761; ABS1763; ABS1765; ABS1767, respectively), or Δ<i>ydcH</i> (ABS1820; ABS1821; ABS1822; ABS1823, respectively), and to P_<i>ydc1</i> or P_<i>ydc2</i> in Δ<i>ydcF</i> (ASEC297; ASEC333) or Δ<i>ydcG</i> (ASEC301; ASEC335) background. B. Expression of a P_<i>ydc1</i> <i>luxABCDE</i> transcriptional fusion in cells grown in LB medium, in a wild type (red; ABS2005) or mutant for <i>ydcF</i> (green; ASEC325), <i>ydcG</i> (purple; ASEC327) or <i>ydcH</i> (blue; ASEC329) background. Note that the Δ<i>ydcH</i> data are relative to the upper part of the ordinate axis (in blue). Growth curves are presented as dotted lines and correspond to the optical density at 600nm while luciferase activities (plain lines) are relative luminescence units normalized by the OD_600nm.</p

YdcH binds specifically to inverted repeats in the promoter region of <i>ydcFGH</i>.

<p>A. Sequence of the region upstream of the <i>ydcFGH</i> operon. The two identified IR are indicated as green arrows. The transcriptional upshift previously identified is indicated as “up”, putative -35, -10 and rbs sequences are underlined, and the <i>ydcF</i> orf is boxed. B. EMSAs (right panels) showing the specific binding of PamR to DNA fragments corresponding to the wild type (wt) and mutated (IR1*) <i>ydcF</i> promoter, and schematic representation of the corresponding area (left panel). IRs are drawn as facing triangles, plain for the wild types and hollowed for the mutated. The quantity of YdcH (in pmol) incubated with 0.1 pmol of labeled target DNA is indicated above each lane.</p