Gery Thibault
email: thibault.sana@epfl.ch

Rodrigo Sana

Maxime Lomas

Aurélie Rémi Gimenez

Chantal Laubier

Claire Soscia

Ana Chauvet

Romé Conesa

Bérengère Voulhoux

Sophie Ize

Bleves
email: bleves@imm.cnrs.fr

Sana Tg

Gimenez Mr

Chauvet

Sophie Bleves

Differential modulation of Quorum-Sensing signaling through QslA in Pseudomonas

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pseudomonas aeruginosa is an ubiquitous Gram-negative opportunistic pathogen responsible for various nosocomial infections in immunocompromised or intubated-ventilated patients, as well as chronic respiratory infections in cystic fibrosis sufferers [START_REF] Govan | Microbial Pathogenesis in Cystic Fibrosis: Mucoid Pseudomonas aeruginosa and Burkholderia cepacia[END_REF]. Its genome encodes a myriad of virulence factors and regulatory mechanisms that allow the pathogen to adapt efficiently to various hostile environments and to cause distinct infections [START_REF] Stover | PAO1, an opportunistic pathogen[END_REF]. Virulence gene expression in P. aeruginosa is under a sophisticated and dynamic regulation network, and responds to largely unidentified environmental signals. This complex regulatory network involves alternative sigma factors, dozens of two-component systems, and Quorum-Sensing (QS) systems [START_REF] Balasubramanian | A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence[END_REF].

QS is a cell density-based interbacterial communication system that involves the diffusion of small autoinducer molecules that are used to coordinate gene expression [START_REF] Fuqua | A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite[END_REF][START_REF] Williams | Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules[END_REF][START_REF] Schuster | Acyl-Homoserine Lactone Quorum Sensing: From Evolution to Application[END_REF].

Once a crucial threshold concentration of signal molecules has been reached, QS target genes can be either activated or repressed. In P. aeruginosa there are two QS systems based on acyl homoserine lactones (HSL) signaling: the LasR/3-oxo-C12-HSL and RhlR/C4-HSL systems.

The las system is placed above the rhl system in the HSL-dependent QS hierarchy, since LasR/3-oxo-C12-HSL activates both rhlR and rhlI [START_REF] Latifi | A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhlR (VsmR) to expression of the stationary-phase sigma factor RpoS[END_REF][START_REF] Pesci | Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa[END_REF]. These two QS systems control the expression of about 6% of genes encoded on P. aeruginosa genome [START_REF] Schuster | Identification, Timing, and Signal Specificity of Pseudomonas aeruginosa Quorum-Controlled Genes: a Transcriptome Analysis[END_REF][START_REF] Wagner | Microarray Analysis of Pseudomonas aeruginosa Quorum-Sensing Regulons: Effects of Growth Phase and Environment[END_REF][START_REF] Vasil | DNA Microarrays in Analysis of Quorum Sensing: Strengths and Limitations[END_REF]. Among them, QS activates the expression of many genes encoding virulence factors, like the elastase LasB secreted by the Type II Secretion System (T2SS) [START_REF] Gambello | Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression[END_REF][START_REF] Chapon-Herve | Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa[END_REF], two Type VI Secretion Systems (T6SS) named H2-and H3-T6SS [START_REF] Lesic | Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis[END_REF][START_REF] Sana | The Second Type VI Secretion System of Pseudomonas aeruginosa Strain PAO1 Is Regulated by Quorum Sensing and Fur and Modulates Internalization in Epithelial Cells[END_REF][START_REF] Sana | Divergent Control of Two Type VI Secretion Systems by RpoN in Pseudomonas aeruginosa[END_REF], the pyocyanin [START_REF] Brint | Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family[END_REF] and biofilm maturation [START_REF] Fazli | Regulation of biofilm formation in P seudomonas and B urkholderia species: Regulation of biofilm formation[END_REF]. In addition to the HSL-mediated QS, P. aeruginosa also produces the Pseudomonas quinolone signal (PQS: 2-heptyl-3-hydroxy-4-quinolone) [START_REF] Pesci | Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa[END_REF]. The quinolone signaling system is intertwined in a hierarchical manner to the HSL-based QS systems of P. aeruginosa, as LasR and RhlR respectively positively and negatively control the levels of PQS by binding the promoter region of pqsR regulator gene [START_REF] Diggle | The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR: PQS regulation of rhl-dependent phenotypes[END_REF][START_REF] Mcgrath | Dueling quorum sensing systems in Pseudomonas aeruginosa control the production of the Pseudomonas quinolone signal (PQS)[END_REF]. In regards to regulation of the QS systems, there are several QS regulators described in the literature, such as MvaT [START_REF] Diggle | Advancing the Quorum in Pseudomonas aeruginosa: MvaT and the Regulation of N-Acylhomoserine Lactone Production and Virulence Gene Expression[END_REF], CdpR [START_REF] Zhao | Structural and Molecular Mechanism of CdpR Involved in Quorum-Sensing and Bacterial Virulence in Pseudomonas aeruginosa[END_REF], the IQS system [START_REF] Lee | A cell-cell communication signal integrates quorum sensing and stress response[END_REF], QscR [START_REF] Ledgham | Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR: Interactions of QscR[END_REF], QteE [START_REF] Siehnel | A unique regulator controls the activation threshold of quorum-regulated genes in Pseudomonas aeruginosa[END_REF], and QslA [START_REF] Seet | Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa: Quorum sensing anti-activator QslA[END_REF]. QscR, QteE, and QslA are thought to play crucial roles in determining the activation threshold of QS [START_REF] Ledgham | Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR: Interactions of QscR[END_REF][START_REF] Siehnel | A unique regulator controls the activation threshold of quorum-regulated genes in Pseudomonas aeruginosa[END_REF][START_REF] Seet | Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa: Quorum sensing anti-activator QslA[END_REF].

In laboratory settings, two clinically isolated strains of P. aeruginosa, named PAO1 and PA14, are principally studied and are noteworthy for their differences in virulence. While the PAO1 strain displays moderate virulence in most model systems, PA14 is highly virulent in mouse, nematode, and plant models of infection [START_REF] Rahme | Common virulence factors for bacterial pathogenicity in plants and animals[END_REF]; for the historical discovery of PA14 strain, see [START_REF] Mathee | Forensic investigation into the origin of Pseudomonas aeruginosa PA14 -old but not lost[END_REF]). Studies aimed at identifying the determinants mediating PA14 hypervirulence have found that the PA14 genome has two pathogenicity islands (108 kb and 11 kb in size), called PAPI-1 and PAPI-2 (P. aeruginosa Pathogenicity Island-1 and 2 respectively) [START_REF] He | The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes[END_REF].

These pathogenicity islands encode virulence factors such as the type III secretion system (T3SS) effector ExoU, a potent and detrimental cytotoxin producing rapid cell death [START_REF] Finck-Barbançon | ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury[END_REF][START_REF] Hauser | PepA, a secreted protein of Pseudomonas aeruginosa , is necessary for cytotoxicity and virulence[END_REF]. Nonetheless, enhanced PA14 virulence is not only a consequence of genomic acquired virulence determinants [START_REF] Lee | Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[END_REF]. In addition to the pathogenicity islands, the PA14 strain has a mutated version of the ladS gene [START_REF] Mikkelsen | The Pseudomonas aeruginosa Reference Strain PA14 Displays Increased Virulence Due to a Mutation in ladS[END_REF], encoding a sensor of the RetS/LadS/GacS signaling cascade, which is associated with virulence and the switch between acute and chronic infections of P. aeruginosa [START_REF] Ventre | Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes[END_REF][START_REF] Chambonnier | The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa[END_REF]. This mutation leads to derepression of the T3SS regulon and thus higher cytotoxicity towards mammalian cells [START_REF] Mikkelsen | The Pseudomonas aeruginosa Reference Strain PA14 Displays Increased Virulence Due to a Mutation in ladS[END_REF]. More recently, differences in the expression of the three T6SSs have also been highlighted in PA14 compared to PAO1 and PAK backgrounds, presumably because of the ladS mutation [START_REF] Allsopp | RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa[END_REF]. Finally, PA14 strain has been shown to secrete high levels of pyocyanin compared to PAO1, which is another important virulence factor of P. aeruginosa [START_REF] Dietrich | The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa[END_REF].

To better understand the divergent virulence profiles between PAO1 and PA14 and determine if there are other unknown players that are involved in this complex mechanism, we chose to perform an unbiased transcriptomic approach using high-throughput RNA sequencing (RNAseq). From RNAseq data, we found significant differences in the expression of many QS targets; overall, QS-activated genes were overexpressed in PA14, while QS-repressed genes were overexpressed in PAO1 strain. Interestingly, the expression of qslA, encoding a QS inhibitor [START_REF] Seet | Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa: Quorum sensing anti-activator QslA[END_REF], is only detectable in PAO1 strain. QslA (QS LasR-anti-activator) is known to bind LasR, and disrupt LasR dimerization, preventing its binding to target promoters [START_REF] Seet | Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa: Quorum sensing anti-activator QslA[END_REF][START_REF] Fan | QsIA disrupts LasR dimerization in antiactivation of bacterial quorum sensing[END_REF].

Since many virulence factors are activated by QS in P. aeruginosa, we hypothesized that decreased expression of qslA in PA14 strain would drive increased QS gene expression and subsequently overexpression of QS target genes, thus leading to the hyper-virulent phenotype of PA14. To test this hypothesis, we first demonstrated that the QslA protein is indeed readily produced in PAO1 strain while it is undetectable in PA14. We further confirmed that genes encoding QS systems are upregulated in PA14 strain compared to PAO1, and that it is due to lower level of QslA. Next, we provided evidence that many QS activated genes encoding important P. aeruginosa virulence factors are differentially regulated between these two strains, such as pyocyanin production, H2-T6SS gene expression, XcpP production, and Xcpdependent secretion of the elastase LasB. Finally, we observed that whereas biofilm formation and cytotoxicity towards macrophages were higher in PA14, this was independent of qslA expression. Altogether, the level of the QslA protein and presumably the expression level of the qslA gene is a key player in QS target genes expression, which consequently contributes to higher pathogenic potential of the PA14 strain.

Results

Global comparison of PAO1 and PA14 RNAseq transcriptomes

To determine if virulence factor genes are differentially expressed between the PAO1 and PA14 P. aeruginosa strains, we performed RNAseq on mRNAs extracted from cultures grown in rich medium at the transition between exponential and stationary phase, in which most of the virulence factors are expressed (see Materiel and Methods). 2029 genes were identified as significantly and differentially regulated between the two strains (Figure 1A and Table 1), which represents about 39% of the P. aeruginosa genome, suggesting a massive gene expression variation between both isolates. Among the genes differentially expressed, PA3431/PA3432 expression levels were increased by respectively 108.7 and 105.3-fold in PAO1 (Table S1). However, we decided not to follow up on these genes encoding hypothetical proteins here. Further work will be required to address this interesting observation, more particularly as PA3431/PA3432 may constitute a holin-/antiholin-like system as PF04172 and PF03788 domains, are respectively present in PA3431 and PA3432, suggesting a potential role in bacterial lysis [START_REF] Saier | Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications[END_REF]. Two other genes whose expression was very different between the two strains drew our attention: PA4685 and PA1244 (qslA). Expression of PA4685 was not detectable in PAO1, while expression of qslA was barely detectable in PA14 (Table S1 andTable 1).

Since we did not detect any PA4685 transcript in our PAO1 transcriptomic data (Table S1), we first decided to test whether the gene was present in the PAO1 genome. PAO1 is one of the most commonly P. aeruginosa strain used in research and is derived from the original Australian PAO isolate, isolated from a wound 50 years ago in Melbourne, and distributed worldwide to laboratories. Over decades, discordant phenotypes of PAO1 subcultures emerged [START_REF] Klockgether | Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains[END_REF]. Notably, there is a 1 kb deletion within the PA4684 and PA4685 genes present in the Washington Genome Center PAO1 strain compared to the published PAO1 sequence [START_REF] Dötsch | Evaluation of a microarray-hybridization based method applicable for discovery of single nucleotide polymorphisms (SNPs) in the Pseudomonas aeruginosa genome[END_REF] (which served as a reference for our in silico analysis). To test whether this locus deletion is present in the PAO1 strain used in this study (named "PAO1 Marseilles"), PCR was performed using pairs of oligonucleotides hybridizing to the coding sequence of PA4685 (TSO116-TSO117) or outside thereof (CCO1, CCO2) (Figure S1A). As shown, our results revealed that PA4685 is absent in the PAO1 Marseilles strain, but present in the PA14 strain used in this study (Figure S1B). This explains the non-expression of PA4685 in PAO1 Marseilles strain observed in our RNAseq transcriptome.

The expression of qslA gene is increased by 55.8-fold in the PAO1 strain (Table 1).

Interestingly, this gene encodes an anti-activator of QS, named QslA [START_REF] Seet | Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa: Quorum sensing anti-activator QslA[END_REF], and led us to hypothesize that the expression of QS-regulated genes may be different between the two isolates. As a result, we decided to look at the expression of all of the QS regulated genes in our RNAseq transcriptomic data. Interestingly, among the 353 genes known to be regulated by QS in P. aeruginosa (315 activated and 38 repressed (9)), 212 were significantly and differentially regulated between both strains (Figure 1A). Overall, QS activated genes were overexpressed in PA14 (shown as yellow dots) whereas QS repressed genes were overexpressed in PAO1 (shown as red dots), suggesting a major differential QS-dependent expression pattern (Figure 1A). All together these observations led us to hypothesize that the difference in QS target genes expression between these two strains could be mediated by the modulation of qslA expression levels.

To first demonstrate a correlation between levels of qslA expression and QslA production, we engineered chromosomally encoded QslAV5 translational fusions in both the PAO1 and PA14 strains. The production of QslAV5 could be monitored and analyzed by Western blotting and immunodetection with an anti-V5 antibody (Figure 1B). The results for the PAO1 strain reflected a constant level of QslA during growth, whereas QslA was not detected under the same conditions in PA14, which coincides with our transcriptomic data.

qslA expression level drives gene expression of HSL-based QS regulators

Then, we asked whether this QS-dependent global differential expression could be due to differential expression of qslA. We reasoned that abolishing qslA expression in the PAO1 background may mimic the PA14 profile whereas QslA overproduction in the PA14 strain may resemble the PAO1 pattern. Our transcriptomic data indicate an increased expression of genes encoding the two HSL-based QS systems in PA14. Indeed, rhlR, rhlI, lasR, and lasI were respectively 2.5, 1.8, 1.9, and 1.5-fold more expressed in the PA14 strain (Table 1).

Therefore, we decided to measure the relative levels of lacZ transcriptional fusions to lasR, rhlR and rhlI promoters [START_REF] Latifi | A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhlR (VsmR) to expression of the stationary-phase sigma factor RpoS[END_REF] in strains PAO1, PA14, PAO1ΔqslA, PAO1ΔqslA carrying plasmid pTS51 encoding qslA to complement the qslA deletion, and PA14 overproducing QslA from the pTS51 (Table 2). In PAO1, deletion of qslA gene increased the expression level of QS regulator genes by 2 to 3-fold (Figure 2A, C, &E). WT level expression was restored by complementation of the mutation in trans (compare columns 3 and 4, repression and activation respectively of qslA, in graphs 2A, 2C & 2E). Overproduction of QslA caused a significant decrease (1.5 to 2.5-fold) in β-galactosidase activities measured for all three of the transcriptional fusions made in the PA14 strain (Figure 2B, D, &F). Thus, the expression levels of lasR, rhlR, and rhlI were correlated in a dose-dependent manner with QslA production in both P. aeruginosa strains. Interestingly, the increased fold expression in PA14 measured for lasR, rhlR, and rhlI (Figure 2), recapitulates the increased fold expression of these genes in our transcriptomic data between two strains (Table S1), suggesting that modulation of qslA expression is solely responsible for this differential expression of QS genes.

Biosynthesis of pyocyanin depends on qslA expression levels

We next hypothesized that differential expression of qslA could explain the differential expression of QS-regulated genes between PAO1 and PA14 strains. To test this hypothesis, we chose to compare the expression level or production profile of several QS activated virulence factors gene in both strains, producing or not QslA.

Pyocyanin is a blue, redox-active phenazine that contributes to P. aeruginosa virulence by inhibiting the oxidative burst of host phagocytic cells, by inducing apoptosis in host cells and through antibiotic activities. This respiratory pigment also participates in the reduction of iron and functions as an intracellular redox buffer [START_REF] Jayaseelan | Pyocyanin: production, applications, challenges and new insights[END_REF]. As the regulation of pyocyanin expression is both mediated by HSL-and PQS-based QS [START_REF] Brint | Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family[END_REF][START_REF] Diggle | The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR: PQS regulation of rhl-dependent phenotypes[END_REF], the study of its production is then a perfect example of the intrinsic and complex relationship between regulations by HSLand PQS-based QS. Furthermore, in our transcriptomic data, expression of the operon phz encoding the genes involved in the production of pyocyanin, were 10 to 20-fold increased in PA14 (Table S1).

The production of pyocyanin was monitored after 3.5 and 24 hours of growth (Figure 3). Pyocyanin concentrations were higher in PA14 than in PAO1 reflecting the lack of expression of qslA. Moreover, the overproduction of QslA in PA14 led to a major decrease in pyocyanin production (5.2-and 22.4-fold at 3.5 and 24 hours) whereas the qslA deletion in PAO1 produced a significant increase (2-and 1.6-fold at 3.5 and 24 hours) that could be restored to WT PAO1 level by complementation. Therefore, the increased pyocyanin production in PA14 strain [START_REF] Dietrich | The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa[END_REF] is due to low qslA expression in this strain.

H2-T6SS expression is driven by QslA levels

The H2-T6SS machinery is a virulence factor of P. aeruginosa that is known to be activated by QS in both PA14 and PAO1 strains [START_REF] Lesic | Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis[END_REF][START_REF] Sana | The Second Type VI Secretion System of Pseudomonas aeruginosa Strain PAO1 Is Regulated by Quorum Sensing and Fur and Modulates Internalization in Epithelial Cells[END_REF]. Our transcriptomic data indicates a 1.5-fold increased expression of the first gene of the H2-T6SS operon (PA1656) in PA14 (Table 1). P. aeruginosa utilizes H2-T6SS to invade epithelial cells by manipulating the microtubular network and host kinase pathways [START_REF] Jiang | A Pseudomonas aeruginosa Type VI Secretion Phospholipase D Effector Targets Both Prokaryotic and Eukaryotic Cells[END_REF][START_REF] Sana | Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network[END_REF][START_REF] Sana | The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting[END_REF] and to promote autophagy [START_REF] Jiang | A Pseudomonas aeruginosa Type VI Secretion Phospholipase D Effector Targets Both Prokaryotic and Eukaryotic Cells[END_REF][START_REF] Jiang | The Pseudomonas aeruginosa Type VI Secretion PGAP1-like Effector Induces Host Autophagy by Activating Endoplasmic Reticulum Stress[END_REF][START_REF] Russell | Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors[END_REF][START_REF] Burkinshaw | A type VI secretion system effector delivery mechanism dependent on PAAR and a chaperone-co-chaperone complex[END_REF]. H2-T6SS also mediates antibacterial activity [START_REF] Jiang | A Pseudomonas aeruginosa Type VI Secretion Phospholipase D Effector Targets Both Prokaryotic and Eukaryotic Cells[END_REF][START_REF] Jiang | The Pseudomonas aeruginosa Type VI Secretion PGAP1-like Effector Induces Host Autophagy by Activating Endoplasmic Reticulum Stress[END_REF][START_REF] Russell | Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors[END_REF][START_REF] Burkinshaw | A type VI secretion system effector delivery mechanism dependent on PAAR and a chaperone-co-chaperone complex[END_REF], making it a trans-kingdom cell targeting machinery [START_REF] Russell | Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors[END_REF][START_REF] Bleves | Game of Trans-Kingdom Effectors[END_REF]. We used a lacZ transcriptional fusion of H2-T6SS promoter region to evaluate its expression in these strains (Figure 4). Strain PAO1∆qslA presented a 1.8fold increase in the expression of H2-T6SS compared to the PAO1 WT strain. Moreover, production of QslA in the mutant strain was able to decrease H2-T6SS expression to comparable level than the WT strain. Similarly in PA14, overproduction of QslA significantly reduced the expression of H2-T6SS. The expression of H2-T6SS machinery genes was thus controlled at a transcriptional level by QslA in both strains.

Production of XcpP depends on qslA expression levels

XcpP is an inner membrane protein of the P. aeruginosa Xcp T2SS machinery, known to secrete various virulence factors [START_REF] Bleves | Protein secretion systems in Pseudomonas aeruginosa: A wealth of pathogenic weapons[END_REF]. XcpP interacts with the outer membrane secretin XcpQ, which forms the pore through which the secreted protein reaches the extracellular medium [START_REF] Bleves | Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa[END_REF][START_REF] Douzi | Deciphering the Xcp Pseudomonas aeruginosa Type II Secretion Machinery through Multiple Interactions with Substrates[END_REF][START_REF] Douzi | Unraveling the Self-Assembly of the Pseudomonas aeruginosa XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics[END_REF] and is therefore an essential component of the T2SS process. The two operons encoding the Xcp machinery are activated by HSL-based QS [START_REF] Chapon-Herve | Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa[END_REF], and our transcriptomic data indicates that xcp genes are upregulated in PA14 in comparison to PAO1 in transition phase (Table S1). We thus asked whether QslA levels could modulate XcpP production, using immunodetection at different time of the growth (Figure 5). Whereas XcpP could be readily detected after 2 hours of growth of the PA14 strain, the same level of proteins was obtained two hours later in PAO1. Moreover, the deletion of the qslA gene in PAO1 led to an earlier synthesis of XcpP and to higher levels of the protein that can be restored to a PAO1 WT profile by complementation. Accordingly, overproduction of QslA in the PA14 strain showed a PAO1like pattern of XcpP. These data corroborated our hypothesis that the T2SS machinery is dependent on QslA for an optimal production.

Elastase secretion is dependent on qslA expression level

The elastase is an Xcp T2SS-secreted protease, encoded by the lasB gene. It degrades elastin, a major component of lung tissue, and cleaves a surfactant protein (SP-D), involved in several immune functions [START_REF] Kuang | Pseudomonas aeruginosa Elastase Provides an Escape from Phagocytosis by Degrading the Pulmonary Surfactant Protein-A[END_REF]. The expression of elastase coding gene is activated by QS [START_REF] Gambello | Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression[END_REF], and we measured an 7.7 fold increased expression of lasB in PA14 in comparison to PAO1during early stationary phase in our RNAseq analysis (Table 1). To measure the impact of QslA on this T2SS substrate, the secretion into the extracellular medium and the extracellular activity of elastase were monitored (Figure 6). The secretion of LasB was analyzed by Coomassie blue staining of the extracellular fraction at different growth times (Figure 6A). As expected, the deletion of qslA in PAO1 altered the kinetics, allowing earlier LasB secretion; after 4 and 6 hours of growth, LasB secretion was significantly higher in the mutant PAO1ΔqslA than in the WT PAO1 strain. This phenotype is restored to a WT phenotype by trans complementation since the protein profile observed for the PAO1ΔqslA strain producing QslA was identical to that observed for the WT PAO1 strain (Figure 6A, compare first and fourth lines). In contrast in PA14, overproduction of QslA led to a delayed and decreased secretion of LasB compared to the PA14 WT strain (Figure 6A, compare fifth and sixth lines). These data were also confirmed by monitoring the LasB protease activity by observing the formation of a protein degradation halos on skim-milk plates (Figure 6B). We noticed an earlier halo in PA14 strain compared to PAO1 (Figure 6B, compare first and fourth lines). In addition, the delay of halo formation in PAO1 can be visualized in a qslA mutant strain (Figure 6B, compare first and second lines). As negative controls, we used mutants in T2SS Xcp machinery for both strains (Figure 6B, third and fifth lines). As shown, the halo was weak after 35 hours.

Altogether these data demonstrated that the functionality of the Xcp T2SS machinery measured by the secretion and extracellular activity of the protease LasB is higher in the PA14 strain, due to the decreased expression of qslA in this strain.

QslA does not control biofilm formation by P. aeruginosa P. aeruginosa ability to form biofilm is a crucial virulence determinant, mainly regulated by QS [START_REF] Fazli | Regulation of biofilm formation in P seudomonas and B urkholderia species: Regulation of biofilm formation[END_REF]. To assess if QslA has a role during biofilm formation, bacterial adherence ). However, profiles of the PAO1 strains, WT or ΔqslA, mutant and of the PA14 strains, WT or overproducing QslA, were the same, suggesting that QslA is dispensable for biofilm formation (Fig. 7A and7B). Therefore, these results tend to suggest that QslA is not implicated in controlling biofilm formation of P. aeruginosa.

Cytotoxicity towards macrophages is not dependent on QslA

Finally, we tested the cytotoxicity of the various strains on J774 macrophages. Indeed while PA14 is highly cytotoxic due to the T3SS effector ExoU [START_REF] Finck-Barbançon | ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury[END_REF], PAO1 is considered an invasive and poorly cytotoxic P. aeruginosa strain. Nevertheless both strains display T3SSmediated antiphagocytic functions and the T3SS machinery and effector encoding genes are regulated by HSL-mediated QS [START_REF] Bleves | Quorum Sensing Negatively Controls Type III Secretion Regulon Expression in Pseudomonas aeruginosa PAO1[END_REF] and PQS. Due to the hyper-cytotoxic phenotype of the PA14 strain, we used two infection conditions according to the two backgrounds (PAO1 and PA14) (Fig. 8). As expected even with longer infection times, the PAO1 strain is poorly cytotoxic compared to PA14 (Fig. 8, compare line 1 and 3). However, the cytotoxicity levels of the PAO1 strains, WT or ΔqslA mutant and of the PA14 strains, WT or overproducing QslA, were the same suggesting that the cytotoxicity of P. aeruginosa is not dependent on QslA.

Discussion

PAO1 and PA14 are two stains of P. aeruginosa that are broadly studied throughout the Pseudomonas research community. Interestingly, the PA14 strain is more virulent than the PAO1 strain in different model infection systems [START_REF] Rahme | Common virulence factors for bacterial pathogenicity in plants and animals[END_REF][START_REF] Lee | Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[END_REF][START_REF] Choi | Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis[END_REF][START_REF] Tan | Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis[END_REF]. Several studies have been conducted to try to decipher why PA14 strain exhibits higher pathogenicity compared to PAO1.

While previous groups have found that pathogenicity island-I and II are present only in the PA14 genome and encodes virulence factors [START_REF] Lee | Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[END_REF], and the ladS gene is mutated in PA14 strain, making T3SS and T6SSs more effective [START_REF] Mikkelsen | The Pseudomonas aeruginosa Reference Strain PA14 Displays Increased Virulence Due to a Mutation in ladS[END_REF][START_REF] Allsopp | RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa[END_REF], we speculated that other determinants might be involved in the differential in virulence between these two strains.

As a result, we decided to use a global transcriptome RNA sequencing approach to highlight new factors that might explain the virulence divergence between these two strains.

The transcriptomic data revealed lower expression of the QS regulator qslA in PA14 strain than in PAO1, which we further confirmed at a protein level. Given the fact that QslA is highly produced in PAO1 (Figure 1B) and plays a crucial role in determining the activation threshold of QS [START_REF] Seet | Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa: Quorum sensing anti-activator QslA[END_REF], we hypothesized that QS regulon expression will occur earlier and would be higher in PA14 in comparison to PAO1. To test this hypothesis, we constructed a deletion mutant of qslA in PAO1 as well as a plasmid allowing overexpression of qslA from a PBAD promoter in PA14 strain (Table 2). This allowed us to investigate whether deletion of qslA in PAO1 can mimic the higher QS activation found in PA14, and conversely, if overproduction of QslA in PA14 will lead to the lower QS activation observed in PAO1. We then focused our study on well-known QS-regulated virulence factors. We were able to confirm that the expression level of genes encoding QS system as well as QS gene targets are differentially regulated depending on the expression level of qslA in both strains, except for biofilm and cytotoxicity phenotypes.

Considering that a large set of genes encoding virulence factors are regulated by QS in P. aeruginosa, and that their expression levels are increased in PA14 compared to PAO1, this could partially explain the hyper-virulent phenotype of PA14. Moreover, a qslA mutant in PAO1 strain is more virulent than the wild-type PAO1 strain in a Caenorhabditis elegans model [START_REF] Seet | Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa: Quorum sensing anti-activator QslA[END_REF]. These results combined with our findings suggest that the high expression of qslA in PAO1 strain is one of the major factors of its decreased virulence compared to PA14, and vice versa for the PA14 strain.

From our results, we questioned whether such mechanisms could be conserved amongst other isolates of P. aeruginosa. To that end, a previous study showed significant differences in QS regulon expression between seven clinical and environmental strains of P. aeruginosa, this supporting the notion that different isolates of P. aeruginosa have defined regulation networks [START_REF] Chugani | Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon[END_REF]. These findings indicate a role for QS in the extension of the range of habitats in which a species can thrive, including the host. Based on this, one can ask whether the differential regulation of QS between the different strains of P. aeruginosa is causing different levels of virulence that can be observed between different isolates [START_REF] Lee | Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[END_REF]. If so, it would be interesting to investigate qslA expression levels in different clinical isolates of P. aeruginosa. However, our data concerning biofilm formation and cytotoxicity towards macrophages suggest that the QslA-regulon does not overlap completely the QS-regulon. One explanation could be that another QS regulator is important for controlling biofilm and cytotoxicity.

Interestingly, the expression of other known QS regulator genes such as qscR, rsaL, mvaT, cdpR and qteE was respectively 1.2, 2.1, 1.6, 3.9, and 1.9-fold increased in PA14 (Table 1), which is not comparable to the 55.8 fold increased expression of qslA in PAO1. This, along with our data concerning the differential modulation of QS, H2-T6SS, pyocyanin, Xcp-T2SS, and LasB, suggests that the modulation of QS between two strains is mostly due to the differential expression of qslA. One could ask what is the molecular mechanism underlining this differential expression of qslA? We performed a simple BLASTn comparing the upstream 500 bp sequence of qslA in PAO1 and PA14, but there was only one nucleotide difference, hardly explaining the differential expression of qslA in both strains. Further studies will be needed to elucidate the exact mechanism of qslA expression in these two strains. Altogether, we propose that differential expression of qslA in PAO1 and PA14 leads to modulation of QS signaling. Since QS is a major regulator of virulence factors in P. aeruginosa, it is tempting to speculate that QslA could be a key player in the hyper-virulence phenotype of PA14, along with LadS (34), PAPI-1 and PAPI-2 [START_REF] He | The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes[END_REF].

Material and Methods

Bacterial strains, culture conditions, plasmids and oligonucleotides

The bacterial strains, plasmids and oligonucleotides used in this study are described in Table 2. LB (Lysogeny broth), PIA (Pseudomonas isolation agar), and TSB (Tryptic soy broth) broths and agar were used for the growth of P. aeruginosa and Escherichia coli strains at 37°C.

Cultures were inoculated at an optical density at 600 nm (OD600) of 0.1 with overnight cultures, and strains were grown at 37°C with aeration in TSB. Recombinant plasmids were introduced into P. aeruginosa strain PAO1 and PA14 by conjugation using pRK2013 mobilization properties, as described previously [START_REF] Sana | Gene Transfer: Conjugation[END_REF]. The antibiotic concentrations were as follows: for E. coli, ampicillin (50 µg ml-1), kanamycin (25 µg ml-1), tetracycline (15 µg ml-1), gentamicin (10 µg ml-1), streptomycin (100µg ml-1); for P. aeruginosa, tetracycline (200 µg ml-1 for plates or 50 mg ml-1 for liquid growth), gentamicin (50 µg ml-1), streptomycin (1000µg ml-1). Expression of the qslA gene under the control of a PBAD promoter in pTS51 was induced or repressed by addition of arabinose or glucose respectively at a final concentration of 0.5% after 1h30 of growth.

Transcriptional analysis of Pseudomonas aeruginosa PA14 and PAO1

To determine if virulence factor genes were differentially expressed between the PAO1 and PA14 P. aeruginosa strains, we performed RNAseq on mRNA extracted from cultures of both strains grown in rich media (LB) to early stationary phase. Total RNA was isolated from cell pellets equivalent to 2 OD600 units of bacterial culture using SV total RNA isolation system (Promega) following the manufacturer's instructions. Once isolated and prior to the library preparation, the integrity of RNA samples was assessed with a Bioanalyzer system. Barcoded, strand-specific, cDNA libraries were constructed, pooled and sequenced in an Illumina HiSeq 2000, single-end 50 bp reads by BGI-Hong Kong. Illumina reads were mapped to the P. aeruginosa genome PAO1 (AE004091.2 (61)) and PA14 (NC_008463.1 [START_REF] Lee | Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial[END_REF]) by Bowtie (version Bowtie1 v0.12.9 (62)), indicating strand-specific sequencing. Quantification of gene expression was determined by the HTSeq package (63) using the GeneBank P. aeruginosa PA14 and PAO1 annotations files and discarding multimapped reads. Data were normalized by RPKM and filtered to the 5,263 orthologous genes conserved between the P. aeruginosa strains PA14 and PAO1. Two biological replicates were performed per condition. Differential expression analysis was analyzed using the Bioconductor package NOISeq version 2.22.1 (64), a non-parametric approach suitable for lowly replicated data, and using a q-value of 0.99 for strong control of false positives.

cDNA library preparation

The RNA was fragmented with RNase III. Then the 5'PPP structures were removed from the RNA samples using RNA 5' polyphosphatase (Epicentre) and subsequently, the RNA was poly was mobilized in the wild type P. aeruginosa strain PAO1 or PA14. The strains, in which the double recombination events occurred and resulted in the chromosomic tagging of qslA with V5, were verified by PCR using primers TSO58 and TSO107.

lacZ reporter fusion and β-galactosidase assay

For PH2-T6SS-'lacZ, the promoter fragment was integrated at the CTX phage attachment site in PAO1 or PA14 and isogenic mutants using established protocols [START_REF] Hoang | Integration-Proficient Plasmids for Pseudomonas aeruginosa: Site-Specific Integration and Use for Engineering of Reporter and Expression Strains[END_REF]. For PrhlR-'lacZ, PrhlI-'lacZ, PlasR-'lacZ, plasmids carrying promoter fragment were conjugated in PAO1 or PA14 and isogenic mutants using established protocol [START_REF] Sana | Gene Transfer: Conjugation[END_REF]. Overnight culture, grown in TSB supplemented with tetracyclin, was diluted in TSB to OD600= 0.1. Growth and ß-galactosidase activity were monitored by harvesting samples at different time intervals. ß-galactosidase activity was measured according to Miller, as described previously [START_REF] Sana | Divergent Control of Two Type VI Secretion Systems by RpoN in Pseudomonas aeruginosa[END_REF], and based on onitrophenyl-b-D-galactopyranoside hydrolysis. ß-galactosidase activities were expressed in Miller units.

Pyocyanin production

Pyocyanin was extracted from the extracellular medium by adding an equal volume of chloroform (CHCl3) and vigorous vortexing as previously described [START_REF] Essar | Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications[END_REF]. The lower pyocyanin-containing organic layer was then taken and vortexed with an equal volume of 0.2 M HCl. The pink pyocyanin-containing aqueous layer resulting from the previous step was then taken, and its absorbance at 520 nm (OD520) was read. Concentrations expressed as micrograms of pyocyanin produced per ml of culture supernatant were determined by multiplying the OD520 by 17.072.

SDS-PAGE and immunoblotting

Proteins from the extracellular medium were loaded at an equivalent of 1.0 OD600 unit, while proteins from cellular extracts were loaded at an equivalent of 0.1 OD600 unit. Proteins were then separated on SDS gels containing 11 or 15% acrylamide depending on the size of the proteins being further detected.

For Western blotting, proteins were transferred from gels onto nitrocellulose membranes. After 30 min to overnight saturation in Tris-buffered saline (TBS) (0.1 M Tris, 0.1 M NaCl, pH7.5), 0.05% (v/v) Tween 20 and 5% (w/v) skim milk, the membrane was incubated for 1 h with anti-V5 (diluted 1:2500), anti-XcpP (1/2000) or anti-DsbA (1:25000); washed three times with TBS, 0.05% Tween 20; incubated for 45 min with goat anti-rabbit immunoglobulin G (IgG) antibodies (Sigma) diluted 1:5000; washed three times with TBS, 0.05% Tween 20; and then revealed with a Super Signal Chemiluminescence system (Pierce).

Adherence assay on inert surfaces and exopolysaccharide production

The adherence assay was performed as previously described in [START_REF] Vallet | The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation[END_REF] with some modifications.

Cultures inoculated at OD600=0.05 with O/N cultures were grown at 37°C with aeration in LB with appropriate antibiotics. Induction of the promoter arabinose (PBAD) was done at OD600=0.6 with 0.2% arabinose for 1.5 hours. 24-well polystyrene microtitre plates were then inoculated at OD600=0.2 in 1 ml of MM63 medium supplemented with casamino acids 0.5%, 1 mM MgSO4 and glucose or arabinose 0.2% for 2 to 48 hours at 30°C. Bacteria were stained with 0.1% crystal violet for a period of 10 min and washed twice with water. The stain was then dissolved in ethanol and absorbance was measured at 595 nm.

Congo red assay was performed as previously described to measure the production of exopolysaccharides [START_REF] Bordi | Regulatory RNAs and the HptB/RetS signalling pathways finetune Pseudomonas aeruginosa pathogenesis: P. aeruginosa signalling via HptB, RetS and sRNA[END_REF].

Cytotoxicity towards macrophages

The cytotoxicity of P. aeruginosa strains grown to mid-log phase in LB broth was assayed using J774 macrophages as described in [START_REF] Soscia | Cross Talk between Type III Secretion and Flagellar Assembly Systems in Pseudomonas aeruginosa[END_REF] except that macrophages were infected for 45 min or 2.5 hr at a multiplicity of infection (MOI) of 20 for the PAO1 and PA14 strains respectively.

The percentage of LDH release was calculated relatively to that of the uninfected control, which was set at 0% LDH release, and that of uninfected cells lysed with Triton X-100, which was set at 100% LDH release.

Statistical analysis

For multigroup comparisons, a main P value was calculated by ANOVA (Stat Plus software) and unpaired Student's t tests were performed using Excel software (Microsoft) for two-group comparisons. On the figures, asterisks indicate statistical significance.

  to abiotic surface of various strains was visualized (Fig 7A) and quantified using crystal violet staining (Fig 7B). The production of exopolysaccharides, a main biofilm component, was also visualized on Congo red-containing plates (Fig 7C). As expected from the literature, the ability of the PA14 strain to form biofilm was higher than for PAO1, the exopolysaccharide staining (Fig 7C, the PA14 strain is red on Congo-red plates) being consistent with adherence assay (Fig 7A and 7B). A clear difference in the adherence capacities was observable after 8h (Fig 7A, compare line 1 with line 3 at 8h) and was even greater at 48h (Fig 7A, compare line 1 with line 3

BInvitrogenFigure 1 :

 1 Figure 1: Transcriptome of PAO1 and PA14 reveals differential QS target genes and qslA

Figure 2 :

 2 Figure 2: β-galactosidase activities of transcriptional fusions PlasR-'lacZ (A,B), PrhlI-'lacZ

Figure 3 :

 3 Figure 3: Pyocyanin quantification in the extracellular medium. Pyocyanin was extracted

Figure 4 :

 4 Figure 4: β-galactosidase activities of transcriptional fusions PH2-T6SS-'lacZ. Ratio of

Figure 5 :

 5 Figure 5: Immunodetection of the T2SS Xcp machinery component XcpP in cellular

Figure 6 :

 6 Figure 6: (A) Detection of elastase in the extracellular medium. Commassie blue stained

Figure 7 :

 7 Figure 7: (A) Bacterial adherence to plastic to infer the ability of strains to form biofilm.

Figure 8 :

 8 Figure 8: Cytotoxicity towards J774 macrophages evaluated using a LDH assay after 2

Figure S1 :

 S1 Figure S1: Validation of the absence of PA4685 gene in PAO1 strain by PCR. (A) Loci of PA4685

Figure S2 :

 S2 Figure S2 : BlastP of QslA from PAO1 and PA14 strains.

Table 1 :

 1 Highlights from Table S1 of genes of interest

			Ratio expression
	Gene name	Function	PA14/PAO1 PAO1/PA14 Activated by QS?
	qslA	QS regulator	55.8	/
	qscR	QS regulator	/	1.2
	rsaL	QS regulator	/	2.1
	mvaT	QS regulator	/	1.6
	cpdR	QS regulator	/	3.9
	qteE	QS regulator	/	1.9
	rhlR	Rhl QS system	2.5	/	X
	rhlI	Rhl QS system	1.8	/	X
	lasR	Las QS system	1.9	/	X
	lasI	Las QS system	1.5	/	X
	PA1656	H2-T6SS machinery	1.5	/	X
	lasB	LasB Elastase	7.7	/	X

Table 1 :

 1 Highlights from TableS1of genes of interest. Presentation of genes of interests regarding their ratio of expression between strains PAO1 and PA14.

Table 2 Strains, plasmids and oligonucleotides used in this study 728

 2

	Strain, plasmid or	Genotype, description or sequence	Source and/or reference
	oligonucleotide
	E. coli strains
	TG1	supE, hsdDR, thiD(lac-proAB), F' (traD36, proAB+, lacIq, lacZDM15)
	CC118(λpir)	(lpir) D (ara-leu), araD, DlacX74, galE, galK, phoA-20, thi-1, rpsE,
		rpoB, Arg(Am), recA1, Rfr (lpir)
	TOP10F'	F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1
		araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str R ) endA1 λ -

(A)-tailed using poly (A) polymerase. Then an RNA adapter was ligated to the 5´-phosphate of the RNA fragments. First-strand cDNA synthesis was performed using an oligo (dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 30 ng/µl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).

Acknowledgements

We are very grateful to Kyler A. Lugo and Lilian H. Lam for careful reading of the manuscript.

We also thank Steve Garvis for advice on RNA preparation and Vertis Biotechnologie AG (Freising-Weihenstephan, Germany) for RNA sequencing. We are grateful to I. Bringer, A. Brun and O. Uderso for technical assistance. T.G.S. and M.R.G. were supported by a Ph.D. fellowship from the French Research Ministry and with a "Teaching and Research" fellowship from Aix-Marseille University. A.L. was financed with a PhD fellowship from "Vaincre La Mucoviscidose" (RF20120600685 and RF20130500911). This work was supported by recurrent funding from the CNRS and Aix-Marseille University, and from "Pathomics" ERAnet PATHO Grant ANR-08-PATH-004-01. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. C.C., and B.I. performed the experiments. T.G.S. and S.B. supervised the execution of the experiments. T.G.S., R.L., A.C., B.I. and S.B. analyzed the data and discussed with M.R.G., A.L and R.V. T.G.S. and S.B. wrote the paper with contribution from B.I. and reading from R.L., A.C., M.R.G. and R.V.

This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

Construction of the ∆qslA mutant

To generate qslA deletion strain, 500 bp upstream and 500 bp downstream of the qslA gene were amplified by overlapping PCR with High Fidelity DNA polymerase (Roche Applied Science) using TSO108, TSO109, TSO110 and TSO111 primers (see Table 2). The PCR product was cloned in pCR2.1 (TA cloning kit; Invitrogen) giving pTS48, which was then sequenced (GATC) and subcloned in pKNG101 suicide vector giving the mutator pTS50. pTS50, maintained in the E. coli CC118 λpir was further conjugated in P. aeruginosa strain PAO1 using protocol previously described [START_REF] Sana | Gene Transfer: Conjugation[END_REF]. The mutant, in which the double recombination events occurred and resulted in the nonpolar deletion of qslA gene were verified by PCR using external primers TSO112-TSO113.

Construction of the chromosomic qslAV5 recombinant gene

PAO1 strains chromosomally encoding QslAV5 translational fusion was engineered by exchanging the qslA stop codon with the sequence encoding the V5 tag followed by a stop codon. This was achieved by overlapping PCR of the 500 bp upstream and downstream regions of the native stop codon of qslA with the High Fidelity DNA polymerase (ROCHE) using primers TSO103, TSO104, TSO105 and TSO106 (Table 2). The PCR product was cloned in pCR2.1 (TA-cloning KIT, Invitrogen), which was then sequenced (GATC) and subcloned in pKNG101 suicide vector giving the pTS52. pTS52, maintained in the E. coli CC118λpir strain,

Author contributions


Reinis Osis

François Laurent

René Poccard-Chapuis

SPATIAL RELATION BETWEEN NATURAL RESOURCES AND LAND USE DYNAMICS IN THE AMAZON AGRICULTURAL FRONTIER

Slash-and-burn agriculture

Photo: Osis, R. ( 2016)

Brazil

Legal Amazon South America 1 -ESO Laboratory -Le Mans Université, France (reinis.osis.etu@univ-lemans.fr). 2 -ESO Laboratory -Le Mans Université, France (francois.laurent@univ-lemans.fr). 3 -Cirad UMR SELMET, France (rene.poccard-chapuis@cirad.fr).

The research was conducted with the financial support of the Brazilian National Council of Technological and Scientific Development (CNPq), process nº 207592/2014-4. Agence Nationale de la Recherche (ANR) (ECOTERA project, ECOefficiences et développement TERritorial en Amazonie brésilienne; ANR-13-AGRO-0003) and Cirad. In the G4 cluster, areas with Loamy sand are preferred for deforestation.

Method:

We define groups of farms with similar relative area of land use over time and natural resources/constraints. 4 clusters were defined for farms larger than 220 ha and 5 clusters for farms smaller than 220 ha (220 ha governmental threshold to define smallholders).

-Algorithm Simple EM (expectation maximisation): EM assigns a probability distribution to each instance which indicates the probability of it belonging to each of the clusters [6]. In the P5 cluster, Loamy sand are also preferred for deforestation (but less than the cluster G4), while the Belterra clay returns to have more forest.

Cluster analysis on farm size, land use changes and the proportion of natural resources or constraints

Method:

Weights of Evidence (WofE) [7].

Data:

-MODIS land use classifications between 2004 and 2013 [START_REF] Perrier | Caractérisation et cartographie de l'éco-efficience des pâturages amazoniens à l'aide d'images MODIS[END_REF].

-Farm boundaries [START_REF] Semas | Base de dados de imóveis rurais (Cadastro Ambiental Rural do Pará -CAR). Governo do Pará[END_REF].

Examples of changes in the intesity of the weights of evidence in three periods, for trasition from forest to pasture

While there was an increasing preference for deforestation to plant pasture in the loamy sand, between 2004 and 2013, the mottled clay and duricrust were less and less deforested for these purposes. In the Belterra clay, pastures were recovered by forest. In the context of the central area the soil has acquired a higher importance in decision rules.

Phases

The weights of evidence vary over time, which is a reflection of the change in the logic of natural resource management

Considering the central area of the county, near the paved roads and the city, dominated by large farms with soybean crops:

Considering the eastern area dominated by smallholders:

Relation between the farm size and the relative area of stepped valleys

Why? Small farms are usually located along rivers.

Consequences: the natural resources of small-sized farms differ from those of large-sized ones, which contributes to diferent management practices. For instance, the high proportion of steeped valleys (50-500 m wide) in small farms generates limitations such as lower proportion of mechanizable areas.

Availability of natural resources or constraints

Depending on the farm size, the natural factors area may vary proportionately, which impacts the farm management Data: -Pedomorphological units [START_REF] Thales | Paragominas pedomorphological units. Shapefile vector data[END_REF].

-Farm borders [START_REF] Semas | Base de dados de imóveis rurais (Cadastro Ambiental Rural do Pará -CAR). Governo do Pará[END_REF].

• The natural resources or constraints available in the farm influence the farmer's land use decision rules.

• Groups (clusters) of farms with similar natural resources or constraints and similar land use dynamics can be identified and may reflect classes of decision rules.

• The decision rules have been changing since 2004.

• The spatialization of these groups can help in the creation of a model of land use change for the county, as a subsidy for the creation of future land-use scenarios.

Conclusions

[ 6 ]

 6 FRANK, E.; HALL, M. A.; WITTEN, I. H. (2016). The WEKA Workbench. Online Appendix for "Data Mining: Practical Machine Learning Tools and Techniques", Morgan Kaufmann, Fourth Edition, 2016. [7] KEMP, L.D.; BONHAM-CARTER, G.F.; RAINES, G.L. (1999). Arc-WofE: Arcview extension for weights of evidence mapping. http://www.ige.unicamp.br/wofe. [8] SOARES-FILHO, B.; CERQUEIRA, G. C.; PENNACHIN, C. L. (2002) "DINAMICA -A stochastic cellular automata model designed to simulate the landscape dynamics in an Amazonian colonization frontier," Ecol. Modell., vol. 154, no. 3, pp. 217-235. [9] HASAN, A. (2017) Map of burned forests of Paragominas. Shapefile vector data. Unpublished. Paragominas county, Pará, Brazil Area: 19,342 km² Population in 2016: 108,547 Creation of the municipality: 1965 Approximately 2,200 registered properties, 84% with more than 1,000 ha Pioneer in actions against deforestation Typology of dynamics of land use Evidence (WofE) [7] : probability of occurrence of a transition from a land use to another depending on a spatial variable: processed with Dinamica EGO platform [8] Data: Soil texture [5].

  WofE of the variable soil texture in the transition forest to pasture (deforestation) between 2004 and 2015

Farm

  hydric conditions and relief are among the variables which farmers consider in decision-making process.OBJECTIVE: UNDERSTAND THE ROLE OF NATURAL RESOURCES IN THE SPATIAL DISTRIBUTION AND TEMPORAL DYNAMICS IN CONTEXT OF LAND USE IN THE EASTERNIn the smallholders area, the preference for deforestation to plant pasture in the mottled clay reduced over period. The soil texture has now lower importance in the decision rules.


Sindhu Preetham Burugupally

Analysis of a mesoscale thermal actuator in open and closed operating cycles

Keywords: Thermal actuator, compliant architecture, open and closed operating cycles, mesoscale

Thermal-based actuators are known for generating large force and displacement strokes at mesoscale (millimeter) regime. In particular, two-phase thermal actuators are found to benefit from the scaling laws of physics at mesoscale to offer large force and displacement strokes; but they have low thermal efficiencies. As an alternative, a combustion-based thermal actuator is proposed and its performance is studied in both open and closed cycle operations. Through a physics-based lumped-parameter model, we investigate the behavior and performance of the actuator using a spring-mass-damper analogy and taking an air standard cycle approach. Three observations are reported: (1) a closed cycle operation results in higher thermal efficiency compared to an open cycle due to the absence of pumping losses in the closed cycle; (2) an increase in heat input to the actuator results in increasing the thermal efficiency of both open and closed cycles; and (3) for a specific heat input, both the open and closed cycle operations respond differently -different stroke lengths, peak pressures, and thermal efficiencies.

Introduction

Mechanical actuation via thermal actuators has been a topic of interest due to their ability to generate forces O(10) 1 N and displacement strokes O(10) -4 m at mesoscale (millimeter) regime [START_REF] Bell | MEMS actuators and sensors: observations on their performance and selection for purpose[END_REF].

Most thermal actuators employ either bimorph [START_REF] Henriksson | Integrated long-range thermal bimorph actuators for parallelizable Bio-AFM applications[END_REF], solid expansion [START_REF] Butler | Average power control and positioning of polysilicon thermal actuators[END_REF], fluid expansion [START_REF] Lu | Optimal design of a flexural actuator[END_REF], state change (liquid-to-vapor) [START_REF] Bardaweel | MEMS-based resonant heat engine: scaling analysis[END_REF], gas expansion/compression [START_REF] Nakajima | Study on micro engines-miniaturizing Stirling engines for actuators and heatpumps[END_REF], or combustion based mechanisms [START_REF] Preetham | Modeling of a resonant heat engine[END_REF]. Among them, state change and combustion based mechanisms are found to be suitable for large displacement and force generation at mesoscale as they benefit from the scaling laws of physics [START_REF] Bardaweel | MEMS-based resonant heat engine: scaling analysis[END_REF][START_REF] Preetham | Modeling of a resonant heat engine[END_REF]. In particular, combustion-based miniature heat engines offer high power density to weight ratio and fast response, thus making ideal for applications that require high speed, large force, and large displacement strokes.

These actuators have applications in exoskeleton [START_REF] Ouyang | Development of a novel compact hydraulic power unit for the exoskeleton robot[END_REF], locomotion [START_REF] Raibert | the rough-terrain quadruped robot[END_REF], and rehabilitation [START_REF] Bradley | NeXOS -The design, development and evaluation of a rehabilitation system for the lower limbs[END_REF].

Unfortunately, thermal actuators or heat engines with sliding-piston architecture at mesoscale suffer from high friction, leakage, and heat losses, resulting in poor thermal efficiencies [START_REF] Formosa | Scaling laws for free piston Stirling engine design: Benefits and challenges of miniaturization[END_REF][START_REF] Burugupally | Power Generation via Small Length Scale Thermo-Mechanical Systems: Current Status and Challenges, a Review[END_REF]. A method to improve the thermal efficiency is by eliminating the crankshaft assembly and associated friction losses [START_REF] Preetham | Modeling of a resonant heat engine[END_REF]; this can be accomplished by implementing a compliant architecture (Fig. 1) instead of a sliding-piston architecture, which also offer other advantages -simpler construction and lower fabrication costs [START_REF] Preetham | Estimation of parasitic losses in a proposed mesoscale resonant engine: Experiment and model[END_REF][START_REF] Preetham | Mathematical modeling of a four-stroke resonant engine for micro and mesoscale applications[END_REF].

Research on the fundamental design of thermal actuators (heat addition/rejection cycle [START_REF] Preetham | Modeling of a resonant heat engine[END_REF]) or heat engines (Otto cycle) [START_REF] Preetham | Estimation of parasitic losses in a proposed mesoscale resonant engine: Experiment and model[END_REF][START_REF] Preetham | Mathematical modeling of a four-stroke resonant engine for micro and mesoscale applications[END_REF] that employ compliant architecture are being studied through models [START_REF] Preetham | Mathematical modeling of a four-stroke resonant engine for micro and mesoscale applications[END_REF], prototype fabrication and experiments [START_REF] Preetham | Estimation of parasitic losses in a proposed mesoscale resonant engine: Experiment and model[END_REF]. First-principle based mathematical models are being developed to assess the performance of the compliant actuators/engines for a range of physical and operating parameters in both open and closed cycle operations. However, it is unclear whether such a thermal actuator/heat engine be analyzed or designed to operate in an open or a closed cycle.

Our present work builds on the growing interest in thermal actuators through the exploration of complaint architecture at the mesoscale regimes. This work builds on our prior physics-based modeling efforts of a resonant heat engine for portable power applications [START_REF] Preetham | Modeling of a resonant heat engine[END_REF][START_REF] Preetham | Estimation of parasitic losses in a proposed mesoscale resonant engine: Experiment and model[END_REF][START_REF] Preetham | Mathematical modeling of a four-stroke resonant engine for micro and mesoscale applications[END_REF]. In this study, through a physics-based lumped-parameter model, similar to our previously developed models on free piston engines [START_REF] Burugupally | Design and performance of a miniature free piston expander[END_REF][START_REF] Burugupally | The effect of working fluid properties on the performance of a miniature free piston expander for waste heat harvesting[END_REF][START_REF] Preetham | Investigations of a new free piston expander engine cycle[END_REF], we will investigate the behavior and performance of a mesoscale thermal actuator using a spring-mass-damper analogy and taking an air standard cycle approach. Here, using the model, we will predict the actuator performance for a range of operating conditions and report on the important differences between the open and closed cycle operations.

Thermal Actuator: Design and Operation

The proposed actuator is similar to a traditional internal combustion engine, where fuel-air mixture is burnt in a piston-cylinder arrangement to produce force and displacement strokes. However unlike an internal combustion engine where the piston motion is constrained by the crankshaft assembly, the piston of the actuator is unconstrained and free to move (Fig. 1). Specifically, the traditional piston-cylinder assembly is replaced by a flexible cavity (or compliant chamber) with a piston at one end. Hence the volume change occurs through deformation of the flexible cavity.

Due to the elimination of a traditional sliding seal and crankshaft assembly, the frictional losses are substantially reduced. This concept shares some features with free piston engines which employs a rebound device to store energy, the flexible cavity itself performs the task of a rebound device thus enabling a reduction in actuator size. The actuator can be operated in both open and closed operating cycles. In the open cycle, the actuator exchanges both heat and mass with the surroundings, while in the closed cycle only exchange of heat takes place.

The actuator when operated in the open cycle comprises of compression (1 → 2), heat addition

(2 → 3), expansion (3 → 4), exhaust (4 → 5 → 6), and intake (6 → 7 → 1) processes as depicted in Fig. 2. The cycle begins with the compression process 1 → 2 where the piston compresses the working fluid to state 2. Then, the heat addition at state 2 'Top Dead Center' (TDC) results in the pressurization of the CV and initiation of the expansion process 3 → 4 where the piston moves towards 'Bottom Dead Center' (BDC). Next, the valve opens and the exhaust process begins where the expended working fluid is discharged out of the CV to the ambient pressure P o (4 → 5 → 6). During the exhaust process, the piston travels from BDC to TDC scavenging the working fluid from the CV. Finally, the intake process 6 → 7 → 1 results in the introduction of fresh working fluid into the actuator cavity due to the suction created by the piston-cavity motion. This completes one operating cycle of the actuator.

Note the compression (CR) and expansion (ER) ratios are defined as CR= V 1 V 2 and ER= V 4 V 3 . It is worth noting that, unlike the open cycle, the closed cycle terminates with the heat rejection process (4 → 1)

as depicted in Fig. 2.

Model

A physics-based lumped parameter model of the actuator is derived to investigate the actuator behavior and performance for both the open and closed cycle operations. The actuator is modeled as a spring-mass-damper system by taking an air standard cycle approach (Fig. 1b). Here, the hollow cavity CV contains the working fluid. In this study, the working fluid is air with constant properties whose specific heat ratio γ = 1.4 and obeys ideal gas law. The heat addition to the actuator normally associated with combustion is modeled as constant volume heat addition process into the cavity of nominal volume V o for a short duration t q →0. The heat rejection from the actuator at the end of the expansion process can occur via mass transfer to the surroundings or by applying a cooling pulse for a short duration t q →0. Mathematical statements of these principles are:

m S 2 ∆ V + b + b p S 2 ∆ V + s S 2 ∆V = ∆P (1)
Ṁ = Ṁi -Ṁe (2) h∆T + d (Mc v T) dt + P∆ V = q + Ṁi c p T i -Ṁe c p T (3)
PV = MRT (4)
Ṁi,e = -k∆P [START_REF] Bardaweel | MEMS-based resonant heat engine: scaling analysis[END_REF] where M is the mass of the working fluid in the CV at any given time t, h is a coefficient that models conduction/convection heat losses from the CV to the surroundings, R is the mass-specific gas constant of working fluid, k is the mass flow rate coefficient, and c v and c p are the constant volume and constant pressure heat capacities of the working fluid, respectively and are assumed constant over the temperature range. The overdot denotes time derivative, while the subscripts 'i' and 'e' denote intake and exhaust processes, respectively. The damping coefficient b p is zero for cases where no external work is done on the actuator during the intake process, and/or for other processes in the actuator's operating cycle.

The nonlinear model, Eqs. [START_REF] Bell | MEMS actuators and sensors: observations on their performance and selection for purpose[END_REF][START_REF] Henriksson | Integrated long-range thermal bimorph actuators for parallelizable Bio-AFM applications[END_REF][START_REF] Butler | Average power control and positioning of polysilicon thermal actuators[END_REF][START_REF] Lu | Optimal design of a flexural actuator[END_REF][START_REF] Bardaweel | MEMS-based resonant heat engine: scaling analysis[END_REF] is first nondimensionalized using the scales in Eq. 6

∆ V = ∆V V o , ∆ P = ∆P P o , ∆ T = ∆T T o , q = q q H
, and t = tω

where ω = γP o S 2 mV o is a reference frequency and the overbar indicates a nondimensional independent or dependent variable. Thus obtained nondimensionalized equations are written in state space format as shown below:

d∆ V d t = ∆ V (7) d∆ V d t = ∆ P γ - b + b p mω ∆ V - s mω 2 ∆ V (8) ∆ Ṫ = 1 Mc v T o ω q + Ṁi T o R + c p ∆ Ti -c v ∆ T -hT o ∆ T -Ṁe RT o (1 + ∆ T) -P o V o ω (1 + ∆ P) ∆ V (9) ∆ Ṗ = 1 P o V o ω (1 + ∆ V) Ṁi -Ṁe RT o (1 + ∆ T) + MRT o ω∆ Ṫ -P o V o ω (1 + ∆ P) ∆ V (10)
Here, it is noteworthy to mention that Eq. 10 is obtained by taking a first derivative of ideal gas law (Eq. 4) with respect to the nondimensional time t. If the heat added per cycle and initial conditions ∆T(0), ∆P(0), and ∆V(0) are specified, equations [START_REF] Bell | MEMS actuators and sensors: observations on their performance and selection for purpose[END_REF][START_REF] Henriksson | Integrated long-range thermal bimorph actuators for parallelizable Bio-AFM applications[END_REF][START_REF] Butler | Average power control and positioning of polysilicon thermal actuators[END_REF][START_REF] Lu | Optimal design of a flexural actuator[END_REF][START_REF] Bardaweel | MEMS-based resonant heat engine: scaling analysis[END_REF] constitutes a nonlinear model for the determination of the intermediate thermodynamic state variables ∆T(t), ∆P(t), and ∆V(t). As the heat added per cycle is periodic, it is assumed that the dependent variables ∆T(t), ∆P(t), and ∆V(t)

will also be periodic at steady state.

(a) (b)

t

Solving the open cycle model

Figure 3a shows the heat-rate function q used for modeling the open cycle. The heat addition pulse associated with combustion phenomenon is assumed impulsive for a duration t q → 0. The heating pulse q at a constant rate of q H is applied at the end of the compression stroke at t = t 1 .

When t q → 0, lim t q →0 q H t q = E, where E is the finite energy supplied to the actuator per cycle during the heat addition process. The heat addition process is followed by expansion, exhaust, and intake processes. The heat rejection from the actuator occurs through the valve via mass transfer during the exhaust process. The first phase of the exhaust, the blowdown process is assumed to be an instantaneous and isentropic, and occurs at the end of the expansion stroke (at zero piston velocity).

The second phase of the exhaust, the displacement process, occurs for a time duration t 4 . At the time t = t 1 + t 2 + t 3 + t 4 = t p , the cycle repeats, hence t p is the time period of the actuator oscillation. The time lapsed in each process need not be equal.

To generate a pressure-volume diagram, the model Eqs. [START_REF] Preetham | Modeling of a resonant heat engine[END_REF][START_REF] Ouyang | Development of a novel compact hydraulic power unit for the exoskeleton robot[END_REF][START_REF] Raibert | the rough-terrain quadruped robot[END_REF][START_REF] Bradley | NeXOS -The design, development and evaluation of a rehabilitation system for the lower limbs[END_REF] is numerically integrated in the order 1→2→3→4→5→1 (Fig. 2) starting with state 1: ∆ V1 , ∆ P1 , ∆ T1 , ∆ V1 = 0. A proper choice of state variables at state 1: ∆ V1 , ∆ P1 , ∆ T1 are necessary to obtain a steady state solution (see Ref. [START_REF] Burugupally | Power generation via small length scale thermo-mechanical systems: current status and challenges, a review[END_REF] for further details). The resulting volume ∆ V2 , pressure ∆ P2 , and temperature ∆ T2 from the compression process, 1→2 is computed by numerically integrating Eqs. [START_REF] Preetham | Modeling of a resonant heat engine[END_REF][START_REF] Ouyang | Development of a novel compact hydraulic power unit for the exoskeleton robot[END_REF][START_REF] Raibert | the rough-terrain quadruped robot[END_REF][START_REF] Bradley | NeXOS -The design, development and evaluation of a rehabilitation system for the lower limbs[END_REF] from the initial condition ∆ V1 , ∆ P1 , ∆ T1 , ∆ V1 = 0 for a time t1 such that the piston velocity V2 = 0. The resulting volume ∆ V3 , pressure ∆ P3 , and temperature ∆ T3 during the heat addition process, 2→3 is computed from equations 8 and 9 by assuming it to be instantaneous (∆ V3 = ∆ V2 and ∆ V3 = ∆ V2 ). The volumes, pressures, and temperatures ∆ V4 , ∆ P4 , ∆ T4 ; ∆ V6 , ∆ P6 , ∆ T6 ; and ∆ V1 , ∆ P1 , ∆ T1 from the expansion 3→4, exhaust (displacement phase) 5→6, and intake processes 6→7→1 are computed by numerically integrating Eqs. [START_REF] Preetham | Modeling of a resonant heat engine[END_REF][START_REF] Ouyang | Development of a novel compact hydraulic power unit for the exoskeleton robot[END_REF][START_REF] Raibert | the rough-terrain quadruped robot[END_REF][START_REF] Bradley | NeXOS -The design, development and evaluation of a rehabilitation system for the lower limbs[END_REF] from the initial conditions ∆ V3 , ∆ P3 , ∆ T3 , ∆ V3 = 0; ∆ V5 , ∆ P5 , ∆ T5 , ∆ V5 = 0; and ∆ V6 , ∆ P6 , ∆ T6 , ∆ V6 = 0 for times t2 , t3 , and t4 respectively such that the piston velocity ∆ V4 = ∆ V5 = 0 and ∆ V6 = ∆ V7 = 0. It is worth nothing that the times t1 , t2 , t3 , and t4 are 'a priori' and are determined during the integration. It is assumed that the blowdown process 4→5 is instantaneous, isentropic, and occurs at zero piston velocity (∆ V4 = ∆ V5 = 0) and ambient pressure such that ∆ P5 = 0. Therefore, the temperature of the working fluid at the end of the exhaust (blowdown phase) process 4→5, ∆ T5 is computed using the adiabatic relation given by Eq. 11.

∆ T5 = (1 + ∆ T4 ) 1 + ∆ P4 1 + ∆ P5 1-γ γ -1 (11)

Solving the closed cycle model

Figure 3b shows the heat-rate function q used for modeling the closed cycle. The heat addition pulse associated with combustion phenomenon is assumed impulsive for a duration t q → 0. The heating pulse q at a constant rate of q H is applied at the end of the compression stroke at t = t 1 . When t q → 0, lim t q →0 q H t q = E, where E is the finite energy supplied to the actuator per cycle during the heat addition process. The heat addition process is followed by expansion and heat rejection processes.

The heat rejection from the actuator takes place by applying a cooling pulse at the end of the expansion stroke (at zero piston velocity) for a short duration t q →0. At the time t = t 1 + t 2 = t p , the cycle repeats, hence t p is the time period of the actuator oscillation. The time lapsed in each process need not be equal. Since no mass transfer can occur in the closed cycle operation, the valve shown in Fig. 1 is sealed. Therefore, the cavity is modeled as sealed cavity. The heat transfer to and from the actuator normally associated with combustion and exhaust is modeled by the heat rate q in and out of the sealed cavity of volume V o . Contrary to open cycle, the negative damping coefficient b p (<0) that models the work done on the actuator during the intake stroke is set to zero, as intake and exhaust processes does not exists here.

To generate a pressure-volume diagram, the model Eqs. [START_REF] Preetham | Modeling of a resonant heat engine[END_REF][START_REF] Ouyang | Development of a novel compact hydraulic power unit for the exoskeleton robot[END_REF][START_REF] Raibert | the rough-terrain quadruped robot[END_REF][START_REF] Bradley | NeXOS -The design, development and evaluation of a rehabilitation system for the lower limbs[END_REF] is numerically integrated in the order 1→2→3→4→1 (Fig. 2) starting with state 1: ∆ V1 , ∆ P1 , ∆ T1 , ∆ V1 = 0. A proper choice of state variables at state 1: ∆ V1 , ∆ P1 , ∆ T1 are necessary to obtain a steady state solution (see Ref. [START_REF] Burugupally | Power generation via small length scale thermo-mechanical systems: current status and challenges, a review[END_REF] for further details). The resulting volume ∆ V2 , pressure ∆ P2 , and temperature ∆ T2 from the compression process, 1→2 is computed by numerically integrating Eqs. [START_REF] Preetham | Modeling of a resonant heat engine[END_REF][START_REF] Ouyang | Development of a novel compact hydraulic power unit for the exoskeleton robot[END_REF][START_REF] Raibert | the rough-terrain quadruped robot[END_REF][START_REF] Bradley | NeXOS -The design, development and evaluation of a rehabilitation system for the lower limbs[END_REF] from the initial condition ∆ V1 , ∆ P1 , ∆ T1 , ∆ V1 = 0 for a time t1 such that the piston velocity V2 = 0. The resulting volume ∆ V3 , pressure ∆ P3 , and temperature ∆ T3 during the heat addition process (2→3) is computed from equations 8 and 9 by assuming it to be instantaneous (∆ V3 = ∆ V2 and ∆ V3 = ∆ V2 ). The volume, pressure, and temperature ∆ V4 , ∆ P4 , ∆ T4 from the expansion process 3→4 is computed by numerically integrating

Eqs. [START_REF] Preetham | Modeling of a resonant heat engine[END_REF][START_REF] Ouyang | Development of a novel compact hydraulic power unit for the exoskeleton robot[END_REF][START_REF] Raibert | the rough-terrain quadruped robot[END_REF][START_REF] Bradley | NeXOS -The design, development and evaluation of a rehabilitation system for the lower limbs[END_REF] from the initial conditions ∆ V3 , ∆ P3 , ∆ T3 , ∆ V3 = 0 for time t2 such that the piston velocity

∆ V4 = ∆ V1 = 0.
It is worth nothing that the times t1 and t2 are 'a priori' and are determined during the integration. It is assumed that the heat rejection process 4→1 is instantaneous and occurs at zero piston velocity (∆ V4 = ∆ V1 = 0).

Results and Discussion

The non-linear mathematical model, Eqs. to a prototype actuator that were experimentally determined in Ref. [START_REF] Preetham | Estimation of parasitic losses in a proposed mesoscale resonant engine: Experiment and model[END_REF]. Standard temperature T o =295 K and P o =93.4 kPa are chosen for the ambient state and for the thermodynamic properties of air. The properties of air used are R=286.9 J/kg-K and c v =717.25 J/kg-K.

Pressure-Volume diagram

A relationship between the pressure and volume for open and closed cycle operations is shown in Fig. 4. It is obtained using the model at steady state operation by impulsively supplying a heat E at the end of the compression stroke 1→2 that corresponded to lean combustion (equivalence ratio φ=0.51 [START_REF] Heywood | Internal Combustion Engine Fundamentals[END_REF]) of octane in the actuator. The peak pressure predictions in the actuator cavity after the heat addition from open and closed cycles are off by a factor of 1.3 (Fig. 4). Because the equivalence ratio for both the cycles are equal (φ=0.51), the temperature rise in both the cycles are equal (∆T 23 =2064 K). However, the pressure rise ∆P 23 among the cycles is different, as the density of air at the thermodynamic state 2 in both the cycles are unequal -for the closed cycle, the density at state 2 is 1.10 kg/m 3 , while for the open cycle, the density at state 2 is 1.87 kg/m 3 . In the closed cycle operation, the pressure in the cavity at the end of heat rejection process is below the ambient pressure (-11 kPa gage), while for an open cycle the pressure equilibrates with the ambient pressure modeled by the blowdown process (P 5 =0 kPa gage). This is because the open cycle interacts with the surroundings via mass transfer that allows the pressure to quickly equilibrate to the ambient conditions, while in the closed cycle there is no mechanism (no mass transfer or heat transfer h=0 W/K) to equilibrate the cavity pressure to the ambient conditions.

To determine the work done per cycle for the reference actuator and φ=0.51, a numerical integration of the PV diagram (Fig. 4) for one operating cycle is performed and found to be 16. and expansion ratios for closed cycle are found to be 5.05, while the compression and expansion ratios for the open cycle are found to be 1.76 and 4.13, respectively (Table 1). This result indicates that despite the same equivalence ratio φ the open and closed cycles respond differently with different performance metrics quantified in terms of stroke lengths, peak pressures, and indicated work. while for the open cycle the stroke lengths are typically not equal and depends on the process. The corresponding CR and ER values for the reference actuator with φ=0.51 are presented in Table 1.

Equivalence ratio and heat input relationship

For a specific fuel, an equivalence ratio is dependent on the mass of fuel injected (or associated heat input) and the mass of air inducted in to the actuator [START_REF] Pulkrabek | Engineering Fundamentals of the Internal Combustion Engine[END_REF].

Efficiency and volume ratios

For a fixed equivalence ratio (say φ=0.5), a difference of about 10% in indicated thermal efficiency is observed between the open and closed cycles (Fig. 7). The higher efficiencies of the closed cycle operations are because of ignoring the pumping losses in the actuator associated with the exhaust and intake processes. Furthermore, as can be seen in figure, an increase in the heat input or equivalence ratio φ increases the volume ratio (ER or CR). This is because a higher heat input or equivalence ratio φ will result in the higher pressure at the thermodynamic state 3 (Fig. 4), resulting in pushing the piston further outward and causing larger volume ratios (ER or CR).

Figure 1 .

 1 Figure 1. Proposed thermal actuator (a) schematic sketch and (b) lumped model. CV denotes the cavity or control volume with state parameters: pressure P, volume V, and temperature T at time t. The valve on the left is used for the intake or exhaust of the working fluid (air). The valve is connected to the ambient atmosphere (P o , T o ) during the intake and exhaust processes. For the closed cycle, the valve remains closed over the entire operating cycle.

Figure 2 .

 2 Figure 2. Ideal pressure-volume diagram of the actuator in open and closed cycle operations. Processes 1→2: Compression; 2→3: Heat addition for duration t q ; and 3→4: Expansion. For closed cycle, process 4→1 is heat rejection. For open cycle, processes 4→5: Blowdown phase of exhaust; 5→6: Displacement phase of exhaust; 6→7→1: Intake. Note the piston positions at states 6 and 7 are 'Top Dead Center' (TDC) and states 4 and 5 are 'Bottom Dead Center' (BDC), respectively.

  The pressure inside the cavity represented by bellows configuration oscillates about standard atmospheric pressure P o . The deformable corrugations of total stiffness s allow horizontal displacement x(t) of the piston (mass) m. The spring with stiffness s helps restore this horizontal position x(t) of the piston m. When the displacement of the piston m is zero, the cavity (CV) volumeV o is V o = LS,where L is the nominal cavity length and S is the cross-sectional area of the cavity. The movement of the piston m is impeded by the dampers b and b p . Damper b models energy conversion which is the sum of both friction work and useful work, and b p (<0) models work done on the actuator during the intake process. For a steady state operation, the working fluid temperature, T o + ∆T(t), pressure, P o + ∆P(t), and cavity volume, V o + ∆V(t) undergo cyclic variation, where the nominal or ambient and time-varying cyclic components are denoted with the 'o' subscript and ∆, respectively. The lumped parameter model of the actuator shown in Fig. 1 is derived by applying Newton's second law for the piston m, conservation of mass, conservation of energy, an ideal gas model to the cavity CV containing the working fluid, and a linear mass flow-rate equation for the valve.

Figure 3 .

 3 Figure 3. Heat-rate function q for modeling the (a) open and (b) closed operating cycles.

[ 1 - 5 ]

 15 is used to predict the open and closed cycle operations of the actuator. The actuator parameters h=0 W/K, b= 21.5 N-s/m, k=6.8 ×10 -7 kg/s-Pa ± 20%, V o =21.8 cc (2.08 cm in diameter and 1.6 cm of nominal length), s=3050 N/m, m=0.18 kg, and clearance volume V c =0.01V o are treated as the reference. The numerical values of k, V o , s, and m belong

Figure 4 .

 4 Figure 4. Open and closed cycle P-V diagrams for b=20 N-s/m, h=0 W/K, and φ=0.51.

Figure 5 .

 5 Figure 5. Open and closed cycle P-V diagrams for b=27.5 N-s/m, b p =-40 N-s/m, h=0 W/K, and φ=0.51. For the reference actuator, the addition of extra load (an increment by 1.5 N-s/m), results in shorter displacement strokes, and the addition of external work modeled by b p = -40 N-s/m during the intake process 7→1 results in significantly altering the PV diagram for the open cycle (comparing Figs. 4 and 5). It is worth noting that in the closed cycle operation, the stroke lengths are equal (ER=CR),

Fig. 6 Figure 6 .

 66 Figure 6. Heat input and equivalence ratio relation for the reference actuator for open and closed cycle operations.

Figure 7 .

 7 Figure 7. Efficiency and volume ratios for the reference actuator for open and closed cycles.

4. 4 .

 4 Frequency of operationIt is evident from Fig.8that the operating frequency of the actuator depends on the equivalence ratio. For the closed cycle, the operating frequency decreases monotonically with increasing equivalence ratio. In contrast, for the open cycle, the operating frequency first decreases and later increases with equivalence ratio due to collision occurring at top dead center (state 6). For the reference actuator, the operating frequency for a closed cycle is 37.03 Hz while for an open cycle is 13.37 Hz.The difference in frequencies is a result of varied stiffness of the cavity, and the number of strokes involved in the open and closed cycles. In a closed cycle, the cavity is sealed hence its stiffness and frequency remain constant through out the operating cycle. But in the open cycle, the stiffness varies and depends on the process. In addition, for a closed cycle only two strokes are involved in calculating the frequency, while in an open cycle all the four strokes are included.

Figure 8 .

 8 Figure 8. Open and closed cycle operating frequencies for the reference actuator.

Table 1 .

 1 Comparison of the performance metrics for the reference actuator in closed and open cycles with equivalence ratio φ=0.51. Mass of air in cylinder (kg/cycle) 2.41 × 10 -5 9.28 × 10 -5

	Performance Parameter	Closed cycle Open cycle
	Indicated thermal efficiency (%)	47.66	38.41
	Brake thermal efficiency (%)	44.33	34.64
	Heat added per cycle (J/cycle)	35.6	138.5
	Cycle frequency (Hz)	37.03	13.37
	Compression ratio (CR)	5.05	1.76
	Expansion ratio (ER)	5.05	4.13
	Average piston speed (m/s)	4.75	4.77
	Power output (W)	584.7	641.6

Conflicts of Interest:

The author declares no conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:

ER