Elia Riquelme

Sabina Omarova

Bérengère Ize

David O'callaghan
email: david.o-callaghan@umontpellier.fr

Analysis of the Brucella suis twin arginine translocation system and its substrates show that it is essential for viability

Bacteria use the Twin Arginine Translocator (Tat) system to export folded proteins from the cytosol to the bacterial envelope or to the extracellular environment. As with most Gramnegative bacteria, the Tat system of the zoonotic pathogen Brucella is encoded by a three gene operon; tatABC. Our attempts, using several different strategies, to create a Brucella suis 1330 tat mutant were all unsuccessful. This suggests that, for B. suis, Tat is essential and is in contrast to a recent report for B. melitensis. This was supported by our findings that two molecules that inhibit the Pseudomonas aeruginosa Tat system also inhibit B. suis, B. melitensis and B. abortus growth in vitro. In a bioinformatic screen of the B. suis 1330 proteome, we identified 28 proteins with putative Tat signal sequences. We used a heterologous reporter assay based on export of the Tat dependent amidase AmiA using the Tat signal sequences from the Brucella proteins to confirm that 20 of the 28 candidates can engage the Tat pathway.

Introduction

Translocation of proteins across membranes is essential for all aspects of the physiology of bacteria, ranging from basic metabolic processes to pathogenic/symbiotic interactions with a host. Gram negative bacteria use two pathways to translocate proteins across the cytoplasmic membrane; the Sec system, which exports unfolded proteins, and the Tat system, which exports folded proteins. The Tat pathway is found in many bacteria, including most animal and plant pathogens. It has the unique ability to transport fully-folded proteins, including a subset that binds redox cofactors, across the cytoplasmic membrane [START_REF] Palmer | The twin-arginine translocation (Tat) protein export pathway[END_REF]. Tat substrates are involved in a large array of bacterial processes such as respiratory metabolism, cellular division, motility, adaptation of bacteria to particular environments, or biofilm formation [START_REF] Buck E De | The importance of the twin-arginine translocation pathway for bacterial virulence[END_REF][START_REF] Gimenez | Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates[END_REF][START_REF] Ize | Role of the Escherichia coli Tat pathway in outer membrane integrity[END_REF][START_REF] Ochsner | Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis[END_REF].

Proteins that are targeted to the Sec or Tat pathways have specific N-terminal signal peptides that allow their recognition by their respective machineries. Sec and Tat signal peptides have the same overall tripartite organization (n-, h-and c-region), but Tat signal peptides are longer than their Sec counterparts (27 to 35 aa for Tat signal peptides versus 17 to 24 aa for Sec signal peptides). One of the main features of Tat signal peptides is the presence of a distinctive twin arginine motif S/TRRxFLK, in the n-region, where the arginine pair is almost invariant and other amino acids are present at a frequency >50%. Another characteristic of Tat signal peptides is that their h-region tends to be less hydrophobic than the corresponding region of Sec signal peptides [START_REF] Cristóbal | Competition between Sec-and TATdependent protein translocation in Escherichia coli[END_REF]. These characteristics have allowed the development of three prediction programs that recognize the features of Tat targeting sequences [START_REF] Bagos | Combined prediction of Tat and Sec signal peptides with hidden Markov models[END_REF][START_REF] Bendtsen | Prediction of twin-arginine signal peptides[END_REF][START_REF] Dilks | Prokaryotic utilization of the twinarginine translocation pathway: A genomic survey[END_REF].

While in most bacteria, tat mutants are viable, in a few organisms a functional Tat system appears to be essential under standard laboratory conditions. These include Haloferax volcanii [START_REF] Dilks | Genetic and biochemical analysis of the twinarginine translocation pathway in halophilic archaea[END_REF], Sinorhizobium meliloti [START_REF] Pickering | The twin arginine transport system appears to be essential for viability in Sinorhizobium meliloti[END_REF] and Mycobacterium tuberculosis [START_REF] Saint-Joanis | Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases β-lactam susceptibility and virulence[END_REF]. In other bacteria, including Burkholderia thailandensis [START_REF] Wagley | The twin arginine translocation system is essential for aerobic growth and full virulence of Burkholderia thailandensis[END_REF], Myxococcus xanthus [START_REF] Kimura | Myxococcus xanthus twin-arginine translocation system is important for growth and development[END_REF], Mycobacterium smegmatis [START_REF] Mcdonough | The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial β-lactamases[END_REF] and Shewanella oneidensis [START_REF] Luo | Mislocalization of Rieske Protein PetA Predominantly Accounts for the Aerobic Growth Defect of tat Mutants in Shewanella oneidensis[END_REF], a functional Tat system is needed for optimal growth in aerobic conditions. Conversely, for E. coli O157:H7 [START_REF] Pradel | Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7[END_REF], Pseudomonas aeruginosa [START_REF] Ochsner | Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis[END_REF], Agrobacterium tumefaciens [START_REF] Ding | Agrobacterium tumefaciens twin-arginine-dependent translocation is important for virulence, flagellation, and chemotaxis but not type IV secretion[END_REF], and Ralstonia solanacearum [START_REF] González | Using the Ralstonia solanacearum tat secretome to identify bacterial wilt virulence factors[END_REF] the Tat system is required for anaerobic growth.

Brucella are facultative intracellular pathogens. Intracellular survival and multiplication of Brucella are essential to the establishment, development and chronicity of the disease.

Brucella enters cells via lipid rafts, a route that ensures their intracellular survival by avoiding fusion of their membrane-bound compartment with lysosomes. Brucella then uses its VirB type IV secretion system [START_REF] O'callaghan | A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis[END_REF] to secrete effector proteins into the infected cells which direct the maturation of the vacuole into a replication-permissive organelle derived from the endoplasmic reticulum [START_REF] Celli | The Intracellular Life Cycle of Brucella spp[END_REF].

The genome sequences of all Brucella species contain genes that encode a Tat system and proteins have been annotated as 'Tat dependent'. A recent report describes the construction and characterization of a B. melitensis tat mutant , suggesting that the Tat system is required for stress resistance and virulence [START_REF] Yan | The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis[END_REF]. Here we report that we are unable to create a Brucella suis tat mutant and we demonstrate that two inhibitors of the P. aeruginosa Tat system inhibit the in vitro growth of several strains of Brucella in a dose dependent manner, suggesting that Tat is essential for viability. A bioinformatic screen for proteins with Tat signal sequences allowed us to identify a set of potential Tat substrates for B. suis, and we were able to confirm that the signal peptides of 20 of these engage the Tat pathway using a heterologous translocation reporter system.

Materials and Methods

Bacterial strains, growth conditions, plasmids.

The bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains were routinely grown in Lysogeny broth (LB) or on LB plates and Brucella on Trypticase Soy (TS).

When required, the medium was supplemented with ampicillin (Amp; 100 μg/mL), Kanamycin (Kan; 25μg/mL) or Chloramphenicol (Cm; 30μg/mL). N-Phenyl maleimide and Bay 11-7082 were from Sigma. Plasmid constructions were generated using standard methods or synthesised commercially (Genwiz).

In silico prediction of Tat substrates and signal sequence peptide sites.

A BLASTP search with the conserved SRRXFLK motif was used to identify putative Tat substrates encoded by the B. suis 1330 genome. Candidate substrates were analysed using the three existing programs for Tat signal prediction: TatP (http://www.cbs.dtu.dk/services/TatP/ [START_REF] Bendtsen | Prediction of twin-arginine signal peptides[END_REF] ), TATFIND (http://signalfind. org/tatfind.html [START_REF] Dilks | Prokaryotic utilization of the twinarginine translocation pathway: A genomic survey[END_REF])

and PRED-TAT (http://www.compgen.org/ tools/PRED-TAT/ [START_REF] Bagos | Combined prediction of Tat and Sec signal peptides with hidden Markov models[END_REF]).

For signal peptide domain prediction the SignalP 3.0 (HMM) and Phobius servers were used.

Most likely signal peptide cleavage sites were inferred from Sec, Tat and lipoprotein signal peptide predictions algorithms (SignalP 3.0, LipoP 1.0, TatP, TATFIND and PRED-TAT).

Plasmid constructions for the different strategies to obtain a tat mutant in Brucella

Clean deletion of the tatABC operon The genomic sequences immediately upstream (535bp) and downstream (512bp) of the tatABC operon were amplified from B. suis 1330 genomic DNA as template with Pfu DNA polymerase (Life technologies, USA) using primer pairs tatA-F1FW/tatA-F1REV and tatC-F2FWD/tatC-F2REV. Primers used for the constructions in this study are described in Supplemental Table S1. The PCR products were cloned into pUC29 and, after sequence verification (MWG Operon Eurofins, Germany), excised using the restriction introduced in the primers and ligated together in the SacB dependent sucide vector pIN60, generating pIN365.

Non polar deletion and marked tatA mutants. The same strategy was used to create a non polar deletion of tatA, with the same DNA fragment downstream of tatA and a 472 bp region upstream of tatA designed so that the ATG of tatA is followed by the second codon of tatB.

This was amplified with the tatA2UPstream-F1FWD/tatA2UPstream-F1FWD primer pair.

Again, the two fragments were ligated into pIN60 to give pIN367. To allow positive and visual selection of an insertion, we introduced replaced the tatA sequence with a chloramphenicol resistance cassette and gfp, amplified from pIN310 with primers eGFPXbaI/CmRMfeI giving a 1583bp. After sequence verification this was cloned into pIN367 as a XbaI and MfeI fragment to give pIN368.

Conditional mutant in the Tat system An anydrotetracycline-inducible tatABC operon was created by inserting the tetR repressor sequence and the Tn10 tetA promoter/operator (tetp/o) to drive expression of the tat operon. This was used to replace the native promoter of the tat operon, allowing us to control its expression by adding or removing anhydrotetracycline. We designed two constructs, the first contains 400bp upstream of the tatABC operon (F1) and the kanamycin resistance gene (810bp). The second construct had the tetR repressor gene and the promoter of the divergent tetA resistance gene followed by a 499bp sequence containing tatA and part of tatB. These two sequences were synthetized commercially (Genewiz) and cloned together in a SacB dependent suicide vector (pIN11) to generate plasmid pIN371.

N-Phenyl maleimide and Bay 11-7082 inhibition assay

Brucella strains were growth overnight in TS broth. Stock solutions of N-Phenyl maleimide or Bay 11-7082 at 100mM were made in ethanol and DMSO respectively. Serial 2 fold dilutions of each inhibitor were made in TS broth starting at 200 µM. One mL of each dilution was inoculated with B. suis, B. abortus and B. melitensis at an OD600 of 0.05 and incubated at 37C

with or without shaking (200rpm). After 24h (shaking) or 48h (static), the OD600 was measured in a spectrophotometer. Results are expressed as the % of the OD of a culture with no inhibitor, experiments were repeated three times.

Heterologous system to detect predicted Brucella Tat substrates

DNA sequences where the sequence encoding the signal sequence of the E. coli AmiA protein was replaced by the signal sequences of the 28 predicted Brucella Tat substrates were synthesised commercially (Genwiz) and cloned into plasmid pssAmiA-AmiAH [START_REF] Ball | Contribution of the Twin Arginine Translocation system to the exoproteome of Pseudomonas aeruginosa[END_REF]. A total of 32 plasmids were constructed to account for alternative cleavage sites (Table S2). E. coli strains (MC4100, MC4100 ΔssamiAC, and MC4100 ΔtatC) were transformed with these plasmids. Strains with no plasmid or transformed with a plasmid encoding wild type AmiA were used as controls.

Results

Characterisation of the Tat system in Brucella

All Brucella have the typical tatABC operon structure [START_REF] Nuñez | The twin-arginine translocation pathway in αproteobacteria is functionally preserved irrespective of genomic and regulatory divergence[END_REF]. Alignment of the tatABC genes from different Brucella species showed a high degree of homology: tatA shows 99.54%

identity, tatB at least 94.53 % and tatC 99.88% across the genus (data not shown). At the amino acid level,the annotated TatA sequences in Genbank have protein sequences of either 72 or 80 residues, however this is probably just an annotation error with an incorrect start codon used in the longer versions. The 72 amino acid sequences show100% identity to each other. TatC also shows 100% sequence identity. TatB sequences vary in length from 184 to 203 aa. While this is partly due to different predicted start sites, TatB displays a greater degree of heterogenicity than TatA and C; there are numerous deletions in a proline rich region in the TatB proteins of B. ovis, B. vulpis and several atypical isolates, as well as two shorter deletions at the C-terminus of B. ovis TatB (Fig. 1).

The Twin arginine system is essential for Brucella

To investigate the role of the Brucella Tat system in virulence, we tried several strategies to construct a B. suis tat mutant.

tatABC deletion. We first attempted to create a mutant with an unmarked deletion of the whole region encoding the tat operon. The genomic flanks (aproximately 500bp) immediately upstream and downstream of the tat operon were amplified using B. suis 1330 genomic DNA and cloned together in a SacB dependent suicide vector to give pIN365. Plasmid pIN365 was then electroporated into B. suis 1330 and Kanamycin-resistant (Kan R ) clones were selected;

PCR analysis confirmed that the plasmid had integrated into the tat locus. A second homologous recombination to get the allelic replacement with excision of the tat operon and integrated plasmid, was selected using sucrose sensitivity. We performed PCR on 200 sucrose resistant, kanamycin sensitive colonies and were unable to detect a colony where the tatABC operon had been deleted; all had reverted to wild type. This suggests that, for B. suis, the Tat system is essential.

Inframe deletion of tatA. Data from a Tn-seq screen performed on B. abortus [START_REF] Sternon | Transposon sequencing of Brucella abortus uncovers essential genes for growth in vitro and inside macrophages[END_REF] showed a very low number of transposon insertions in tatABC (Fig 2). Analysis of the Tnseq profile in the tatABC operon region shows that there are no insertions in the tatC region, however some insertions could be seen in the tatA region. Therefore, we reasoned that a deletion of tatA may be easier than deletion of the entire operon. Primers were designed to amplify upstream and downstream sequences which, when ligated together, replaced the ATG start codon of tatA with that of tatB. Here again we used a SacB dependent suicide vector to select a first integration but were again unable to identify a clone where the tatA gene had been deleted by the second recombination event (300 colonies tested).

Disruption of tatA with a chloramphenicol cassette and GFP marker. Due to the unsuccessful attempts in identifying an unmarked, inframe deletion mutant of the whole operon or the tatA gene, we decided to use a strategy that would allow us to positively select mutants by inserting two selectable markers, chloramphenicol resistance and GFP, into the tatA sequence. Here, attempts to isolate clones with even a single-crossover event were unsuccessful. No kanamycin resistant colonies were isolated after several attempts at electroporation.

Construction of conditional mutant in tat system. As a final strategy to generate a tat mutant, we attempted to construct a conditional mutant where tatABC expression was inducible. We used the anhydrotetracycline (ATC) inducible tetracycline resistance promoter from Tn10, which has already been used with success in Brucella, [START_REF] Smith | Postreplication roles of the Brucella VirB Type IV secretion system uncovered via conditional expression of the VirB11 ATPase[END_REF] to control expression of the tatABC operon.

To determine the opitmal ATC concentrations to induce expression of the tatABC genes from the tetA promoter, we used a plasmid carrying the gfp gene under control of the tetA promoter (pJC45). We establish by flow cytometry (FACS) that the minimal concentration for induction was 0.2 µM, GFP expression was maximal at 5 µM, and there was a reduction of fluorescence at 10 µM (data not shown). After electroporation with pIN371, colonies were grown on plates with different concentrations of ATC (0.2, 0.5, 1, 1.5, 2.0, 2.5, 5 and 10 µM)

and the appropriate antibiotics. From the many conditions tested, colonies resulting from the first crossover were only obtained when 0.2 µM ATC was present in both the liquid medium used during the 4-hour recovery post electroporation and in the plates. The colonies obtained were smaller than usual. This suggests that the integration of the plasmid affects expression of the Tat system which has a negative effect on the bacterium. Again, attempts to isolate second crossover events, where the wild type promoter was replaced by the tet promoter

were not successful and the first crossover colonies were lost after a few passages on agar.

Taken together, the results of this work provide evidence that the twin arginine transport system of B. suis is essential for viability under all the conditions that we tested.

Inhibitors of the Pseudomonas aeruginosa Tat system inhibit Brucella growth in vitro

As we could not genetically inactivate the Tat system, we tested two compounds previously reported to inhibit some bacterial Tat systems: N-Phenyl maleimide and Bay 11-7082. Both compounds have been proposed as potent Tat inhibitors and have been shown to inhibit the secretion of Phospholipase C, a Tat dependent substrate, in P. aeruginosa [START_REF] Vasil | Identification and evaluation of twin-arginine translocase inhibitors[END_REF]. We tested the effects of the two compounds on the growth B. suis, B. abortus and B. melitensis. In initial disc diffusion experiments with B. suis on plates, no growth inhibition was seen (not shown).

However, when tested in liquid culture both compounds appeared to inhibit bacterial multiplication for all three Brucella strains. Similar effects were seen in shaking and non shaking conditions with both inhibitors. Bay 11-708 showed a stronger capacity to inhibit Brucella growth than N-Phenyl maleimide. We sought to test the effects of these compounds on Brucella in cell infection assays. However, we found that both were highly cytotoxic at concentrations below 50 μM (not shown) meaning that they could not be used.

In silico prediction of Tat substrates in the Brucella proteome

Proteins are targeted to the Tat machinery through specific N-terminal signal peptides that have a distinctive twin arginine motif. A BlastP search with the conserved motif (SRRXFLK) was used to identify potential Tat substrates in B. suis 1330 proteome and then analysed with three programs for Tat signal prediction: TatP, TATFIND and PRED-TAT. We considered that a protein contained a putative Tat signal if it was predicted by at least two of the programs: a total of 28 proteins were identified (Table 2 and Table S2).

Heterologous system to confirm predicted Brucella substrates

To confirm the Tat-dependence of our 28 putative substrates, we used a heterologous reporter assay in E. coli. This assay is based on the Tat dependent export of the AmiA amidase which allows the identification of Tat signal peptide. When fused to a Tat signal peptide, AmiA is exported into the periplasm which rescues the SDS sensitive phenotype of E. coli MC400

ΔssamiAC that lacks periplasmic AmiA and AmiC activities. AmiA does not rescue SDS sensitivity when fused to a Sec signal peptide or when expressed in a tat mutant [START_REF] Gimenez | Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates[END_REF]. DNA sequences encoding signal sequence-AmiA fusions were synthesised and cloned into pssAmiA-AmiAH comercially [START_REF] Gimenez | Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates[END_REF][START_REF] Ball | Contribution of the Twin Arginine Translocation system to the exoproteome of Pseudomonas aeruginosa[END_REF]. If a putative Tat substrate had more than one predicted cleavage site, all were tested; we thus tested 33 putative signal peptides. We found that 20 of the 33 putative signal sequences restored SDS resistance of the ΔssamiAC but not of the ΔtatC strain (Fig. 4 and Fig S1). These data show that the B. suis genome encodes at least 20 bona fide Tat substrates.

Discussion

There is a high degree of conservation in genome organization between tatA, tatB and tatC genes across the Brucella genus and groups of highly related bacteria such as Ochrobactrum spp. and the well characterised tatABC operon of E. coli. While TatA and TatC are highly conserved (other than a two possible start sites for TatA), TatB shows the greatest degree of variability with deletions in a proline rich region and at the C-terminus. The NMR structure of the E. coli TatB protein suggests that this region is non structured [START_REF] Zhang | Solution structure of the TatB component of the twinarginine translocation system[END_REF]. These regions do not interact with TatC or the signal sequence of the transported substrate, but may interact with the substrates [START_REF] Ulfig | The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding[END_REF].

We attempted to create a tat mutant by deleting or interrupting the chromosomally encoded tat genes using several different strategies. None were sucessful, strongly suggesting that the Tat system is essential. The essentiality of the system is further suggested by Tn-seq analysis that was performed in B. abortus [START_REF] Sternon | Transposon sequencing of Brucella abortus uncovers essential genes for growth in vitro and inside macrophages[END_REF]. It could be possible that our attempts to disrupt the tat operon had also disrupted expression of serS which is also essential (Fig 2 ). However, the inhibitory effects of N-phenyl maleimide and Bay 11-7082, reported to inhibit Tat function in P. aeruginosa [START_REF] Vasil | Identification and evaluation of twin-arginine translocase inhibitors[END_REF] again suggest the essentiality of the Tat system. Unfortunately, these molecules were cytotoxic for both HeLa and J774 cells, which will limit their use as antimicrobial agents or for use as experimental Tat inhibitors in cell culture based infection models. Yan et al described the construction and characterisation of a B. melitensis mutant with a deletion of the tatA gene [START_REF] Yan | The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis[END_REF]. The reasons why construction of a tat mutant was possible in B. melitensis, while it appears essential in both B. suis and B. abortus and both inhibitors had an effect B. melitensis 16M are not clear. However, it may be possible that the B. melitensis strain used in that study has acquired unknown compensatory mutations that make inactivation of Tat possible (as recently reported for the central carbon metabolism in B. suis [START_REF] Machelart | Convergent evolution of zoonotic Brucella species toward the selective use of the pentose phosphate pathway[END_REF]). While many of the Tat substrates we identified are important to Brucella physiology, and some contribute to Brucella virulence, there are none that could explain the essentiality of the B. suis Tat system; however it is possible that this is due to a cumulative effect of loss of more than one Tat substrate.

Our bioinformatic screen identified 28 putative Tat substrates in B. suis 1330, two more than in the study by Nunez et al [START_REF] Nuñez | The twin-arginine translocation pathway in αproteobacteria is functionally preserved irrespective of genomic and regulatory divergence[END_REF]. Using the amidase heterologous reporter screen we were able to confirm that 20 were true Tat substrates. Four of the remaining candidates were able to export AmiA in a tat mutant, indicating that the fusions are at least partially routed to the Sec pathway. There have been reports that some Tat and Sec signal sequences can show a degree of promiscuity [START_REF] Tullman-Ercek | Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides[END_REF] and that a small number of AmiA fusions can restore SDS resistance to a tatC mutant [START_REF] Huang | Signal peptide hydrophobicity modulates interaction with the Twin-Arginine translocase[END_REF].

Yan et al described a similar bioinformatic screen in B. melitensis [START_REF] Yan | The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis[END_REF]. This study also tested translocation using the amidase reporter assay and constructed and analysed mutants in the predicted Tat substrates, suggesting that 6 contribute to virulence. As expected, given the high degree of sequence identity between the two strains, there were many similarities, however there were some differences in the list of predicted Tat substrates and the results from the amidase reporter assay they presented were very different to those of our study. While some so the differences are due to annotation anomalies, many cannot be explained. A comprehensive comparison of the two data sets is shown in supplemental data (Table S3).

Several of the identified proteins in our study, including a multicopper oxidase, the Rieske protein, MsrP, NosX, NosZ and NirK are are well known Tat substrates in other organisms.

Others, however, are not 'classical' Tat substrates; these include the two DsbA proteins, the L,D transpeptidases, the periplasmic binding proteins plus several proteins with unknown function. We identified homologues of all the confirmed substrates encoded by the genomes of closely related alpha proteobacteria (including Ochrobactrum, Rhizobiales and Agrobacterium) using BlastP and bioinformatically confirmed the presence of Tat signal sequences for all except the homologues of BR_RS10515 (data not shown). This suggests that the alphaproteobacterial have a repertoire of Tat substrates that differs from those in the enterobacteria and Pseudomonas. As seen with P. aeruginosa [START_REF] Gimenez | Genome wide identification and experimental validation of Pseudomonas aeruginosa Tat substrates[END_REF], a large number of the Tat substrates are predicted to contain co-factors (metal ions, iron-sulphur clusters..) and are predicted to be involved in electron transport, respiration and other oxidoreduction reactions.

These include the Rieske Fe/S protein, an essential subunit of mitochondrial and bacterial cytochrome bc1 complexes, NosX, NosZ, and NirK. Several of the Tat substrates we identified are involved in resistance to oxidative stress and survival at low oxygen concentrations (Brucella is generally classed as a strict aerobe, but can use alternative terminal electron acceptors to survive anaerobiosis), characteristics that have been associated with Brucella virulence [START_REF] Haine | NnrA is required for full virulence and regulates several Brucella melitensis denitrification genes[END_REF][START_REF] Ronneau | Brucella, nitrogen and virulence[END_REF], although they are not essential as nosX is a pseudogene in B. ovis [START_REF] Tsolis | Genome degradation in Brucella ovis corresponds with narrowing of its host range and tissue tropism[END_REF].

Transport of a wide range of substances across bacterial envelopes, including the uptake of many nutrients, is performed by ABC transporters. Brucella has an exceptionally large number of predicted ABC transporters [START_REF] Jenner | ATP-binding cassette systems of Brucella[END_REF]. The uptake systems generally work with periplasmic binding proteins that concentrate the substrate in the periplasm and deliver it to the inner membrane transport components. We identified 7 Tat dependent periplasmic binding proteins. This seems to be common in the α-proteobacteria since 10, 14 and 23 Tat dependent periplasmic binding proteins were predicted for Ochrobactrum anthropi, Agrobacterium tumefaciens and Sinorhizobium meliloti, respectively [START_REF] Ding | Agrobacterium tumefaciens twin-arginine-dependent translocation is important for virulence, flagellation, and chemotaxis but not type IV secretion[END_REF][START_REF] Nuñez | The twin-arginine translocation pathway in αproteobacteria is functionally preserved irrespective of genomic and regulatory divergence[END_REF][START_REF] Pickering | Characterization of the twin-arginine transport secretome in Sinorhizobium meliloti and evidence for host-dependent phenotypes[END_REF].

Many of the Tat substrates are important for the formation and maintenance of the bacterial envelope. The L,D transpeptidases are involved in peptidoglycan remodeling and biosynthesis. The Brucella genome encodes over 80 predicted lipoproteins, which play an essential role in envelope integrity and, in some cases, bacterial virulence [START_REF] Goolab | Analyzing the molecular mechanism of lipoprotein localization in Brucella[END_REF]. Two lipoproteins were identified as Tat substrates. It has been suggested that Tat dependent lipoproteins, often fast folding hydrolases and oxido-reductases, have evolved in extremophiles, environemental bacteria such as Streptomyces coelicolor, L. pneumophila, and

Haloferax volcanii [START_REF] Shruthi | TAT-Pathway-dependent lipoproteins as a nichebased adaptation in prokaryotes[END_REF]. This fits with our observations that Brucella have evolved from soil bacteria by a process of genome reduction [START_REF] Wattam | Comparative phylogenomics and evolution of the brucellae reveal a path to virulence[END_REF]. Formation of disulphide bridges in the periplasm by the Dsb system is essential for the correct folding of envelope proteins. We identified two DsbA proteins encoded by B. suis as Tat substrates. Transposon mutagenesis studies have shown that DsbA proteins are both required for full virulence [START_REF] Köhler | The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell[END_REF]. While it is possible to mutate both genes individually, a double mutation has not been tested, and may be lethal. Their role in virulence may be due to reduced fitness or due to incorrect folding of virulence proteins, as reported for Legionella pneumophilia, where a DbsA protein is required for folding of Dot/Icm proteins [START_REF] Jameson-Lee | DsbA2 (27 kDa Com1-like protein) of Legionella pneumophila catalyses extracytoplasmic disulphide-bond formation in proteins including the Dot/Icm type IV secretion system[END_REF]. Disulphide bond formation systems vary in prokaryotes probably reflecting specific requirements for survival in diverse ecological niches [START_REF] Landeta | Disulfide bond formation in prokaryotes[END_REF]. In E.

coli, the DsbA signal sequence directs it to an unusual pathway depending on SecY, SecA and SRP, possibly because it folds rapidly in the cytoplasm [START_REF] Schierle | The DsbA Signal Sequence Directs Efficient, Cotranslational Export of Passenger Proteins to the; Escherichia coli Periplasm via the Signal Recognition Particle Pathway[END_REF][START_REF] Shimohata | Peculiar Properties of DsbA in Its Export across the; Escherichia coli Cytoplasmic Membrane[END_REF]. Analysis of the genomes of other alpha proteobacteria closely related to Brucella suggests that their DsbA are also Tat substrates (not shown), suggesting the DsbA proteins fold too rapidly in the cytoplasm to engage the SecY, SecA and SRP pathway.

All together our data show that the Tat system is essential for B. suis survival and the originality of its substrates might account for this essentiality. More work will be needed to understand why Tat is essential and if, as for most pathogens, it has a role in virulence. or 48h (static) culture, the OD600 were measured. Data is expressed as % of the OD600 of a culture with no inhibitor. Data represents the mean ± SD of three independent experiments.

Statistical analysis was performed using a One-way ANOVA followed by the Sidak's test.

pIN365 pIN60 with upstream

  and downstream regions flanking the tat operon. To create a clean deletion of the operon. This study pIN367 pIN60 with sequences encoding eGFP and CmR between upstream and downstream regions flanking tatA. To create a GFP CmR marked tatA deletion. This study pIN371 pIN11 derivative carrying 400bp upstream of the tatABC operon, KmR, tetR repressor, tatA under control of the tetA promoter..To replace the tat the control of the E. coli tat promoter or inducible expression from the upstreamT7 promoter (ApR)[START_REF] Jack | Coordinating assembly and export of complex bacterial proteins[END_REF] pssAmiA-AmiAH ssAmiA fused to mature AmiA carrying a C-terminal hexa-histidine tag in pUNI-

Fig 1 Fig 2

 12 Fig 1 TatB alignment

  Statistical differences are denoted with * * * * P ≤ 0.0001;*** = P ≤ 0.001; ** = P ≤ 0.01;* = P ≤ 0.03).

Fig 4 .Fig 1 Fig 2 Fig 3 .

 4123 Fig 4. Analysis of predicted Brucella Tat signal sequences in the amidase reporter assay.

Fig 4 .

 4 Fig 4. Analysis of predicted Brucella Tat signal sequences in the amidase reporter assay.

Table 1 Bacterial strains and Plasmids

 1

	Bacterial Strains Description	Source/Reference
	Brucella	suis		ATCC 23444 T
	1330
	Brucella abortus		Laboratory collection
	2308
	Brucella			ATCC 23456 T
	melitensis 16M
	E. coli DH5α		Φ80lacZΔM15 Δ(lacZYA-argF) U169	Invitrogen
			recA1 endA1 hsdR17 (rk-, mk +) phoA
			supE44 thi-1 gyrA96 relA1 λ-
	E. coli MC4100	F-∆lacU169 araD139 rpsL150 relA1 ptsF	Laboratory collection
			rbs flbB5301
	E. coli MCDSSAC MC4100 amiAΔ2-33, amiCΔ2-32	[4]
	E. coli B1LKO		MC4100 ΔtatC	[46]
	Plasmids
	pIN60		SacB dependent suicide vector. (KanR)	[47]
	pIN11		SacB dependent suicide vector. (CmR)	[47]
	pJC45		pBBR1 based, expresses GFP under	[26]
			control of plasmid tetR/tetA promoter.
			(KanR)
	pIN310		pUC29 expressing eGFP and CmR	Laboratory strain

Table 2 B. suis 1330 proteins predicted to be Tat substrates

 2

			Tat
	B. suis locus	Annotation 1	dependent 2
	BR_RS00235 L,D-transpeptidase	Yes
	BR_RS01455 CAP domain-containing protein/alkaline phosphatase	Yes
	BR_RS01605 ABC transporter substrate-binding protein	Yes
	BR_RS01705 DsbA family protein	Yes
	BR_RS02290 DsbA family protein	Yes
	BR_RS03515 L,D-transpeptidase	Yes
	BR_RS07125 Rieske Fe-S protein.	Yes
	BR_RS10155 Molybdopterin-dependent oxidoreductase	Yes
	BR_RS10515 ABC transporter periplasmic substrate-binding protein UF1007 Yes
	BR_RS11255 NirK:Putative multicopper oxidases	Yes
	BR_RS11325 NosZ : Nitrous-oxide reductase	Yes
	BR_RS11350 NosX protein/FAD:protein FMN transferase	Yes
	BR_RS12255 ABC transporter periplasmic substrate-binding protein	Yes
	BR_RS13015 Amidohydrolase family protein	Yes
	BR_RS13330 CueO : Multicopper oxidase	Yes
	BR_RS13635 FrpC	Yes
	BR_RS13715 ABC transporter periplasmic substrate-binding protein	Yes
	BR_RS13835 ABC transporter periplasmic substrate-binding protein	Yes
	BR_RS14675 MsrP : TMAO/DMSO reductase	Yes

Acknowledgements

We thank Xavier Debolle for extracting data from their RNAseq study and providing Figure 2 and Annette Vergunst for helpful discussions. Elia Riquelme was the recipient of a PhD fellowship from Conacyt Sabina Omarova was financed by a PhD fellowship from the Ministry of Research and Education of the Republic of Azerbaijan This work was supported by institutional funding from the Université de Montpellier, Université Aix-Marseille, INSERM, CNRS and Sante Publique France.

 S22 Results of the amidase SDS resistance assay. See supplemental Table S2 and Figure S1 for full data sets