Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms. Author Summary Pseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.


Author Summary
Pseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections.Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate.The biofilm extracellular matrix incorporates extracellular DNA (eDNA).This stabilizes biofilm architecture and helps confer tolerance to antibiotics.Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane.Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling.This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles.
Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction.Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

Introduction
Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections including lung, urinary tract and wound, bacteremia and infections associated with medical devices [1].It is notorious for its tolerance to antimicrobial agents, a property that is largely a consequence of its ability to form biofilm communities [1,2].Bacterial exoproducts including cell surface appendages, extracellular polymeric substances, biosurfactants and secondary metabolites all contribute to P. aeruginosa biofilm formation and maturation [3][4][5][6][7].
Apart from exopolysaccharides such as Psl, Pel and alginate, the extracellular polymeric matrix of P. aeruginosa biofilms incorporates proteins, rhamnolipids, membrane vesicles (MVs) and extracellular DNA (eDNA) [5,[8][9][10].Rhamnolipid biosurfactants are required during the initial stages of micro-colony formation and are involved in the migration-dependent formation of the caps of mushroom-shaped micro-colonies formed in flow-cell grown biofilms [10].They also aid the maintenance of channels between multicellular structures within biofilms and contribute to biofilm dispersal [5].With respect to the biofilm micro-colonies that characteristically form in flow-chambers fed with glucose minimal medium, eDNA is present at high concentrations in the outer layers of microcolonies in young biofilms.
However, in mature biofilms, eDNA is primarily located in the stalks at the borders between micro-colony caps and stalks [8].
The release of eDNA occurs via the lysis of a sub-population of bacterial cells [10][11][12][13][14].It is involved in attachment, aggregation and stabilization of biofilm microcolonies.eDNA can act as a nutrient source, chelate metal cations and confer tolerance to antibiotics such as the polymyxins and aminoglycosides [10,12,13].
eDNA also binds other biopolymers (exopolysaccharides and proteins) stabilizing biofilm architecture and conferring protection against adverse chemical and physical .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted March 2, 2021.; https://doi.org/10.1101/2021.03.01.433341 doi: bioRxiv preprint challenges [12,13].By intercalating with eDNA, secondary metabolites such as phenazines enhance biofilm integrity [12,15].Pyocyanin for example can contribute to DNA release through the formation of reactive oxygen species such as hydrogen peroxide that damage cell membranes [12].Although the mechanism(s) responsible for eDNA release has not been fully elucidated both eDNA and MVs can be generated via explosive cell lysis mediated via a cryptic prophage endolysin encoded within the R-and F-pyocin gene clusters [14].
Consequently, P. aeruginosa mutants with defects in this cell-to-cell communication system form aberrant, flat undifferentiated biofilms [10].In P. aeruginosa, the QS regulatory network consists of a hierarchical cascade incorporating the overlapping las, rhl and pqs pathways that employ N-acylhomoserine lactones (AHLs) and 2alkyl-4-quinolones (AQs) as signal molecules [1,16,17].All three QS systems contain auto-induction loops whereby activation of a dedicated transcriptional regulator by the cognate QS signal molecule induces expression of the target synthase such that QS signal molecule production can be rapidly amplified to promote co-ordination of gene expression at the population level.
PQS is formed through the oxidation of HHQ by PqsH [25] while formation of the AQ N-oxides such as 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) requires the alternative mono-oxygenase PqsL [18].The PqsE protein has dual functions; while it is not essential for AQ biosynthesis, it is required for the AQ-independent production of several factors that contribute to biofilm maturation including pyocyanin, rhamnolipids and lectin A [26].While activation of RhlR-dependent genes depends on PqsE [27], the AQ-independent, thioesterase-independent mechanism by which PqsE acts has not yet been elucidated [17,22].
Therefore, by analogy with other QS systems, HHQ and PQS can both act as autoinducers by generating a positive feedback loop that accelerates their biosynthesis.However, in contrast to HHQ which only regulates the pqsABCDE operon [17], PQS is a ferric iron chelator [31] that not only drives AQ biosynthesis via PqsR but also the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways [17].In addition, PQS can act as a cell-sensitizing pro-oxidant [32] and is required for MV production via a direct physical interaction with lipopolysaccharide (LPS) within the outer membrane [33].The packaging of PQS within MVs also provides a means for trafficking this hydrophobic QS signal within a P. aeruginosa population [34].
With respect to biofilm development, the pqs system is of particular interest because pqs biosynthetic mutants fail to produce eDNA, rhamnolipids, pyocyanin and MVs, and form thin defective biofilms containing little eDNA [8,27,33,35].The mechanism involved in PQS-mediated DNA-release in biofilms is not understood but has been suggested to be linked to phage induction causing cell lysis [8,[36][37][38][39][40].Although explosive cell lysis releases eDNA in biofilms and generates MVs through vesicularization of shattered membrane fragments, pqsA mutants are not defective for explosive lysis [14] and therefore this phenomenon is unlikely to account for PQSdependent eDNA release.
In the present study we sought to identify additional factors involved in eDNA release by screening a transposon (Tn) mutant library for eDNA-release defective mutants.
Apart from pqs biosynthetic mutants, we obtained Tn insertion mutants within the twin-arginine translocation (Tat) pathway that exhibited reduced levels of eDNA release, fail to produce rhamnolipids or pyocyanin and form defective, eDNA-poor, antibiotic susceptible biofilms.Since mutation or deletion of tat resulted in altered AQ production, reduced pyocyanin, rhamnolipid and MVs, and as the tat mutants were refractory to exogenously supplied PQS, the aberrant biofilm phenotype observed could be accounted for by perturbation of PQS autoinduction.By screening a library of P. aeruginosa Tat substrate mutants, we identified the Rieske sub-unit of the cytochrome bc1 complex as being required for pqs-dependent QS and hence eDNA release and biofilm maturation.

Transposon mutagenesis screen for P. aeruginosa mutants exhibiting reduced DNA release
To identify P. aeruginosa genes that contribute to eDNA release, a mariner transposon (Tn) mutant library was generated in strain PAO1.Approximately 10,000 mutants grown in microtitre plates were assayed for reduced eDNA release using propidium iodide (PI) to quantify eDNA because it is unable to penetrate live bacteria and because its fluorescence is enhanced 30-fold on binding DNA [41].From the initial screen, 84 Tn insertion mutants were selected and re-screened to eliminate strains with double Tn insertions and to confirm their eDNA phenotype.For 34 of the remaining mutants, the regions flanking each Tn insertion were sequenced and the corresponding genes identified.For many of the eDNA-release deficient mutants, the Tn insertions were located within genes required for AQ biosynthesis (pqsC and pqsH) or regulation (pqsR) (Fig. 2) These data confirm our previous work that first uncovered a role for PQS signalling in eDNA release [8].Apart from the pqs mutants, two mutants were obtained with insertions in the tatA and tatB genes respectively (Fig. 2).These code for components of the twin-arginine translocation (Tat) system that mediates the export of folded proteins and was originally named with respect to the presence of an Arg-Arg motif in the signal sequence of Tatexported products also called Tat substrates [42,43].In P. aeruginosa diverse proteins involved in phosphate and iron metabolism, virulence and energy transduction are exported to the periplasm, or secreted via the Tat export system and tat mutants exhibit pleiotropic phenotypes [44,45].Fig. 2 shows that genetic complementation of the P. aeruginosa tatA mutant with a plasmid-borne copy restored eDNA release.

The Tat pathway contributes to biofilm development in P. aeruginosa
Since eDNA makes an important contribution to biofilm development and architecture [10,12,13], biofilm formation by the P. aeruginosa tatA mutant in flowchambers was investigated.After 4-days growth, the P. aeruginosa wild-type and complemented tatA/pTatA mutant formed biofilms with mushroom-shaped structures whereas the tatA mutant formed thin, flat biofilms (Fig. 3A).Ethidium bromide staining of the latter revealed that they contained much less eDNA than the structured biofilms formed by the wild-type and complemented tatA mutant strain (Fig. 3A).Consistent with this biofilm phenotype, exposure of the biofilms formed by each of the three strains to tobramycin showed that the tatA mutant biofilm was more sensitive to tobramycin than either the wild-type or tatA/pTatA mutant biofilm (Fig. 3B).

P. aeruginosa tatA mutants are defective in the production of rhamnolipids, pyocyanin and MVs
Since rhamnolipids, pyocyanin and MVs are all important components of P. aeruginosa biofilms, their production was quantified in the tatA Tn insertion mutant and in a ΔtatABC deletion mutant.Fig. 4 (A and B) shows that both tat mutants produce substantially less pyocyanin and rhamnolipid than the parent or tatA complemented strain.Furthermore, MV levels (Fig. 4C) were reduced by ~50% in the tatA mutant compared with the wild type and could be restored by genetic complementation.

Inactivation of the tat pathway by mutation or small molecule-mediated inhibition perturbs PQS signalling
The reductions in eDNA release, rhamnolipids, pyocyanin and MVs noted in the tat mutant as well as its biofilm phenotype are similar to those observed for P. aeruginosa strains with mutations in pqs genes such as pqsA, the first gene in the AQ biosynthetic pathway (see Figs. 2 and Fig 4 and [8,40]).These data suggested that the tat mutant biofilm phenotype was likely, at least in part, to be due to a defect in PQS signalling.
To investigate the impact of the tatA mutation on the expression of pqsA, a CTX::pqsA'-lux fusion was introduced into the chromosomal CTX site of both the wild type and tatA mutant.Fig. 5A shows that pqsA expression in the tatA mutant is reduced ~4 fold compared with the wild type strain and restored by genetic complementation of the mutant.In agreement with these data, the Tat inhibitor, Bayer 11-7082, identified by Vasil et al [46] reduced pqsA expression in the wild type PAO1 strain by ~4 fold consistent with the reduction noted for the CTX::pqsAlux fusion in the tatA mutant (Fig. 5B).Bayer-11 7082 had no effect on growth or light output in P. aeruginosa expressing the lux genes from a derepressed lac promoter (S1 Fig. ).In addition, the concentrations of PQS in whole culture extracts of P. aeruginosa after growth in LB medium as determined by LC-MS/MS was respectively ~56% lower in the tatA mutant compared with the wild type and complemented tat mutant (Fig. 5C).
Since pqsA expression and hence AQ production is also PqsR/MvfR-dependent, we compared the expression of pqsR in the ΔtatABC with the parent strain and found no difference (S2 Fig. ).
Since mutation of tatA results in reduced pqsA expression, it is possible that the auto-induction of PQS signalling via PqsR/MvfR is compromised.To uncouple the autoinduction of AQ production, the pqsABCD genes were introduced into the P. aeruginosa ΔpqsA and ΔpqsAΔtatA mutants respectively on a plasmid (pBBRMCS5::pqsABCD) that constitutively expresses the pqsABCD genes [47].S3 suggesting that in the absence of autoinduction in the tat mutant background.P.
aeruginosa is unable to synthesize AQs.
Exogenous PQS does not restore PQS signalling in a P. aeruginosa tatA mutant QS systems are characteristically autoinducible such that exogenous provision of the cognate signal molecule induces expression of the signal synthase and hence downstream target genes [48].When the tatA mutant was provided with exogenous PQS, eDNA release did not increase (Fig. 2).To investigate this finding further, either PQS or HHQ was exogenously supplied to the wild type, tatA mutant or the complemented tatA mutant strains carrying chromosomal pqsA'-lux fusions.The data presented in Fig. 6A show that the response of the tatA mutant to PQS or HHQ respectively at 5 or 20 µM with respect to pqsA expression was at least 2-fold lower than the controls.Since both wild type and tatA mutant still produce AQs endogenously, the experiments were repeated in the P. aeruginosa ΔpqsA and tatA ΔpqsA mutants since no AQs are produced in these genetic backgrounds.Fig. 6B shows that the response to both PQS and HHQ is substantially reduced (e.g.~4 fold at 5 µM PQS) in the absence of tatA in the P. aeruginosa tatA ΔpqsA double mutant.
To determine the consequences of perturbed PQS signalling on the expression of the rhamnolipid (rhlA) and pyocyanin biosynthetic genes (P. aeruginosa has two, almost identical redundant 7 gene phenazine biosynthetic operons termed phzA1-G1 and phzA2-G2; [49]), the corresponding miniCTX-lux promoter fusions for rhlA and phzA1 respectively were constructed and introduced onto the chromosomes of the wild type, ΔpqsA and tatA ΔpqsA mutants respectively.Fig. 7A shows the expression profiles of rhlA'-lux as a function of time.Both the wild type and ΔpqsA mutant show an ~2 fold increase in rhlA expression when supplied with exogenous PQS (20 µM) and share similar profiles over the growth curve.In contrast, the rhlA'lux fusion in the tatA mutant does not show the same expression profile or response to exogenous PQS as the wild type and ΔpqsA mutant strains.The rhlA'-lux expression profile in the tatA mutant supplied with exogenous PQS is however clearly restored when the mutation is complemented by pTatA (Fig. 7A).

PqsE restores pyocyanin in the tat mutants
Although PqsE is not essential for AQ biosynthesis, it is required for the production of pyocyanin, rhamnolipids and biofilm maturation and its function is independent of PQS, HHQ or PqsR [27,50].Consequently, the expression of certain PQS signalling-dependent exoproducts such as pyocyanin can be restored in a pqsA negative (and hence AQ-negative) mutant by expressing pqsE.To determine whether expression of pqsE alone could restore phzA1 expression and pyocyanin production in the tat mutants, pqsE was introduced on pUCP18.Fig. 7B shows that phzA1 expression can be restored in the tatA ΔpqsA mutant in the absence of AQs while Fig. 8 confirms that plasmid-borne pqsE restores pyocyanin production.

The reduction in rhamnolipid production in the tat mutants does not account for the perturbation of PQS signalling
In P. aeruginosa biofilms, rhamnolipids provide protective shielding against neutrophils [51,52] and contribute to the effectiveness of PQS signalling by enhancing the solubility and bioactivity of PQS [53].In Fig. 4B, we show that rhamnolipid production is substantially reduced in the P. aeruginosa tat mutant backgrounds.To determine whether the perturbation of PQS signalling in the tat mutants is a consequence of reduced rhamnolipid production, we investigated the impact of exogenous rhamnolipids on pqsA expression.S5 Fig.
shows that the addition of purified rhamnolipids (10 or 50 µg/ml) to the tatA ΔpqsA mutant with or without PQS (40 µM) had little effect on pqsA expression.

Identification of the Tat substrate responsible for perturbation of PQS signalling
Recently Gimenez et al [45] experimentally validated the Tat-mediated export of 34 P. aeruginosa gene products predicted to have Tat signal peptides.To determine which of the exported Tat substrates was responsible for perturbation of PQS signalling, allelic replacement mutants were constructed in P. aeruginosa strain PA14 for each substrate.Before introducing the pqsA'-lux fusion onto the chromosomal CTX site of each Tat substrate mutant, we first confirmed that PQS signalling in a PA14 ΔtatABC mutant was perturbed in a similar manner to that observed for PAO1, the genetic background used so far in the study (Fig. 9A).
Determination of the maximum expression of pqsA'-lux for each of the 34 Tat substrate mutants (Fig. 9A) revealed that although a number of mutants exhibited reduced light output, the greatest reduction was observed for deletion of PA14_57570 (equivalent to PA4431 in PAO1, here designated petA following the nomenclature of orthologues described in other species).This gene codes an ironsulfur cluster protein, the Rieske subunit of the cytochrome bc1 complex.
To confirm that the ΔpetA mutant in common with the P. aeruginosa tat mutants exhibited similar defects as a consequence of perturbed PQS signalling, we compared their AQ production (Fig. 9B

Discussion
Extracellular DNA is one of the most abundant components of the extracellular matrix of bacterial biofilms [10][11][12][13][14].However, the detailed mechanism(s) by which eDNA is released is not fully understood but likely to involve multiple pathways.
P. aeruginosa pqsL and pqsA mutants release more or less eDNA respectively, consistent with the levels of PQS they produce [8].Furthermore, the biofilms formed by pqsA mutants are thin and flat and contain little eDNA consistent with pqs signalling playing an important regulatory role in biofilm maturation [8].To gain further insights into eDNA release, a P. aeruginosa transposon mutant library was screened and two groups of mutants exhibiting reduced eDNA release were identified.The first of these contained Tn insertions in genes involved in pqs signalling including pqsC, pqsH and pqsR.Mutations in pqsC or pqsR in common with pqsA both abrogate AQ biosynthesis while pqsH mutants are unable to produce 3-hydroxy-AQs such as PQS but maintain production of AQs and AQ N-oxides such as HHQ and HQNO respectively [17,54].These data suggest that eDNA release is likely to require PQS/HHQ rather than the effector protein PqsE.This is because although pqsE mutants in common with pqsA mutants form poor biofilms, pqsE pqsA double mutants could not be complemented by pqsE alone to restore biofilm development [26].This contrasts with pyocyanin and lectin A for example, as production of both can be restored by PqsE in the absence of AQ biosynthesis [26].
The transposon insertions in the second group of P. aeruginosa eDNA release mutants obtained were located in tatA and tatB, the first two genes in the tatABC operon that codes for the twin-arginine translocase secretion pathway [42,43].The Tat secretion system was originally discovered in the thylakoid membranes of plant chloroplasts and subsequently identified in bacteria and archaea [42].Tat exports folded proteins out of the cytoplasm and across the cytoplasmic membrane in an ATP-independent manner [42].In bacteria such as E. coli and P. aeruginosa proteins exported by the Tat system are most frequently terminally localized in the periplasm, but some (e.g.phospholipase C) can be transported across the outer membrane by the Xcp type II secretion system, thereby becoming extracellular.Tat signal peptides harbor a conserved S/TRRxFLK consensus motif, where the twin arginine is invariant and in the vast majority of cases essential for export [45].
Using prediction algorithms for Tat substrates, 44 putative P. aeruginosa PA14 Tat signal peptides have been identified, and of these, 34 confirmed experimentally using a novel amidase-based Tat reporter [45].These include phospholipases and proteins involved in pyoverdine-mediated iron-uptake, respiration, osmotic stress defence, motility, and biofilm formation [44,45].However, none of these are known to be involved in AQ biosynthesis, transport or pqs signal transduction.
In our flow-chamber grown biofilms, clear differences between the P. aeruginosa PAO1 wild type and tatA mutant biofilms were apparent.While the wild type formed characteristic mushroom-shaped structures under these conditions, the tatA mutant formed flat, thin biofilms.In addition, eDNA was observed primarily in the stalks of the mushroom-shaped structures in the wild-type biofilms, whereas the tatA mutant biofilms contained no stalks and little extracellular DNA.Moreover, the biofilms formed by the tatA mutant were more sensitive to tobramycin treatment than wildtype biofilms, presumably in part due to the inability of the tatA mutant to release DNA and form differentiated multicellular structures.Indeed, DNA binds positively charged antibiotics and can act as a 'shield' against aminoglycosides such as tobramycin [53].In P. aeruginosa biofilms, exogenously provided DNA integrates into P. aeruginosa biofilms increasing their tolerance toward aminoglycosides [55].
The formation of flat, eDNA deficient, tobramycin-sensitive biofilms and the reduction in rhamnolipid, pyocyanin and MV production by the P. aeruginosa tatA mutant are consistent with the pqsA mutant biofilm phenotype [8] and imply the existence of a link between Tat export and pqs signalling.Since transcriptomic studies of PQS signalling in P. aeruginosa have not provided any evidence that the Tat export system is QS controlled [17,26], we considered it likely that mutation of the tat genes resulted in perturbed PQS signalling.This observation could also account, at least in part, for the reduced virulence of P. aeruginosa tat mutants in a rat pulmonary infection model [44].
To explore this hypothesis, the expression of pqsA and the concentrations of PQS and HHQ produced were compared in P. aeruginosa PAO1 wild type, tatA and complemented tatA mutant strains.Similar results were also obtained for P. aeruginosa strain PA14.Disruption of the tat operon had a pronounced effect on the expression of pqsA, with a nearly 3-fold reduction in pqsA expression.Moreover, addition of the Tat inhibitor, BAY 11-7082 [46] also resulted in a comparable reduction in pqsA expression.LC-MS/MS quantification of PQS levels in early stationary phase after 8 h growth, showed a ~50% reduction, thus correlating with the pqsA'-lux promoter fusion assays.Although PQS and HHQ both serve as PqsR ligands for driving the positive feedback loop for pqsABCDE expression that is central to AQ signalling [28][29][30], exogenous provision of neither PQS nor HHQ could fully restore pqsA expression in the tatA mutant.Since this mutant still produces AQs, albeit at a reduced level, a pqsA mutation was introduced into the tatA mutant background to examine the response to exogenous AQs in an AQ-negative genetic background (tatA ΔpqsA).The data obtained confirmed that the P. aeruginosa tatA ΔpqsA mutant responded only weakly to exogenously added PQS or HHQ, albeit to a much lesser extent than the isogenic ΔpqsA mutant with an intact tat operon.
These data suggest that disruption of tat impairs the ability of P. aeruginosa to fully induce AQ-dependent QS.The consequences of tat-dependent perturbation of pqs signalling are clearly evident in the modified expression profile of the rhlA'-lux fusion in the tatA mutant with or without exogenously supplied PQS.Similar results were also obtained for phzA1 expression in that the reduction in expression observed in a tatA ΔpqsA mutant background was not restored to wild type levels by exogenous PQS.
Since pyocyanin can be produced in the absence of PQS by ectopic expression of pqsE [26], it was possible that the tat phenotype is either a consequence of the inability of PQS/HHQ to activate the pqsABCDE operon via PqsR to generate sufficient PqsE protein or because the activity of PqsE depends on a functional Tat system.To investigate the impact of PqsE on pyocyanin production in the PAO1 tatA mutants, a plasmid borne copy of pqsE was introduced into the tat mutants.
Pyocyanin production was fully restored suggesting that the tat mutant phenotype is not due to the inability of PqsE to function but rather a failure of the pqs autoinduction circuitry to produce sufficient PQS/HHQ to efficiently activate pqsABCDE transcription and hence pqsE expression.
AQs such as PQS are extremely hydrophobic and virtually insoluble in aqueous environments.Hence PQS signalling can be enhanced by increasing the solubility and delivery of PQS.Calfee et al [53] showed that rhamnolipids increase the solubility and bioactivity of PQS in P. aeruginosa with respect to lasB expression.
Since rhamnolipid production is substantially reduced in the P. aeruginosa tat mutants, we added back purified rhamnolipids to the tatA ΔpqsA mutant.However, no effect on pqsA expression was observed indicating that the PQS signalling defect in the tat mutants is not simply due to the loss of rhamnolipid production and an inability to solubilize PQS.
In Escherichia coli, inactivation of tat leads to a characteristic cell envelope defect because of the mis-localization of two amidases involved in cell wall metabolism.
This results in the formation of bacterial chains, leakage of periplasmic proteins and enhanced susceptibility to detergents [56].Since PQS is a signal molecule that must be transported across the cell envelope in both directions, it was possible that an envelope defect in P. aeruginosa tat mutants impacts on the PQS signalling autoinduction cascade.However, Ball et al [57] showed that P. aeruginosa tat mutants do not show the same Tat-dependent envelope defects found in E. coli.In addition, we found no evidence for intracellular accumulation of AQs indicative of a transport defect.
To uncover the mechanistic link between Tat and PQS signalling, we sought to determine whether mutation of tat resulted in the inability to export a specific Tat substrate, the loss of which resulted in perturbation of PQS autoinduction and hence the expression of PQS-dependent genes and the formation of defective biofilms.By screening 34 validated Tat substrate mutants for reduced pqsA expression, we identified the petA gene as responsible for the defect in pqs-dependent QS.This gene codes for the Rieske protein, an iron-sulfur cluster protein sub-unit component of the cytochrome bc1 complex involved in electron transfer and respiration under aerobic conditions and also essential for enabling P. aeruginosa to grow under anaerobic conditions in the presence of nitrite [58].Self-poisoning of cytochrome bc1 occurs via HQNO which is produced via the same biosynthetic pathway as PQS (Fig. 1).HQNO binds to the quinone reduction (Qi) site of the respiratory cytochrome bc1 complex [59].This results in the generation of reactive oxygen species that cause P. aeruginosa cell death and autolysis favouring biofilm formation and antibiotic tolerance.Rieske subunit and cytochrome b1 mutants do not undergo autolysis and are insensitive to exogenous HQNO [59].Similarly, P. aeruginosa pqsL mutants that are unable to produce HQNO also fail to undergo autolysis [59].Our data also show that in common with the tat mutants, the petA mutant produces reduced levels of HQNO (as well as PQS and HHQ).This suggests that lack of eDNA in the tat mutant biofilms is because the cells do not undergo limited autolysis as they lack the self-poisoning mechanism that depends on both an intact cytochrome bc1 and sufficient HQNO.Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms.Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

Bacterial strains and growth conditions
The P. aeruginosa and E. coli strains used are listed in S1 Table and were grown in LB or ABTG [8] at 37 o C unless otherwise stated.P. aeruginosa biofilms were cultivated at 30 o C in flow-chambers irrigated with FAB medium [60] supplemented with 0.3 mM glucose or in M9 medium with succinate (for static biofilms).Selective media were supplemented with ampicillin (Ap; 100 µg ml -1 ), gentamicin (Gm; 60 µg ml -1 ), or streptomycin (Sm; 100 µg ml -1 ).PQS, HHQ and 2-heptyl-4hydroxyquinoline N-oxide (HQNO) were synthesized and characterized in house as described before [19,30] and dissolved in methanol before being added to growth media at the appropriate concentration.

Mutant construction, screening and validation
The P. aeruginosa PAO1 Tn mutant library was constructed using the Mariner transposon vector pBT20 as previously described [61].Transconjugants carrying Tn insertions were picked from the selective plates and inoculated into microtiter plates containing ABTG medium [62] supplemented with propidium iodide (PI) and the level of red fluorescence quantified.Mutants producing reduced levels of eDNA were selected and the sequences flanking the Tn insertion identified by arbitrary PCR essentially as described by Friedman and Kolter [63] but using the specific primers TnM1 and TnM2 (S2 Table ).DNA Sequencing was performed by Macrogen, Seoul, Korea with primer TnMseq (S2 Table ).An in-frame P. aeruginosa tatABC deletion mutant was generated by allelic exchange using the oligonucleotide primers Tat3D-UF, Tat3D-UR, Tat3D-DF and Tat3D-DR (S2 Table ) to introduce the up-and downstream regions of the tatABC locus into the suicide vector pME3087 to generate pME3087::tatABC (S1 Table ).The latter was introduced into P. aeruginosa via conjugation with E. coli S17-1 λpir followed by enrichment for tetracycline-sensitive cells as described by Ye et al [64].The ΔtatABC deletion in P. aeruginosa PAO1 was confirmed by PCR and sequence analysis.To generate the PA14 ΔpetA mutant, 500 bp upstream and downstream of the gene were amplified using respectively primers S.PA4431upFor/S.PA4431upRev and S.PA4431downFor/S.PA4431downRev listed in S2 Table .The PCR product was cloned in pKNG101 suicide vector by onestep sequence and ligation-independent cloning (SLIC) [65] which was then sequenced.The resulting plasmid, pKNGΔpetA, maintained in the E. coli CC118λpir strain, was then mobilized in P. aeruginosa strains.The mutant, in which the double recombination events occurred, was confirmed by PCR analysis.A similar strategy was used to construct allelic replacement mutants for each of 34 validated Tat substrates [45].These strains are summarized in S3 Table and their validation will be described in detail elsewhere.For the generation of the petA cis-complemented strain, PA14∆petA::(CTX1::petA), the petA genes along with a 500 bp fragment corresponding to the putative promoter region for the petA gene were PCR amplified using S-4431CTXFor/S-4431CTXRev and cloned by SLIC into the mini-CTX1 vector yielding pminiCTX1-petA.Transfer of this plasmid in P. aeruginosa ∆petA strain was carried out by triparental mating using the conjugative properties of the helper plasmid pRK2013.The recombinant clones containing the mini-CTX inserted at the attB locus on the P. aeruginosa genome were selected on tetracycline-containing PIA generating PA14∆petA::(attB::petA).

Construction of a tatA complementation plasmid
The tatA gene was amplified by PCR using primers FtatA and RtatA (S2

Bioluminescent reporter gene fusion assays
To investigate the impact of the tat mutation and Tat inhibitor Bayer 11-7082 [46] on PQS signalling, transcriptional fusions between the promoter regions of pqsA, pqsR, rhlA, phzA1 and phzA2 and the luxCDABE operon were constructed using the miniCTX-lux plasmid as previously described [17].In addition, a constitutively bioluminescent reporter using a miniCTX::tac-luxCDABE promoter fusion was constructed as a control for Bayer 11-7082.Bioluminescence as a function of bacterial growth was quantified in 96 well plates using a combined luminometerspectrometer.

Cultivation of biofilms
Biofilms were grown in flow-chambers with individual channel dimensions of 1 x 4 x 40 mm as described previously [66].One hour after inoculation, with bacteria, the growth medium flow (0.2 mm/s corresponding to laminar flow with a Reynolds number of 0.02) was started.When required, eDNA in biofilms was stained with 1 µM ethidium bromide prior to microscopy.Tobramycin (10 µM) was added to the biofilm medium after 4 days of cultivation.After 24 h of tobramycin treatment, propidium iodide (10 µM) was added to the flow cells to visualize the dead cells via confocal laser scanning microscopy.P. aeruginosa PA14 and petA mutant biofilms were grown under static conditions over 48 h at 37 °C on glass slides (Ibidi) incorporating 300 µl chambers.After 48 h incubation, spent medium was removed and the biofilm eDNA stained with YoYo-1 (40 µM).

Microscopy and image processing of flow cell biofilms
All images of flow-chamber-grown and static biofilms were captured with a confocal laser scanning microscope (CLSM) equipped with detectors and filter sets for monitoring green fluorescent protein, Syto9, propidium iodide, and ethidium bromide.Images were obtained using a 63x/1.4objective or a 40x/1.3objective.
Simulated 3-D images and sections were generated using the IMARIS software package (Bitplane AG, Zürich, Switzerland).

AQ, pyocyanin, rhamnolipid and MV analysis
The AQs (PQS, HHQ and HQNO) were quantified by LC-MS/MS after extracting cell free supernatants or whole bacterial cells in triplicate with acidified ethyl acetate or methanol respectively as described by Ortori et al [19].Pyocyanin was extracted with chloroform and quantified spectrophotometrically [26].Rhamnolipids were quantified indirectly using the orcinol method [35].
For PQS solubilization experiments, rhamnolipids were purified as described by Muller et al [67].MVs were harvested by ultracentrifugation, the pellets resuspended in 10mM HEPES buffer and the lipid content quantified using FM4-64 essentially as described previously [68].
MV production was normalized by dividing the lipid fluorescence units by CFU values determined by dilution plating.Assays were performed in triplicate at least twice.

Statistical Analysis.
Significance for differences between wild type and isogenic mutants was determined  The pqsE gene product has dual functions contributing to AQ biosynthesis as a thioesterase and also via an AQ-independent, thioesterase-independent mechanism to e.g.pyocyanin, rhamnolipid, and lectin production and to biofilm maturation.The conversion of HHQ to PQS confers additional functionalities since PQS unlike HHQ induces microvesicle formation and is a potent iron chelator.

Fig.
Fig. shows that PQS, HHQ and HQNO are present in the culture medium of the Fig.) demonstrating that PetA is the Tat substrate required for PQS-dependent eDNA release.

Fig 1 .
Fig 1.The PQS signalling pathway in P. aeruginosa.The PqsABCDE proteins synthesize HHQ, which is converted to PQS by PqsH and also HQNO in conjunction with PqsL.Both HHQ and PQS are released by the cells into the extracellular environment and are taken back up by neighboring cells.Autoinduction occurs when either HHQ or PQS binds to PqsR and amplifies expression of the pqsABCDE operon.

Fig 2 .
Fig 2. Transposon mutant screen for P. aeruginosa strains defective for eDNA release.P. aeruginosa wild-type and mutant strains were grown for 24 h in 96 well microtiter plates containing ABTG medium, after which the relative levels of eDNA in the cultures were determined using a PI binding assay.The means and standard deviations of eight replicates are shown.

Fig 3 .
Fig 3. P. aeruginosa tat mutants form defective biofilms with increased susceptibility to tobramycin.CLSM images showing four-day-old biofilms formed in flow chambers of the gfp-tagged P. aeruginosa wild-type (a), tatA mutant (b) and genetically complemented tatA mutant (c).In (A) biofilms were stained for total biomass with Syto9 (green) and for eDNA with ethidium bromide (red).In (B) biofilms were treated with tobramycin and the medium was supplemented with propidium iodide prior to CLSM such that dead cells appear red while live cells appear green.Each panel shows one horizontal optical section two flanking vertical optical sections.Bars, 20 µm.

Fig 4 .
Fig 4. Production of pyocyanin (A), rhamnolipids (B) and MVs (C) are reduced in P. aeruginosa tat mutants.(A) Pyocyanin levels are shown in the P. aeruginosa wild type, ΔpqsA, tatA, and tatABC deletion mutants and the tatA mutant complemented with plasmid-borne tatA.The impact of exogenous PQS (40 µM) on the tatA and ΔtatABC mutants is also shown.Insert panel shows the absence of green pigment in the tatA mutant compared with the wild type and complemented tatA mutant.(B) Rhamnolipid production in the tatA and ΔtatABC mutants compared with the wild type, ΔpqsA mutant and tatA complemented with plasmid-borne tatA.(C) Comparison of MV production in the tatA mutant, complemented tatA mutant and in a double tatA ΔpqsA mutant compared with the wild type strain.Experiments were repeated in triplicate at least twice.***p < 0.001, **p < 0.01; n.s.not significant.

Fig 5 .
Fig 5. Mutation of tatA or exposure to the Tat inhibitor Bayer 11-7082 inhibits pqsA expression and AQ production.(A) Mutation of tatA or (B) treatment with Bayer 11-7082 supplied at either 20 µM or 40 µM reduces the maximal expression of a P. aeruginosa PAO1 chromosomal pqsA'-lux promoter fusion without affecting growth.(C) LC-MS/MS analysis of PQS production by P. aeruginosa PAO1 wild type compared with the tatA mutant and complemented tatA mutant.Experiments were repeated in triplicate at least twice.***p < 0.001; n.s.not significant.

Fig 6 .
Fig 6.Exogenous AQs do not restore pqsA expression in P. aeruginosa tatA (A) or tatA ΔpqsA (B) mutants.Exogenous PQS or HHQ was added at 5 µM or 20 µM to (A) wild type, the tatA mutant and complemented tatA mutant or (B) ΔpqsA, ΔpqsA tatA or tatA ΔpqsA mutant complemented with tatA.Maximal light output from the chromosomal pqsA'-lux fusion was recorded as a function of growth (RLU/OD600).Experiments were repeated in triplicate at least twice.***p < 0.001, **p < 0.01, and *p < 0.05; n.s.not significant.

Fig 7 .
Fig 7. Rhamnolipid (rhlA) and pyocyanin biosynthetic (phzA1) genes show altered expression profiles in P. aeruginosa tat mutants and fail to respond to exogenous PQS.(A) Light output from a chromosomal rhlA'-lux fusion as a function of growth (RLU/OD) over time when introduced into the wild type, pqsA and tatA mutants or complemented tatA mutant in the absence or presence of exogenous PQS (20 µM).(B) Maximal light output from a chromosomal phzA1'-lux fusion as a function of growth (RLU/OD600) when introduced into the wild type, pqsA, or pqsA tatA mutants in the absence or presence of exogenous PQS (10 or 40 µM) or plasmid-borne pqsE or the pUCP18 vector control.Experiments were repeated in triplicate at least twice.***p < 0.001, **p < 0.01, and *p < 0.05; n.s.not significant.

Fig 8 .Fig 9 .
Fig 8. PqsE restores pyocyanin production in P. aeruginosa tat mutants.Production of pyocyanin by the wild type compared with the tatA and ΔtatABC mutants transformed with plasmid-borne pqsE or the empty vector.Experiments were repeated in triplicate at least twice.***p < 0.001, (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is

Table ) ,
introduced into pUCP22 (S1Table) and electroporated into the P. aeruginosa tatA and tatA ΔpqsA mutants.Transformants were selected on LB plates containing 200 µg ml -1 carbenicillin Exogenous rhamnolipids do not enhance PQS-dependent expression of pqsA in a tatA ΔpqsA mutant.PQS (40 µM) was added with or without purified rhamnolipids (50 µg/ml) to a pqsA mutant or a tatA ΔpqsA mutant carrying chromosomal pqsA'-lux fusions.Maximal light output as a function of growth (RLU/OD600) is presented.Experiments were repeated in triplicate at least twice.S6 Fig Rhamnolipid biosynthesis gene rhlA shows altered expression profiles in P. aeruginosa PA14 ΔtatABC and ΔpetA mutants compared with wild type and fail to respond to exogenous PQS.Bioluminescence from a chromosomal rhlA'-lux fusion as a function of growth (RLU/OD) over time when introduced into (A) the PA14 wild type, (B) ΔtatABC and (C) ΔpetA mutants in the absence or presence of exogenous PQS (20 µM).
S7 FigDeletion of petA in P. aeruginosa PA14 results in a reduction in the eDNA content of biofilms.Biofilms of wild type PA14, the ΔtatABC and ΔpetA mutants and the genetically complemented PA14 mutant (ΔpetA::(CTX::petA)) were grown cultured statically and stained for eDNA with YOYO-1.(A)CLSMimages and (B) eDNA quantification.Experiments were repeated in triplicate at least twice.***p<0.001,**p < 0.01.S1 TableStrainsand plasmids used in this study S2 Table Oligonucleotide primers used in this study S3 Table P. aeruginosa PA14 Tat substrate mutants used in this study