BatR: A novel regulator of antibiotic tolerance inPseudomonas aeruginosabiofilms

BatR: A novel regulator of antibiotic tolerance in Pseudomonas aeruginosa biofilms | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results BatR: A novel regulator of antibiotic tolerance in Pseudomonas aeruginosa biofilms Ainelen Piazza , Harshanie Dasanayaka , Gerhard Saalbach , Carlo Martins , Eleftheria Trampari , View ORCID Profile Mark A. Webber , View ORCID Profile Freya Harrison , Jacob G. Malone doi: https://doi.org/10.1101/2024.04.17.589660 Ainelen Piazza 1 Department of Molecular Microbiology, John Innes Centre , Colney Lane, Norwich, NR4 7UH UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Jacob.malone{at}jic.ac.uk Ainelen.piazza{at}jic.ac.uk Harshanie Dasanayaka 1 Department of Molecular Microbiology, John Innes Centre , Colney Lane, Norwich, NR4 7UH UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gerhard Saalbach 2 John Innes Centre Proteomics Platform Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carlo Martins 2 John Innes Centre Proteomics Platform Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eleftheria Trampari 3 Quadram Institute Bioscience, Norwich Research Park , Norwich, Norfolk NR4 7UQ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mark A. Webber 3 Quadram Institute Bioscience, Norwich Research Park , Norwich, Norfolk NR4 7UQ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mark A. Webber Freya Harrison 4 School of Life Sciences , Gibbet Hill Campus, The University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Freya Harrison Jacob G. Malone 1 Department of Molecular Microbiology, John Innes Centre , Colney Lane, Norwich, NR4 7UH UK 5 School of Biological Sciences, University of East Anglia, Norwich Research Park , Norwich, NR4 7TJ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Jacob.malone{at}jic.ac.uk Ainelen.piazza{at}jic.ac.uk Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Pseudomonas aeruginosa is a multidrug-resistant opportunistic human pathogen. Chronic infections are associated with biofilms, conferring resistance to antibiotics and complicating treatment strategies. This study focuses on understanding the role of the uncharacterized gene PA3049 , upregulated under biofilm conditions. In the context of P. aeruginosa biofilms, PA3049 plays a role in withstanding antimicrobial challenges both in vitro and in clinically validated infection models. Under sub-inhibitory concentrations of antibiotic, the deletion of PA3049 resulted in reduced pyocyanin production and altered abundance of enzymes controlling denitrification, pyoverdine, and hydrogen cyanide biosynthesis. Notably, PA3049 directly interacts with two kinases implicated in stress response, inactivating their active sites. Renamed as the B iofilm a ntibiotic tolerance R egulator (BatR), PA3049 is a key player in P. aeruginosa biofilm maintenance and antimicrobial tolerance. These findings contribute to understanding the complex bacterial lifestyle in biofilms, shedding light on a previously uncharacterized gene with significant implications for combating multidrug-resistant infections. IMPORTANCE P. aeruginosa is a multidrug-resistant ESKAPE pathogen that causes chronic biofilm-based infections and is a leading cause of mortality in cystic fibrosis (CF) patients. Understanding the molecular mechanisms underlying P. aeruginosa biofilm resilience and antimicrobial resistance is crucial for developing effective therapeutic interventions. This study focuses on characterizing the gene PA3049 , now known as the b iofilm a ntibiotic tolerance R egulator ( batR ). BatR plays a central role within P. aeruginosa biofilms, orchestrating adaptive responses to antimicrobial challenges. Our work sheds light on the contribution of batR to biofilm biology and its relevance in lung infections, where subinhibitory antibiotic concentrations make BatR pivotal for bacterial survival. By advancing our understanding of P. aeruginosa biofilm regulation, this study holds significant promise for the development of innovative approaches against biofilm-associated infections to mitigate the growing threat of antimicrobial resistance. INTRODUCTION Pseudomonas aeruginosa is a virulent, opportunistic human pathogen and one of the ESKAPE group of bacteria ( 1 ) that are leading causes of multidrug-resistant, nosocomial bacterial infections. P. aeruginosa presents a significant challenge in healthcare settings where it causes highly persistent, chronic infections, primarily affecting catheter and cannula implants ( 2 ), burn or other major injury victims ( 3 ), chronic wounds ( 4 ), and immunocompromised patients ( 5 ). In 2015, multidrug-resistant P. aeruginosa led to an estimated 4,500 attributable deaths and 68,000 infections in the EEA ( 6 ), while over 30% of P. aeruginosa isolates reported to EARS-Net in 2018 were resistant to at least one regularly monitored group of antimicrobials ( 7 ). P. aeruginosa is also a major respiratory pathogen, causing both acute and chronic lung infections. Pulmonary infections caused by P. aeruginosa are a major cause of mortality and morbidity in people with the genetic disorder cystic fibrosis ( 8 , 9 ). Chronic P. aeruginosa infections are frequently associated with biofilms, which enable it to evade host immune responses and confer broad resistance/tolerance to antimicrobial agents, complicating treatment strategies. Bacterial biofilms exhibit common traits and phenotypic characteristics, including cell-to-cell communication (quorum sensing), the production and deployment of extracellular polymeric substances and extracellular DNA (eDNA), and the spatially structured control of motility, adhesins and c-di-GMP levels ( 10 ). The upregulation of genes linked to stationary phase adaptation, environmental stress and anaerobiosis further underscores the distinct features associated with biofilm growth ( 11 , 12 ). Additionally, the spatial arrangement of cells within the biofilm community, exposed to multiple resource gradients, introduces heterogeneity in cell physiology and metabolism that plays an important role in antibiotic tolerance. This diversity includes significant subpopulations of less metabolically active (dormant) cells, which contribute to the notable tolerance of biofilms to antibiotics designed to target active metabolic processes ( 13 ). For example, dormant cells within oxygen-depleted zones of P. aeruginosa biofilms exhibit lower overall mRNA transcript abundance and increased tolerance to ciprofloxacin and tobramycin ( 13 ). Increased antimicrobial tolerance has also been associated with the global metabolic adaptations that arise in response to the biofilm environment ( 14 ). P. aeruginosa showcases this adaptability through its ability to grow anaerobically using nitrate as an alternative electron acceptor ( 15 ). The relevance of metabolic adaptions in response to antibiotics was underscored by an in vitro screen for tobramycin-resistant P. aeruginosa PA14 mutants that revealed a significant proportion of resistant mutants with transposon insertions in energy metabolism genes ( 16 ). Although a consensus has emerged on the role of many biofilm-associated traits, the large number of uncharacterized genes reported as differentially expressed under biofilm conditions highlights the substantial gaps that remain in our understanding of this complex bacterial lifestyle ( 17 ). Among the minority of upregulated loci in dormant, biofilm-dwelling P. aeruginosa cells is the small, uncharacterised gene PA3049 ( 13 ). PA3049 is predicted to encode a 70-residue cytoplasmic hydrophilin; part of a family of small, extremely hydrophilic, glycine-rich proteins that contribute to desiccation/osmotic stress tolerance in plants and yeast, but whose role in bacteria is less well understood ( 18 ). PA3049 is annotated as a homolog of the Escherichia coli ribosome m odulator factor (RMF) ( 13 ). RMF, a ribosomally associated protein, facilitates ribosome hibernation by associating with 100S ribosome dimers and modulates E. coli translation during the stationary phase ( 19 ). Unlike other ribosome components or associated proteins, rmf expression is inversely dependent on growth rate, indicating its potential involvement in the bacterial stress response ( 19 ). Classified as a hydrophilin, RMF accumulates under hyperosmotic stress conditions ( 18 ). RMF deletion in E. coli is linked to decreased aminoglycoside tolerance, potentially due to the location of its ribosome binding site ( 20 ). However, while RMF is crucial for ribosome hibernation in E. coli ( 21 ), PA3049 apparently does not fulfil a similar function in P. aeruginosa ( 22 ). Deleting PA3049 in P. aeruginosa compromises membrane integrity in biofilm-forming cells, suggesting a potential role in maintaining cell viability for the dormant subpopulation ( 13 ). Nonetheless, despite its conservation in sequenced P. aeruginosa strains and likely functionality in biofilms, the precise role of PA3049 in P. aeruginosa biofilm formation remains unknown ( 22 ). Here, we used a combination of bioinformatics, infection biology and molecular microbiology to determine the distribution of PA3049 across bacterial genomes, its importance for host infection and biofilm formation, its contribution to antimicrobial tolerance and its mechanism of action in P. aeruginosa . PA3049 -like genes are widespread in γ-proteobacteria, with a substantial degree of sequence and structural divergence predicted between the Pseudomonadales and Enterobacterales. PA3049 plays an important role in the formation of biofilm architecture and enabling established biofilms to withstand antimicrobial challenge. These global traits were traced to a narrow set of phenotypic and protein abundance shifts, with PA3049 deletion leading to reduced pyocyanin production and altered abundance of enzymes controlling denitrification, pyoverdine and hydrogen cyanide biosynthesis. Finally, we showed that PA3049 interacts directly with two different kinases; SrkA and GlpK that have recently been linked to antibiotic tolerance, suggesting an in vivo mechanism of action. Given the importance of PA3049 for P. aeruginosa antimicrobial tolerance and biofilm maintenance, we renamed this gene B iofilm a ntibiotic tolerance R egulator (BatR). RESULTS PA3049 homologs are widespread in γ-proteobacteria To uncover the role of PA3049 ( batR ) in P. aeruginosa , we first compared its predicted structure with that of E. coli RMF and assessed the wider distribution of batR -like genes in bacterial genomes. An alignment of BatR and RMF sequences, sharing 49.09% identity, shows a 15-residue C-terminal extension on BatR that is absent from RMF ( Fig. 1a ). AlphaFold three-dimensional protein structure prediction of BatR highlighted a marked difference in the fold of the protein C-terminus compared to its E. coli homolog. BatR contains a predicted α-helix of 12 residues at the C terminus (red boxed) in addition to the two α helices connected by a 13-amino acid linker region that comprise E. coli RMF ( Fig. 1b ). Download figure Open in new tab FIG 1. a. Sequence alignment of BatR and RMF. The amino acid sequences were aligned using ClustalW and black asterisks mark conserved residues in both proteins. b. AlphaFold model of BatR (magenta), overlaid onto the structure of E. coli RMF (cyan). Note the additional alpha-helix predicted for BatR (red boxed) at the C-terminus of the predicted structure. c. The phylogenetic relationship between BatR/RMF homologs. The tree is based on 765 proteins homologous to BatR/ RMF from γ-proteobacteria. A phylogenetic tree of RMF homologs revealed that they are confined to the γ-proteobacteria class ( Fig. 1c ), with genes associated with RMF showing distinct evolutionary paths across diverse bacterial orders. Notably, batR in Pseudomonadales forms a distinct cluster that diverges significantly from other bacterial orders. This divergence is particularly evident for the Enterobacterales, including E. coli and Yersinia pestis ; and Vibrionales including Vibrio cholerae, where RMF homologs have been shown to play roles in ribosome hibernation ( 23 ). While rmf and batR appear to share an ancestral root, their divergent structure and phylogeny, supported by the existing literature ( 22 ) are consistent with an alternative functional role for BatR. batR protects P. aeruginosa biofilms from sub-inhibitory concentrations (SIC) of antibiotics in vitro Given the heightened abundance of batR transcripts in dormant P. aeruginosa cells within biofilms ( 13 ), we assessed the importance of batR in biofilm formation and its effects on antimicrobial tolerance. To do this, we generated a non-polar deletion mutant (Δ batR ) in PAO1 and examined the response of the mutant to antimicrobial agents when grown in liquid and on solid agar. Assays were conducted using several different antibiotic classes, targeting distinct metabolic processes: aminoglycosides (gentamicin, GENT), β-lactams (piperacillin, PIP) and quinolones (ciprofloxacin, CIP). Consistent with previous findings ( 22 ), the deletion of batR did not impact bacterial viability or result in growth impairment in shaking cultures exposed to SIC of tested antimicrobials (see Fig. S1 ). Additionally, the minimal inhibitory concentration ( 24 ) of all three tested antibiotics was unaffected in Δ batR ( Table 1 ). Interestingly however, Δ batR showed a small but significant increase in sensitivity to PIP and CIP (12-17%), when grown on a solid surface ( Fig 2a ). Download figure Open in new tab FIG 2. a. Antibiotic disk diffusion assay. Results are represented as the normalized width of the antimicrobial halo (NW halo), calculated as described in ( 55 ). Differences in inhibition were significant between WT and Δ batR strains (F 5,50 =52.52, P<0.0001) and post hoc analysis showed significant differences between WT and Δ batR strain under PIP and CIP treatments (p<0.0001 ***) and non-significant (ns) effect with GENT. b. Biofilm formation. Crystal Violet absorbance (A 590nm ) after 24-h static growth in LB medium. Treated samples were incubated in the presence of SIC of antibiotics PIP, GENT, and CIP. NT=not treated. ANOVA F 7,32 =49.35; p<0.0001, post hoc analysis showed this was only significant for CIP treatment (p<0.0001 ***) c. Glass Beads Biofilm survival . Bacterial recovery (CFU/bead) from established biofilms grown on glass beads for 24h following treatment with SIC of PIP for 90 min. d . Glass Beads Biofilm survival complementation . Strains: WT PAO1 strain carrying the empty vector pME6032 (WT-pME6032) and Δ batR either carrying the empty vector pME6032 (Δ batR -pME6032) or overexpressing batR (Δ batR- pME- batR ). ANOVA F 2,12 =6.414; p=0.0127, post hoc analysis showed this was significant for WT-pME6032 (PIP) vs Δ batR -pME6032 (PIP) and for Δ batR -pME6032 (PIP) vs Δ batR -pME- batR (PIP) (p<0.05*). View this table: View inline View popup Download powerpoint Table 1. Minimal inhibitory concentration (MIC) The comparison of PAO1 WT and Δ batR cultures grown in liquid medium ( 24 ) under static conditions revealed no measurable effect on biofilm formation ( Fig. 2b ). However, the addition of SIC of CIP led to a significant reduction in biofilm formation for the Δ batR strain compared to WT PAO1 ( Fig. 2b ). Given the initial observation that sensitivity to both PIP and CIP was affected on solid surfaces ( Fig. 2a ), we used an alternative, glass bead biofilm model ( 25 ), to assess the survival of cells within established biofilms challenged with PIP. This model revealed a substantial reduction in the survival of established Δ batR biofilms compared to WT PAO1, for samples exposed to SIC of PIP ( Fig. 2c ). Notably, the Δ batR phenotype could be fully rescued by the heterologous expression of batR ( Fig. 2d ). Considering the different mode of action of the antibiotics tested in this study, our results suggest that the contribution of BatR to P. aeruginosa antimicrobial tolerance may occur through a general, rather than a drug-specific mechanism. A simple explanation for these phenotypes could be a nonspecific change in membrane permeability. However, we discarded this hypothesis after quantifying the intracellular concentration of resazurin, a fluorescent dye used as a permeability and efflux marker ( Fig. S2 ), where little difference was seen in the response of WT and Δ batR strains. BatR affects the ability of P. aeruginosa to withstand antimicrobial challenge in established biofilms and clinically validated infection models To assess the clinical significance of BatR in P. aeruginosa , we employed the established ex vivo pig lung (EVPL) model ( 26 ) to simulate P. aeruginosa biofilm infections in CF bronchioles. This model comprises two environments: the bronchiolar lung tissue surface and the SCFM (Synthetic Cystic Fibrosis Medium) to mimic the luminal mucus in the human CF lung ( 27 ). We first determined the efficiencies of the antibiotics PIP, CIP, and GENT to clear EVPL infections. Under our test conditions, PAO1 biofilms were highly tolerant to PIP treatment ( Fig. S3 ), precluding its use in these assays. Therefore, we assessed survival of the biofilms challenged with CIP at a SIC of 16 µg/ml. We recovered the biofilms at 2 d and 7 d postinfection (PI) to determine CFU per EVPL section. Our results show that BatR not only contributes to CIP tolerance, but also plays a key role in biofilm establishment within the EVPL model at 2 d PI ( Fig. 3a ). However, by 7 d PI, while P. aeruginosa remained viable on the lung tissue, batR did not appear to contribute significantly to biofilm formation or CIP tolerance, although there was a decrease in tolerance to CIP treatment in the WT strain ( Fig. 3a ). These findings suggest a role for BatR in biofilm establishment and antibiotic tolerance in P. aeruginosa infections, particularly in early stages of CF. We further explored these phenotypes in an in vitro biofilm model designed to mimic the conditions found in chronic wounds ( 28 ). Survival comparisons in biofilms without CIP challenge revealed no differences between WT and Δ batR strains. However, significant differences were observed for PAO1 Δ batR biofilms treated with different concentrations of CIP, compared to WT PAO1 in this model ( Fig. 3b ). Next, we examined the biofilm architecture in infected lung pieces, with and without SIC of CIP in the EVPL + SCFM. Replica sets of infected lung pieces were fixed at both 2 d and 7 d PI and sections stained with H & E (Haematoxylin & Eosin) to visualise the total biofilm mass and tissue architecture. At 7 d PI, the EVPL biofilms show the typical “sponge”-like appearance consisting of extracellular matrix punctuated by gaps, resembling CF biofilms observed in vivo ( 29 – 31 ). However, there were clear qualitative differences in the biofilm observed between the Δ batR and WT strains, as shown in Fig. 4 . The matrix covering the biofilm structure appeared thicker in the WT biofilm when challenged with SIC of CIP, something that is not observed in the biofilm formed by the Δ batR strain ( Fig. 4 ). These observations suggest that batR may play a role in influencing biofilm architecture, particularly in response to SIC of antibiotics. Download figure Open in new tab FIG 3. a. Ex Vivo Pig Lung (EVPL) Model . Growth of P. aeruginosa PAO1 strain (WT) and Δ batR , on 15 pieces of EVPL bronchiole (five replicate pieces of tissue infected per strain from five independent lungs) plus Synthetic Cystic Fibrosis Medium (SCFM), with and without 16 µg/ml CIP treatment. Colony forming units (CFU) were retrieved from biofilms after 2 d and 7 d growth at 37°C. Bars denote mean for each genotype across all five lungs, and asterisks denote a significant difference under that condition. ANOVA F 7,112 =19.90; p<0.0001, post hoc analysis showed significant differences for WT 2d vs Δ batR 2d; WT 2d vs Δ batR +CIP 2d; Δ batR 2d vs WT+CIP 2d, (p<0.0001 ***); and for WT 7d vs WT+CIP 7d; WT 7d vs DbatR+CIP 7d (p<0.05 *). b. Synthetic Chronic Wound (SCW) Model . Viability of P. aeruginosa PAO1 (WT) and the Δ batR strain living in established biofilms (24 h), treated with different concentrations of CIP in the cell suspension on top of the matrices. Dots represent the average from three biological replicates and error bars indicate the standard deviation. ANOVA found a significant difference in biofilm survival between WT and Δ batR under 64 µg/ml CIP treatment. Download figure Open in new tab FIG 4. Haematoxylin and eosin (H & E) stained sections of EVPL bronchiolar tissue with SCFM medium infected with P. aeruginosa at 7 d post infection. EVPL was infected with P. aeruginosa PAO1 WT and Δ batR , with uninfected tissue as a negative control. The x20 magnification images from the sections are shown here for non-treated tissues (SCFM) and treated with CIP (SCFM + CIP). The cartilage and tissue surface (lower half of each image) stain pink and the bacterial biofilm stain purple, including the bacterial cells and biofilm matrix. Representative images of phenotypes at day 7 PI are shown here, but the same results were observed for all biological replicates analysed. BatR induces the production of specific virulence factors in the EVPL model under CIP challenge To understand the impact of BatR on P. aeruginosa biology in the EVPL model, we quantified the production of key virulence-associated exoproducts ( 27 ). After washing the lung tissues twice in PBS to remove planktonic cells, we measured virulence factor production by biofilms growing on the lung tissue surface. Despite non-significant changes in siderophore abundance (pyoverdine and pyochelin, Fig. S4 ), a notable increase in blue colouration, indicative of pyocyanin production, was evident in the WT strain compared to Δ batR when treated with CIP on day 2 ( Fig. 5 a,b ). Download figure Open in new tab FIG 5. a. Production of pyocyanin (A 695 ) by P. aeruginosa WT and Δ batR in the EVPL model . Significant differences in pyocyanin production between WT and Δ batR under CIP treatment (ANOVA F 3,32 = 16.10, p<0.0001***), post hoc analysis showed significant differences (p<0.0001***) for WT vs WT (CIP), WT vs Δ batR (CIP); Δ batR vs WT (CIP) and Δ batR vs Δ batR (CIP). b. P. aeruginosa biofilms on squares of bronchiolar tissue. The blue pigmentation is typical of P. aeruginosa and is a mixture of the exoproducts pyoverdine and pyocyanin; note the increased intensity of blue colour in the WT strain grown in SCFM exposed to CIP. BatR induces specific changes in the PAO1 proteome under antimicrobial challenge Next, to investigate the physiological changes associated with BatR during antibiotic challenge in P. aeruginosa biofilms, we conducted a comparative proteomic analysis between the WT and Δ batR PAO1 strains, in the presence and absence of SIC of the antibiotic PIP. High-resolution mass spectrometry following TMT labelling detected an average of 4,581 individual proteins in each sample (S1 Data), representing ∼80% of the predicted total P. aeruginosa PAO1 proteome ( 32 ). BatR was exclusively detected in the WT PAO1 strain. Surprisingly, limited differences were detected between the proteomes of the two strains ( Fig. S5 , S1 Data), suggesting that the impact of batR deletion is quite specific under the conditions tested. Of 21 proteins decreased in the Δ batR strain in the absence of PIP challenge ( Table 2 ), seven (PA0617-PA0633) are components of a predicted bacteriophage. In addition, we saw reduced abundance of proteins involved in transport & metabolism; two heat shock proteins and four proteins of unknown function in the Δ batR strain. Conversely, 14 proteins were increased in the Δ batR strain, including Rubredoxin-1, Type VI secretion system components and the Phenazine-1-carboxylate N-methyltransferase PhzM ( Table 3 ). View this table: View inline View popup Table 2. COG pathway analysis of proteins decreased in the Δ batR strain View this table: View inline View popup Table 3. COG pathway analysis of proteins increased in the Δ batR strain For cells subjected to PIP challenge (S1 Data), batR deletion is associated with significantly decreased abundance of 11 proteins ( Table 4 ), including components of the Hydrogen cyanide (HCN) and Pyoverdine (PVD) synthesis pathways, quorum sensing and transcriptional regulators, and iron transport proteins. Deletion of batR significantly increased abundance of only twelve proteins under PIP challenge. These proteins play roles in energy production and conversion, especially nitrate respiration, primary metabolism, and Type VI secretion ( Table 5 ). View this table: View inline View popup Table 4. COG pathway analysis of proteins decreased in the Δ batR strain under PIP challenge View this table: View inline View popup Download powerpoint Table 5. COG pathway analysis of proteins increased in the Δ batR strain under PIP challenge To further explore the suggested connection between BatR and PVD and HCN production, we grew WT and Δ batR strains in the presence of SIC of the antibiotics PIP, CIP, and GENT, and quantified PVD and HCN production. Contrary to expectations, PVD production was significantly increased in the Δ batR strain under PIP challenge in liquid media ( Fig. 6a ). Likewise, exposure of WT and Δ batR strains to Feigl Anger solution, showed enhanced HCN production in the Δ batR strain when challenged with PIP ( Fig. 6d ). These results confirm the link between BatR and both phenotypes but suggest that the association may be highly dependent on growth conditions. Consistent with results from the EVPL assays, we did not observe a significant increase in siderophore production under CIP or GENT challenge conditions in the Δ batR strain ( Fig. 6 b,c ). These findings suggest that, while batR deletion leads to a general antibiotic/biofilm tolerance phenotype, some of the specific proteomic changes observed are antibiotic-specific. Download figure Open in new tab FIG 6. a, b, c. Pyoverdine production. Fluorescence (PVD) relative to growth (A 600 ) of WT and Δ batR PAO1 cells in liquid medium with and without PIP (a), CIP (b) and GENT (c). PVD: relative fluorescence measured at 460 nm (excitation: 400 nm) for the strains at 65 h. d. HCN production. Feigl-Anger assay showing release of HCN from WT and Δ batR PAO1 strains. Three independent biological replicates are shown. The blue pigmentation corresponds to HCN release; note the increased intensity of blue colour in the Δ batR PAO1 strain when challenged with PIP (blue boxed). BatR interacts with two kinase enzymes To understand the molecular basis of BatR function, we next investigated its interactions with other PAO1 proteins by performing a co-immunoprecipitation (Co-IP) analysis (S2 Data). M2-tagged BatR pulled down several cytoplasmic proteins, indicating potential direct regulatory mechanisms ( Table 6 ). Independent validation of strongly co-precipitating proteins was conducted using Bacterial Two-Hybrid (B2H) analysis. This confirmed specific interactions between BatR and two structurally diverse kinase enzymes: Gl ycerol p hosphate k inase ( GlpK ) and S tress response k inase A ( SrkA ) ( Fig. 7a ). These interactions are noteworthy given the recently established roles of both proteins in antimicrobial tolerance and stress response (33, Liu Y, 2022 #12, 34). Download figure Open in new tab FIG 7. BatR interacts with two kinase-like proteins. a . Representative image of qualitative β-galactosidase assays on agar plates. pKT25 and pUT18C fusions are shown in rows and columns, with the indicated protein/empty vector present in each case. Positive control (+): pKT25-zip and pUT18C-zip encoding the two adenylate cyclase fragments, T25 and T18, each fused to the leucine zipper domain of the yeast transcriptional activator GCN4. b and c are cartoon representations of BatR (magenta) modelled onto the proteins GlpK of SrkA ( 35 ), respectively. The interacting residues are shown in yellow and the W 52 residue is labelled. View this table: View inline View popup Download powerpoint Table 6. BatR interacting proteins To gain additional insight into the potential interaction between BatR and GlpK / SrkA, we used AlphaFold 2 ( 35 ) to create three dimensional models of all three proteins. These models allowed us to dock BatR onto GlpK and SrkA, predicting potential interaction residues and providing valuable structural information. Interestingly, the BatR residue W 52 was common to both predicted interaction interfaces ( Fig. 7 b,c ). This residue is highly conserved among Pseudomonas BatR homologs but is missing from E. coli RMF ( Fig. S6 ). GlpK (PA3582) is an ATP-dependent glycerol kinase that catalyses the first step in the metabolism of glycerol, producing glycerol 3-phosphate (G3P) during aerobic microbial metabolism in P. aeruginosa ( 36 ). G3P accumulation is associated with reduced cell growth, diminished pyocyanin production, lowered tolerance to oxidative stress and increased kanamycin susceptibility in P. aeruginosa ( 34 ). The observed phenotypes in the Δ batR strain align with those linked to G3P accumulation ( Figures 2 , 3 & 5), suggesting a potential role for BatR in suppressing GlpK activity. The interaction between BatR and GlpK is predicted to be electrostatic, with the amino acids from BatR (W 44 , E 46 and W 52 ) and GlpK (V 326 , N 328 and Y 335 ) being involved ( Fig. 7b ). We propose that BatR’s binding suppresses GlpK activity as the binding may interfere with the ATP/ADP predicted binding site (residues 314 & 318, Table 7 ). Consistent with the expected consequences of G3P accumulation, batR deletion significantly affected PAO1 growth and survival when cultured in M9 containing succinate & glycerol, as previously described ( 34 ) ( Fig. 8a ). Download figure Open in new tab FIG 8. BatR interacts with Gl ycerol p hosphate k inase (GlpK) and S tress r esponse k inase A ( SrkA ). a. Growth of P. aeruginosa PAO1 strains with 20 mM succinate and 20 mM glycerol as the carbon sources. The mean growth for 3 biological replicates for strains WT ( 35 ) and Δ batR (blue) is shown as a solid line and standard deviation shown as dotted lines. Cells were grown for 48 h at 37°C under static conditions. b. Hydrogen peroxide (H 2 O 2 ) sensitivity assay. Photographs of the bacterial culture plates 1 d after incubation at 37°C showing halos corresponding to inhibition zones of bacterial growth around the H 2 O 2 disks, indicating bacterial sensitivity. Phenotypes of the WT and Δ batR (highly sensitive) strains are shown. The concentration of H 2 O 2 on each disk is shown (0.35-6.0 %). c. Biofilm pellicle assay showing 1 d biofilm and pyocyanin production. Photograph of static cultures of the WT and the Δ batR strains overexpressing srkA grown without (-) and with (+) 0.05 mM IPTG demonstrating mature biofilm at air-liquid interface. Two biofilm characteristics were observed: pellicle (arrowed) and pyocyanin production (blue coloration; red boxed). View this table: View inline View popup Table 7. Predicted GlpK (PA3582) active and binding sites (source www.uniprot.org ) SrkA (PA0486) is a eukaryotic-like serine-threonine protein kinase, whose protective role in antimicrobial and environmental stress has been characterised in E. coli ( 33 , 37 ). This protein is linked to a reactive oxygen species (ROS) cascade, and a deficiency of srkA stimulates stress-induced p rogrammed c ell d eath (PCD) even after stress dissipated. The deletion of batR in P . aeruginosa strain PAO1 leads to reduced survival rates following antibiotic treatment in biofilms ( Figure 2 &3), despite no observed changes in MIC values ( Table 1 ), consistent with SrkA dysregulation in E. coli ( 33 ). Additionally, BatR exhibits a protective role against hydrogen peroxide (H 2 O 2 ) stress in PAO1 ( Fig. 8b ), like SrkA in E. coli , supporting a potential connection between BatR and SrkA in stress response pathways. The interaction between BatR and SrkA is also predicted to be electrostatic, involving specific amino acid residues from both BatR (R 26 , W 44 , E 46 and W 52 ) and SrkA (N 32 , Y 34 , P 113 , A 234 , G 235 , Y 276 and F 293 ) ( Fig. 7c ). We propose that the binding of BatR suppresses the kinase activity of SrkA, possibly impacting its role in ATP binding (residue 33 predicted to bind ATP, see Table 8 ). This interaction might enhance cellular resistance to stress. To test this hypothesis, we overexpressed srkA in the WT and Δ batR strains. The overproduction of SrkA in both strains resulted in increased lethality and enhanced pyocyanin production in pellicle biofilms grown in liquid medium ( Figure 8c ). It is noteworthy that pyocyanin production occurs underneath growing pellicles when srkA is overexpressed. Given the lack of characterisation of srkA in P. aeruginosa so far, we propose a potential role in regulating antimicrobial stress and pyocyanin production, two phenotypes associated with batR in this study. View this table: View inline View popup Download powerpoint Table 8. Predicted SrkA (PA0486) active and binding sites (source www.uniprot.org ) DISCUSSION In this study, we characterise the P. aeruginosa hydrophilin BatR and determine its role in antimicrobial tolerance and biofilm formation, and its potential clinical significance. batR homologs exhibit a high degree of conservation within diverse bacteria, particularly in ɣ-proteobacterial species ( 23 ). The identification of a well-supported clade of Pseudomonadales carrying batR homologs that is both genetically and structurally distinct from the characterised Enterobacterales/Vibrionales rmf clade suggests a divergent evolutionary path for these genes, and a distinct cellular function for BatR. The well characterised batR homolog in E. coli ; rmf , contributes to ribosome hibernation and tolerance to the aminoglycoside antibiotics gentamicin and netilmicin ( 20 , 38 ). Consistent with this, the binding site of this hibernation factor is near to those described for these antibiotics, thus potentially interfering with its mechanism of action ( 23 ). Conversely, the contribution of batR to antimicrobial tolerance is not linked to antimicrobial mode-of-action, making it highly unlikely that BatR functions solely via ribosomal inhibition, as previously suggested. BatR contributes to both biofilm formation and antibiotic tolerance in conditions like those in CF infections. Our data suggest that BatR is particularly important during the early stages of P. aeruginosa lung tissue infection at sub-inhibitory concentrations of antibiotics. This is relevant in CF patients since they are typically subject to extended antibiotic regimes, but the drugs do not necessarily reach the entire lung at inhibitory concentrations ( 39 ). Both WT and Δ batR strains formed sponge-like biofilm structures, characteristic of CF infections after 7 days PI in the EVPL model. However, upon challenge with CIP the Δ batR strain formed a less resistant biofilm structure on the surface of the EVPL bronchiolar tissue compared to the WT strain. Interestingly, whilst these differences were visually obvious, similar CFU were recovered from tissue infected by both genotypes at 7 days. This is consistent with a large fraction of dead or unculturable cells in WT biofilms, as previously observed for P. aeruginosa PA14 biofilms ( 27 ). Noteworthy, transcriptomic analysis of P. aeruginosa strain PA14 in EVPL model revealed significant differential expression of batR at 7 days compared with 1 day PI ( 40 ). BatR is also involved in P. aeruginosa biofilm tolerance in the synthetic chronic wound model of diabetic foot infections. This is consistent with the finding that batR expression is differentially increased in burn wound infections ( 41 ), and underscores the versatility of batR in mediating P. aeruginosa pathogenesis across various infection settings. BatR function appears to be consistently associated with pyocyanin production. This molecule was notably reduced in Δ batR following exposure to CIP in the EVPL model ( Figure 5 ). Acting as a potent electron acceptor, pyocyanin influences cellular redox balance, inducing oxidative stress in host cells and ultimately leading to cell damage and lysis ( 42 ). Within the oxygen-limited environment of P. aeruginosa biofilms, pyocyanin is crucial for metabolic continuity and significantly impacts the biofilm’s response to antibiotic treatments ( 43 – 45 ). Our proteomic results align with previous research demonstrating the induction of denitrification enzymes by phenazine deficiency in P. aeruginosa biofilms ( 46 ). Additionally, upregulation of the permease FeoB (Table X) by batR facilitates efficient iron uptake in biofilms, highlighting the intricate interplay between BatR, phenazine metabolism and iron homeostasis ( 47 ). Consistent with our H&E staining results ( Figure 4 ), pyocyanin production has been linked to biofilm architecture and eDNA production in P. aeruginosa ( 48 ), contributing to the observed differences in biofilm structure. Finally, to understand the molecular basis of BatR function we investigated its protein interaction partners within the cell. Through screening, two proteins; SrkA and GlpK, were identified. BatR interaction with SrkA and GlpK control antimicrobial tolerance and virulence factor production of P. aeruginosa PAO1 biofilms. Thus, BatR may have potential therapeutic utility as a target for the control of P. aeruginosa infections. Similarly to E. coli , SrkA in P. aeruginosa may have a regulatory role in stress mediated PCD, mediated by BatR interaction. E.coli SrkA is partially regulated by the Cpx envelope stress-response system, which has both protective and destructive roles that help bacteria make a live-or-die decision in response to stress ( 33 ). Interestingly, transcription of rmf is also induced by the Cpx system in E. coli , suggesting a link between them ( 49 ). Our results also suggest the involvement of SrkA in regulating pyocyanin production in P. aeruginosa , although further research is necessary to fully understand this link. G3P metabolism has been characterized in P. aeruginosa due to its relevance to CF infections ( 34 ): glycerol is released from the cleavage of phosphatidylcholine, the major lung surfactant in CF patients ( 50 ). G3P is involved in maintaining cellular homeostasis, and increased levels of G3P lead to reduced pyocyanin production and resistance to kanamycin ( 34 ). This represents a clear example of bacterial antibiotic resistance closely correlated with physiological metabolism ( 51 , 52 ). However, the specific mechanism by which G3P accumulation induces phenotypic alterations in P. aeruginosa PAO1 remains unclear. Our current view of the mechanism of action of this novel post-transcriptional regulator, BatR, expressed under biofilm conditions that plays a role under antimicrobial stress in the opportunistic human pathogen P. aeruginosa, is sketched in Fig. 9 . Download figure Open in new tab FIG 9. Schematic representation of BatR mechanism of action. a. BatR-GlpK interaction and its impact on G3P metabolism in P. aeruginosa PAO1. G3P, crucial for various cellular processes, can be imported from the extracellular environment or derived from glycerol phosphorylation via GlpK activity. The accumulation of G3P reduces antibiotic resistance, pyocyanin production, oxidative stress tolerance, and twitching motility. GlpK activity is suppressed upon interaction with BatR, potentially resulting in decreased G3P levels and increased pyocyanin and antimicrobial resistance. b . BatR-SrkA interaction and its implications in stress-mediated bacterial PCD and pyocyanin production : SrkA, plays a protective role against stressors such as H 2 O 2 and antibiotics, controls pyocyanin production, and interacts with BatR. This interaction is hypothesised to be crucial for SrkA’s protective function and suggests a novel mechanism for stress response modulation in P. aeruginosa . In conclusion, our findings expand the understanding of molecular mechanisms contributing to antimicrobial tolerance in P. aeruginosa biofilms. These results have broad implications for the functions of uncharacterised proteins induced under biofilm conditions, shedding light on their pivotal role in orchestrating multifaceted processes. This deeper insight enhances our ability to develop targeted therapeutic interventions to combat biofilm-associated challenges. MATERIALS AND METHODS Bioinformatic analysis A phylogenetic tree of RMF proteins was constructed using 765 publicly available protein sequences from the NCBI database. The dataset was curated based on a criterion of 50% sequence identity and 40% query cover to ensure the representation of diverse homologs while maintaining a reasonable level of similarity. The multiple sequence alignment was performed using Clustal Omega (v1.2.4), generating an alignment matrix with 137 columns and 135 distinct patterns. Phylogenetic signal analysis revealed 82 parsimony-informative sites, 33 singleton sites, and 22 constant sites. Manual curation was done to remove the repetitive sequences and false hits. The phylogeny estimation was done using IQ-TREE, (multicore v1.6.12), employing the maximum likelihood (ML) criterion. The tree visualization was done using iTOL (v6.8.1), representation chosen was an unrooted tree with branch lengths proportional to the inferred evolutionary distances between sequences. The clade colours were assigned at order level of the taxonomy. Bacterial strains and growth media Bacterial strains and plasmids used in this study are listed in Table 9 . Unless otherwise stated, P. aeruginosa PAO1 and E. coli DH5α strains were routinely cultured in lysogeny broth (LB (JH, 1972 #6)) at 37°°C solidified with 1.5% w/v agar where appropriate. For Gly + Succinate growth curves, PAO1 strains were grown in M9 salts supplemented with 1 mM MgSO 4 , 1 mM CaCl 2 ( 53 ), adding 20 mM succinate and 20 mM glycerol as the carbon sources. For growth curves, measurements were taken every 30 min for up to 48 h on a FLUOstar nano plate reader (BMG) with the plate being incubated at 37°°C under static or planktonic conditions, as indicated. View this table: View inline View popup Table 9. Strains and plasmids used in this study Carbenicillin (Carb) was used at 100 μg/ml, Kanamycin (Kan) at 50 μg/ml, Tetracycline (Tet) at 12.5 μg/ml for E. coli and 100 μg/ml for P. aeruginosa , IPTG at 0.5 mM, and X-gal at 40 μg/ml. The antibiotics Piperacillin (PIP), Gentamicin (GENT), and Ciprofloxacin (CIP) were employed at concentrations optimized for each experiment, with details provided accordingly. Molecular biology techniques and Genetic manipulation of PAO1 These procedures were performed as previously described ( 54 ). All pTS1 plasmid inserts were synthesised and cloned into pTS1 by Twist Bioscience. The ORF of batR and srkA were amplified by PCR with primers batR_EcoRI_F / batR_XhoI_R and srkA_EcoRI_F / srkA_XhoI_R ( Table 10 ), respectively, and ligated between the EcoRI and XhoI sites of pME6032. For the flag-tagged batR construction in pME6032, primers batR_EcoRI_F and 3xflag_batR_XhoI ( Table 10 ) were used. Bacterial-2-hybrid plasmids were made by Gibson assembly ( glpK ) and restriction cloning ( batR, acpP, srkA, pslC, pilG , and ribA ) into the XbaI and KpnI sites of pKNT25 and pUT18C using the primers indicated in Table 10 . View this table: View inline View popup Table 10. Primers used in this study. MIC determination Minimum inhibition concentrations of antibiotics were determined by the broth microdilution method ( 24 ) following the EUCAST guidelines, using Mueller-Hinton broth. The Sub Inhibitory Concentration (SIC) was defined as being 1/4 of the lowest antibiotic concentration that inhibited visible growth after overnight incubation at 37°C. Inhibition disc assay Bacterial cultures were grown in LB medium at 37°C to mid-log phase, A 600nm =0.5, and 150 µl were spread on each plate. Discs containing the antibiotics/H 2 O 2 were gently placed on the agar and plates were incubated inverted overnight. The normalized width of the antimicrobial “halo” (NW halo ) of each disk was determined after ( 55 ). Glass beads biofilms and Crystal Violet (CV) assays These assays were performed as described elsewhere ( 25 ) with the following modifications. Ten independent biological replicates were included: five PIP-exposed biofilm lineages (challenged with 0.25× MIC of PIP for 90 min) and five unexposed control lineages. Cells recovered from the beads were serial diluted and spotted onto LB plates for CFU counting. For the CV assay, the A 590nm was measured at using a SPECTROstar nano plate reader (BMG Labtech). Membrane permeability assays These assays were performed as described elsewhere ( 56 ). EVPL infection model EVPL was prepared as previously described ( 27 , 57 ). Porcine lungs were obtained from two local butchers (Quigley and Sons, Cubbington; and Taylor’s Butcher, Earlsdon) and dissected on the day of delivery under sterile conditions. Following infection of bronchiolar tissue pieces, 500 μl of SCFM ± 16 μg/ml CIP was added to each well. Tissue pieces were incubated at 37°C for 2 and 7 d. Uninfected controls were included. EVPL biofilm recovery and assessment of bacterial load and virulence factors production were determined as described elsewhere ( 27 ). Haematoxylin & eosin staining H & E staining was assayed as previously described ( 27 ). The infected/uninfected EVPL tissue pieces were fixed and sent to the University of Manchester Histology Core Facility for paraffin wax embedding, sectioning, and mounting. Samples were de-paraffinized and stained in Mayer’s hemalum solution (Merck Millipore) and counterstained in eosin Y solution (Merck Millipore). Images were taken using a Zeiss Axio Imager Z2 light microscope with the Zeiss AxioCam 506 and Zeiss Zen Blue v2.3 pro software. Synthetic chronic wound infection model These assays were performed as previously described ( 28 ). Quantitative Proteomics (TMT) for expression analysis The detailed protocol is presented in the supplemental materials section (S3 Data). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE ( 58 ) partner repository with the dataset identifier PXD050997 and 10.6019/PXD050997. Hydrogen cyanide (HCN) production The Feigl-Anger assay was employed to detect HCN production. These assays were performed as described elsewhere ( 59 ). Co-Immunoprecipitation and mass spectrometry analysis The detailed protocol is presented in the supplemental materials section (S4 Data). Data are available via ProteomeXchange with identifier PXD050995. Bacterial 2 hybrid assays These assays were performed as described elsewhere ( 60 ) with some modifications. The ORFs of batR, acpP, pilG, pslC, ribA, srkA and glpK were cloned into pKT25 and pUT18C using either conventional restriction enzyme cloning or Gibson assembly, as indicated in Table 10 . Data presentation and statistical analyses All graphs and one-way ANOVA followed by post-hoc Tukey’s Multiple Comparison Test, where appropriate, were performed using GraphPad Prism version 5.04 for Windows, www.graphpad.com . SUPPLEMENTAL MATERIAL Download figure Open in new tab FIG S1. Growth curves are shown for strains WT ( 35 ), Δ batR (blue) in LB medium and WT ( 35 ), Δ batR (black) in LB medium supplemented with SIC of CIP, GENT or PIP. The mean growth for 3 biological replicates is shown as a solid line and standard deviation shown as dotted lines. Cells were grown for 24 h at 37⁰C under shaking conditions. Download figure Open in new tab FIG S2. Membrane permeability assays. Drug accumulation as measured by resazurin fluorescence at excitation 544 nm and emission of 590 nm (544,590) is shown. Lines indicate average from 3 biological replicates, with 4 technical replicates for each. Download figure Open in new tab FIG S3. Log of total CFU of P. aeruginosa strain PAO1 WT recovered from the EVPL model following treatment with antibiotics. Each strain was grown on EVPL tissue for 48 h then transferred to antibiotic or PBS as a control for 18 h and the CFU/lung determined. Download figure Open in new tab FIG S4. Production of siderophores by P. aeruginosa WT and Δ batR biofilms in the EVPL model. Units are as follows: for pyoverdine (PVD), fluorescence with excitation/emission of 400±20/460±20 nm per CFU and pyochelin (PCH), fluorescence with excitation/emission of 360±35/460±20 nm per CFU of P. aeruginosa . Graphs a and c show PVD production at 2 d and 7 d PI, respectively. Graphs b and d show PCH production at 2 d and 7 d PI, respectively. The bars denote means and an ANOVA test shows non-significant differences. Download figure Open in new tab FIG S5. Proteomic analysis: Scatterplot representing pairwise comparison of mean log2 protein abundance values for P. aeruginosa WT and Δ batR , with and without PIP challenge. A complete list of genes and information on their predicted functions are given in Table 2 and Table 3 . Download figure Open in new tab FIG S6. Multiple sequence alignment of selected BatR homologs. ClustalW was used to align RMF of E. coli and BatR homologs of different Pseudomonas species. The sequence alignment was visualized using Geneious software. The conserved tryptophan residue at position 52 in Pseudomonas spp. is shown (arrowed). S1 Data. Integrated proteomic data comparing PAO1 WT and Δ batR (±PIP). Contains underlying data for Fig S5. (XLSX). S2 Data. Co-IP data comparing PAO1 WT-pME- batR and pME-3xFLAG- batR . Contains underlying data for Table 6 . (XLSX). S3 Data. Protocol used for Quantitative Proteomics (TMT) for expression analysis. (DOCX) S4 Data. Protocol used for Co-Immunoprecipitation and mass spectrometry analysis. (DOCX) ACKNOWLEDGMENTS The authors would like to Govind Chandra for his feedback on bioinformatic analysis. This work was supported by BBSRC Grants BB/X010996/1, BBS/E/J/000PR9797, and BB/T004363/1. AP’s placement in FH’s lab was supported by the Flexible Talent Mobility Award Scheme (FTMA). REFERENCES 1. ↵ Rice LB . 2008 . Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE . J Infect Dis 197 : 1079 – 81 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Shigemura K , Arakawa S , Sakai Y , Kinoshita S , Tanaka K , Fujisawa M . 2006 . Complicated urinary tract infection caused by Pseudomonas aeruginosa in a single institution (1999–2003) . International Journal of Urology 13 : 538 – 542 . OpenUrl CrossRef PubMed Web of Science 3. ↵ Chitkara YK , Feierabend TC . 1981 . 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Share BatR: A novel regulator of antibiotic tolerance in Pseudomonas aeruginosa biofilms Ainelen Piazza , Harshanie Dasanayaka , Gerhard Saalbach , Carlo Martins , Eleftheria Trampari , Mark A. Webber , Freya Harrison , Jacob G. Malone bioRxiv 2024.04.17.589660; doi: https://doi.org/10.1101/2024.04.17.589660 Share This Article: Copy Citation Tools BatR: A novel regulator of antibiotic tolerance in Pseudomonas aeruginosa biofilms Ainelen Piazza , Harshanie Dasanayaka , Gerhard Saalbach , Carlo Martins , Eleftheria Trampari , Mark A. Webber , Freya Harrison , Jacob G. 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