Functional interplay between (p)ppGpp and RNAP in Acinetobacter baumannii

Functional interplay between (p)ppGpp and RNAP in Acinetobacter baumannii | 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 Functional interplay between (p)ppGpp and RNAP in Acinetobacter baumannii View ORCID Profile Anthony Perrier , View ORCID Profile Aurélie Budin-Verneuil , View ORCID Profile Régis Hallez doi: https://doi.org/10.1101/2025.07.04.663200 Anthony Perrier 1 Bacterial Cell cycle & Development (BCcD), Biology of Microorganisms Research Unit (URBM), Namur Research Institute for Life Science (NARILIS), University of Namur , Namur (5000), Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anthony Perrier Aurélie Budin-Verneuil 2 Université de Caen Normandie , CBSA UR 4312, Caen, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aurélie Budin-Verneuil Régis Hallez 1 Bacterial Cell cycle & Development (BCcD), Biology of Microorganisms Research Unit (URBM), Namur Research Institute for Life Science (NARILIS), University of Namur , Namur (5000), Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Régis Hallez For correspondence: regis.hallez{at}unamur.be Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The (p)ppGpp-dependent stress response is required for pathogenic bacteria to survive both outside and inside the host but the mechanisms behind this survival are mostly unknown. In this study, we characterize the (p)ppGpp metabolism in the opportunistic pathogen multi-drug-resistant Acinetobacter baumannii . We show that two stressful conditions potentially encountered during infection – iron starvation and polymyxin exposure – induce (p)ppGpp production. The absence of (p)ppGpp led to multiple consequences on the physiology of A. baumannii , including an increase of surface motility, a decrease in catalase activity, a poor survival upon nutrient starvation, a rapid killing during desiccation and a strong attenuation in a Galleria mellonella model of infection. Using a motility suppressor screen, we isolated multiple independent stringent mutations in rpoB and rpoC that suppress the (p)ppGpp-dependent phenotypes. By combining the suppressor screen with deep sequencing, we isolated dozens of additional mutants, expanding the list of putative stringent RNAP mutations described so far. Furthermore, our transcriptomic data reveal that (p)ppGpp deeply impacts the transcriptional landscape of A. baumannii on solid surface to induce many stress-related genes, including catalase and hydrophilins critical for tolerance to desiccation. This work highlights the functional interplay between (p)ppGpp and RNAP in the successful survival of A. baumannii in the environment but also during infection. Introduction The stringent response (SR) is one of the mechanisms that facilitates bacterial adaptation to harmful conditions. In 1969, Cashel and Gallant identified two compounds related to the SR, the guanosine tetra-phosphate (ppGpp) and penta-phosphate (pppGpp) collectively referred to as (p)ppGpp ( 1 ). The SR was first referred to the growth arrest of Escherichia coli cells experiencing amino acid starvation due to increasing intracellular levels of (p)ppGpp. Now, the SR refers to any response caused by elevated levels of (p)ppGpp by any means ( 2 ). The intracellular levels of (p)ppGpp are mainly regulated by enzymes of the RSH family (RelA/SpoT Homologue). Most of bacteria possess one long bifunctional RSH, whereas the β- and γ-Proteobacteria harbor two long RSH proteins, one bifunctional (SpoT) and one monofunctional (RelA). Interestingly, based on in silico analyses and experimental evidence, the Moraxellaceae family to which belongs the genus Acinetobacter seems to have two long monofunctional RSH, RelA and SpoT displaying respectively functional synthetase and hydrolase activities ( 3 , 4 ). In addition to long RSH, some bacteria also possess short monodomain RSH with either a synthase or hydrolase activity, respectively called Small Alarmone Synthetase (SAS) or Hydrolase (SAH). Since its discovery more than 50 years ago it has been often hypothesized that (p)ppGpp directly interacts with the bacterial RNA polymerase (RNAP). But it is only during the last decade that substantial progress has been made and two (p)ppGpp binding site (respectively referred to as site 1 and site 2) were mapped on the E. coli RNAP. The site 1 is located at the interface between the ω and β’ subunits ( 5 – 7 ) while the site 2 is created by the interaction of the transcription factor DksA with the β’ subunit ( 8 ). The position of the two binding sites strongly suggests an allosteric regulation of the RNAP by (p)ppGpp. Additionally, (p)ppGpp can also bind to other proteins mostly acting as a competitive inhibitor of guanosine nucleotides ( 9 ). A E. coli K12 strain devoid of (p)ppGpp synthetase – and thus unable to produce (p)ppGpp hereafter referred as (p)ppGpp 0 – harbors several physiological defects including amino acids auxotrophy. Growing a (p)ppGpp 0 strain on minimal medium without amino acids allows the selection of spontaneous suppressors, always mapping to the RNAP, frequently in the β or β’ subunit and rarely in the ω subunit ( 10 ). Such RNAP mutants, known as “stringent RNAP” ( 11 ), can suppress other (p)ppGpp-related phenotypes. Acinetobacter baumannii is a gram-negative γ-Proteobacterium belonging to the Moraxellaceae family. In 2017, the World Health Organization (WHO) established a list of bacterial pathogens of public health importance and ranked carbapenem-resistant A. baumannii (CRAB) amongst the most critical pathogens. More recently in 2024, the list has been revised but CRAB maintained their critical status because of the high mortality following antibiotic-resistant infections as well as the lack of new drug effective against metallo-β-lactamase producing CRAB ( 12 ). Known as a multidrug-resistant opportunistic pathogen, A. baumannii is also highly resistant and tolerant to stressful environments ( 13 ). For instance, clinical isolates of A. baumannii can persist for months on dry surfaces in an extreme dehydrated state ( 14 ). Intrinsically disordered proteins called hydrophilins were shown to promote tolerance to desiccation ( 15 ). With two long monofunctional RSH enzymes and DksA recently proposed to functionally replace the major stress response sigma factor RpoS ( 16 ), A. baumannii stands out as unusual and intriguing amongst γ-Proteobacteria. However, studies on (p)ppGpp in A. baumannii are still scarce ( 17 , 18 ). As seen in many other pathogens, (p)ppGpp 0 strains display virulence defects both in mouse and Galleria mellonella infection models ( 17 , 19 ). Beyond using serine hydroxamate (SHX) as a proxy for amino acid starvation, the specific stresses that trigger the SR in A. baumannii are still unknown. Production of (p)ppGpp has also been described to regulate surface motility with distinct outcomes observed between the reference strains ABUW5075 and ATCC17978 ( 17 , 19 ). This prompted us to further investigate the physiological role of this second messenger in A. baumannii . We were able to identify a third enzyme likely involved in (p)ppGpp metabolism, ABUW_1957 encoding a SAH capable of hydrolyzing (p)ppGpp in vivo both in E. coli and A. baumannii , although its exact function remains to be elucidated. A classical but thorough phenotyping of RSH mutants highlighted a critical role of (p)ppGpp in the survival of A. baumannii cells exposed to nutrient deprivation and desiccation. We also found that iron starvation and exposure to polymyxin B, two stresses possibly encountered during infection, induced (p)ppGpp production. We isolated stringent RNAP mutants that suppress most of the (p)ppGpp-dependent phenotypes, including virulence defects of the (p)ppGpp 0 strain in the G. mellonella model of infection. By combining our suppressor screen with deep sequencing in a Mut-Seq approach ( 20 ), we largely expanded our list of candidate missense mutations within rpoB and rpoC that likely generate stringent RNAP. Finally, we found that ∼9% of the entire transcriptome was significantly modified in (p)ppGpp 0 cells in contact with solid surface and that the expression of the vast majority of these differentially expressed genes was restored in a stringent RNAP suppressor. Together our data show that RNAP and (p)ppGpp jointly regulate several phenotypical traits critical for the survival in stressful conditions and the virulence of A. baumannii . Results A. baumannii harbors three RelA/SpoT homolog enzymes A. baumannii carries two genes coding for long RSH enzymes, spoT ( ABUW_0309 ; ABUW_RS01520 ) and relA ( ABUW_3302 ; ABUW_RS16040 ). RelA Ab is a monofunctional synthetase enzyme with a degenerate and inactive hydrolase domain (S + /H - ) ( 17 ). However, in contrast to E. coli and many other β- and γ-proteobacteria where SpoT is a bifunctional RSH both synthetizing and hydrolyzing (p)ppGpp (S + /H + ), SpoT Ab is a monofunctional hydrolase enzyme with a pseudo-synthetase domain regulating its hydrolase activity (S - /H + ) ( 4 ). In addition to RelA Ab and SpoT Ab , we identified two putative short RSH, one SAH ( ABUW_1957 ; ABUW_RS09520 ) and one SAS ( ABUW_0769 ; ABUW_RS03770 ). First, we investigated the role of the putative SAH hereafter referred to as SahA. In E. coli , spoT is essential in an otherwise wild-type (WT) background since (p)ppGpp produced by RelA accumulates to a toxic level in a Δ spoT background when grown on complex media. In contrast, spoT can be inactivated in a Δ relA background. To test whether SahA could hydrolyze (p)ppGpp, a P1 lysate made on MG1655 Δ relA Δ spoT::kan R was transduced in a WT strain of E. coli MG1655 harboring a pBAD33 plasmid, either empty or expressing spoT Ec , spoT Ab or sahA under the control of the arabinose-inducible pBAD promoter. As shown in Fig. S1A , the endogenous spoT gene could be inactivated in the presence of arabinose and one of the three expression vectors (pBAD33- spoT Ec , pBAD33- spoT Ab or pBAD33- sahA ) but not with the empty pBAD33 plasmid, strongly suggesting that SahA is indeed able to hydrolyze (p)ppGpp in vivo . Then, we tested whether sahA and spoT could compensate each other as (p)ppGpp hydrolases in A. baumannii . For that, we first generated Δ relA , Δ relA Δ spoT , Δ relA Δ sahA and Δ relA Δ spoT Δ sahA knock-out mutants in A. baumannii in which relA is inactivated. Then, a copy of relA Ab under the control of an IPTG-inducible promoter was introduced in all these mutant strains. Interestingly, induction of relA Ab in the Δ relA Δ spoT strain led to a severe growth defect whereas the strain lacking both (p)ppGpp hydrolases (Δ relA Δ spoT Δ sahA ) could barely grow when relA Ab was induced. In contrast, expressing back relA Ab in strains harboring a functional copy of spoT – the single Δ relA or the double Δ relA Δ sahA mutant strain – did not impact the growth. ( Fig. 1 ). These results further suggest that SahA is, to some extent, able to hydrolyze (p)ppGpp in vivo , both in E. coli and A. baumannii . Nevertheless, despite several attempts with different methods, we failed to generate a single Δ spoT mutant in A. baumannii , strongly suggesting that SpoT is the main (p)ppGpp hydrolase in A. baumannii and that SahA cannot compensate for the loss of spoT , at least in the conditions we tested. Download figure Open in new tab Figure 1. SahA can hydrolyze (p)ppGpp in vivo in A. baumannii. Viability of A. baumannii AB5075 Δ relA , Δ relA Δ spoT , Δ relA Δ sahA, Δ relA Δ spoT Δ sahA cells carrying a copy of relA Ab under an IPTG-inducible promoter (P tac - relA Ab ). Overnight cultures were serial diluted (1:10), spotted on LB agar plates with or without IPTG (500 µM) and incubated overnight at 37 °C. Finally, we investigated the role of the putative SAS. The fact that a Δ relA Δ spoT Δ sahA triple mutant in A. baumannii was viable already suggested that (p)ppGpp was unlikely produced by this putative SAS, at least in our lab conditions. A whole genome sequencing of this triple mutant excluded the presence of any suppressor mutations. To further investigate the potential (p)ppGpp production in vivo by this enzyme, E. coli MG1655 WT (hydrolase +) and MG1655 Δ relA Δ spoT (hydrolase -) strains were transformed with a pBAD33 plasmid, either empty or harboring relA Ec or ABUW_0769 , and grown on complex LB medium supplemented with glucose or arabinose. Expressing relA Ec was toxic in the Δ relA Δ spoT background, likely because of (p)ppGpp accumulation whereas the expression of ABUW_0769 did not have any impact on the growth of both strains ( Fig. S1B ). Together with the fact that (p)ppGpp was undetectable in the A. baumannii Δ relA and Δ relA Δ spoT mutant strains ( Fig. S2 ) , these results suggest that no (p)ppGpp was synthesized from ABUW_0769, at least in all the conditions we tested. Absence of (p)ppGpp induces pleiotropic effect in A. baumannii It has been previously described that a Δ relA mutant in the AB5075 background shows a hyper-motile phenotype and forms elongated cells in stationary phase ( 17 ). Considering the high phenotypic heterogeneity among A. baumannii strains and also within the same strain in different labs ( 21 ) we investigated several known (p)ppGpp-dependent phenotypes in our mutants. First, the growth of the mutants was measured in complex medium (LB) and minimal synthetic medium with xylose as sole carbon source (MOPSX) and compared to the WT. Surprisingly, we did not find important differences, except a lower plateau for Δ relA and Δ relA Δ spoT cells grown in LB and a slight growth delay in MOPSX ( Fig. 2A and Fig S3A ). Then, we analyzed the cell morphology of bacteria grown overnight in LB. The Δ relA and Δ relA Δ spoT mutants showed heterogenous filamentation with a median of 2.82 and 2.51 μm, respectively, against 2.12 μm for the WT strain ( Fig. 2B and Fig. S3B ). In contrast, the Δ sahA mutant showed a cell size distribution close to the WT strain with a median of 2.24 μm ( Fig. S3B ). We also looked at surface motility on low agar plates. After 24 h at 37 °C, the Δ relA and Δ relA Δ spoT strains colonized most of the 90 mm Petri dishes, whereas the WT and Δ sahA strains did not move at all, with only growth observed around the inoculation site ( Fig. 2C and Fig. S3C ). Interestingly, the motility behavior correlates well with the colony aspect of the different strains. The WT and Δ sahA strains formed white shiny colonies while the Δ relA and Δ relA Δ spoT strains formed browner colonies on LB agar plates ( Fig. S3C ). Download figure Open in new tab Figure 2. The pleiotropic defects of Δ relA are suppressed by mutations in RNAP. ( A ) Representative growth of WT, Δ relA , Δ relA rpoB R557C , Δ relA rpoB R460C and Δ relA rpoC R436G at 37 °C in complex (LB) or minimal synthetic (MOPSX) medium for 20 h in 96-well plates. ( B ) Microscopic analysis of WT, Δ relA , Δ relA rpoB R557C , Δ relA rpoB R460C and Δ relA rpoC R436G cells grown overnight at 37 °C in liquid complex medium. A representative picture (scale bar = 10 µm) is shown for each strain accompanied by a violin plot showing cell size distribution; the median size (µm) is indicated in red. ( C ) Surface motility of cells described in (B) on low agar (0.5%) complex medium. The appearance and the color of the colonies can also be observed on the picture in the box in top right corners. Pictures for both experiments were taken after 24 h incubation at 37 °C. ( D ) Viability of WT, Δ relA , Δ relA rpoB R557C , Δ relA rpoB R460C and Δ relA rpoC R436G strains on complex or minimal synthetic (M9X) medium with or without supplementation [DIP (150 µM); FeSO 4 (50 µM]. Overnight cultures were serial diluted (1:10), spotted on plates and incubated overnight at 37 °C. Since A. baumannii is characterized by a wide genetic and phenotypic diversity, a Δ relA mutant was also constructed in the well-studied ATCC17978 strain. Similarly to AB5075 Δ relA , ATCC17978 Δ relA formed an heterogenous population of short and longer cells with a median of 2.36 µm when grown overnight in LB compared to 1.75 µm for the ATCC17978 WT strain ( Fig. 3A ). Likewise, we found that in our conditions, WT cells from the ATCC17978 strain were not motile in contrast to the Δ relA mutant which was also able to colonize the entire plate in 24 h ( Fig. 3B ). Download figure Open in new tab Figure 3. rpoB R557C is also a suppressor of Δ relA in A. baumannii ATCC17978 strain. ( A ) Microscopic analysis of WT, Δ relA , Δ relA rpoB R557C cells grown overnight at 37 °C in liquid LB medium. A representative picture (scale bar = 10 µm) is shown for each strain accompanied by a violin plot showing cell size distribution; the median size (µm) is indicated in red. ( B ) Surface motility of cells described in (A) on low agar (0.5%) complex medium grown at 37 °C for 24 h. Finally, we used serial dilution drop assays to investigate the sensitivity of our strains to several stresses described as linked to (p)ppGpp. Figure S3 shows that the Δ sahA strain behaved like the WT strain under all conditions tested. Surprisingly, despite the fact that exposure to SHX – known to induce amino acid starvation – strongly induced (p)ppGpp accumulation ( Fig. S2 ), the strains unable to produce (p)ppGpp (Δ relA and Δ relA Δ spoT ) could grow well on synthetic minimal medium M9 with xylose as sole carbon source (M9X), showing that none of these mutants suffered from amino acid auxotrophy. On the contrary, Δ relA mutants had severe growth defects on plates containing triclosan (fatty acid starvation) or 2,2′-dipyridyl (DIP) (iron starvation), while the addition of 50 μM of iron alleviated the defects induced by DIP ( Fig. 2D and Fig. S3C ). In agreement with our drop assay, we found that DIP led to noticeable ppGpp accumulation. However, to our surprise, neither cerulenin nor triclosan induced (p)ppGpp production in our conditions, suggesting that fatty acid starvation is not a signal triggering (p)ppGpp accumulation ( Fig. 4 and Fig S4 ). We also tested polymyxin B and, as previously described for E. coli ( 22 ), we observed ppGpp accumulation in cells exposed to this last-resort antibiotic used against A. baumannii infection ( Fig. 4 ). Download figure Open in new tab Figure 4. Serine hydroxamate, 2,2’-dipyridyl and polymyxin B induce (p)ppGpp production in A. baumannii. Nucleotides extracted from WT cells either non-treated (Ctrl) or exposed to 3.4 mM serine hydroxamate (SHX), 400 µM 2,2’-dipyridyl (DIP), 250 µg/mL cerulenin (Ceru) or 2 mg/mL polymyxin B (PolyB) for 10 min, were analyzed on thin layer chromatography. Mutations in rpoB or rpoC suppress the (p)ppGpp-dependent phenotypes While characterizing the hyper-motility behaviour of Δ relA on low agar plates, we inadvertently selected suppressors, which appeared after more than 96 h of incubation at 37 °C as white shiny dots on top of the bacterial lawn of motile cells ( Figure S5 ). Mutations in rpoB or rpoC – respectively encoding the β and β’ subunit of the RNAP – were found by whole genome sequencing in all tested non-motile suppressor candidates ( Table 1 ). In E. coli , (p)ppGpp binds to the RNAP to reprogram transcription and mutations in rpoB or rpoC can induce conformational changes that mimic the effect of (p)ppGpp binding ( 23 ). Moreover, some of these so-called stringent mutations were often associated with resistance to rifampicin (Rif R ). However, none of the suppressor mutations ( rpoB R460C , rpoB R557C and rpoC R436G ) that we identified here led to a Rif R phenotype. View this table: View inline View popup Download powerpoint Table 1. List of the putative mutations leading to stringent RNAP in A. baumannii Interestingly, these three mutations could suppress most of the (p)ppGpp-dependent phenotypes observed in Δ relA ( Figure 2 ) except (i) the cell size distribution observed in stationary phase cultures, which was even aggravated for rpoB R460C ( Fig. 2B ) and (ii) the sensitivity to triclosan, with rpoC R436G being less resistant than the WT ( Fig. S3D ). More importantly and despite these differences, we found that the three suppressor mutations could fully restore the virulence of A. baumannii in the Galleria mellonella model of infection. Indeed, the Kaplan-Meier analysis showed that the survival of the larvae infected with each of the suppressors was not significantly different from the WT, while in agreement with previous report ( 4 , 17 ), the larvae infected with Δ relA showed a significantly better survival ( p < 0.0001) ( Figure 5 ). These data suggest that the virulence defect of the (p)ppGpp 0 strain is mainly due to a transcriptional deregulation of virulence factors. In contrast, no significant differences were found when comparing the survival curve of larvae infected with the Δ sahA single mutant to the one infected with WT cells ( p = 0.4022) ( Fig. S6 ), suggesting that sahA is not required for A. baumannii virulence, at least in the G. mellonella infection model. Download figure Open in new tab Figure 5. Mutations in rpoB and rpoC restore the virulence of a Δ relA mutant. Kaplan-Meier survival analysis of Galleria mellonella larvae infected with the WT, Δ relA, Δ relA rpoB R557C , Δ relA rpoB R460C and Δ relA rpoB R446G strains. Each curve represents the average of three independent experiment for a total of 80 larvae per condition. All survival curves are not significantly different from the WT survival curve except the one for the Δ relA mutant (Log-rank (Mantel-Cox) test; p < 0.0001). We also selected motility suppressors for the ATCC17978 ΔrelA mutants by incubating low agar plates for an extended time, until white shiny colonies appeared on top of the bacterial lawn as observed for the AB5075 strain. By using specific primers that anneal to the rpoB R557C allele, we were able to fish out 8 positive clones that were then validated by Sanger sequencing. Again, as for AB5075, rpoB R557C was able to suppress most of the phenotypes displayed by Δ relA in the ATCC17978 strain ( Fig. 3 ). To check to what extent rpoB and rpoC are hotspots for the selection of Δ relA suppressors, we used an unbiased Mut-Seq approach ( 20 ) to map as many mutations as possible in rpoB or rpoC that suppress the hyper-motility phenotype displayed by Δ relA . For that, 284 non-motile Δ relA suppressors were selected on low agar complex medium plates as described above. It is important to note that among them, only 8 were Rif R ( Table 1 ). Then, the pooled gDNA of these 284 clones was used as a template to amplify rpoB and rpoC loci and both PCR products were deep sequenced. By using a confidence threshold of 0.5% (see M&M section), 39 and 18 putative substitutions – leading to 32 and 16 missense mutations respectively in rpoB and rpoC – were identified ( Fig. 6 and Table 1 ). Download figure Open in new tab Figure 6. Putative stringent mutations leading to loss-of-motility in Δ relA are spread in 6 sub-regions within rpoB and rpoC. For a given nucleotide position, only SNPs found by Mut-Seq with a percentage ≥ 0.1 are represented. Confidence threshold of 0.5% is represented by the horizontal dashed line. Blue boxes highlight the sub-regions where most SNPs were found. Together these data strongly support that the (p)ppGpp-dependent transcriptional reprogramming is responsible for most of the phenotypes observed in (p)ppGpp 0 cells and suggest that RNAP is a primary (p)ppGpp target in A. baumannii . A single substitution in rpoB can revert the (p)ppGpp-dependent transcriptomic changes To further characterize the suppressing effects of rpoB R557C in Δ relA , the RNA extracted from the WT, Δ relA and Δ relA rpoB R557C cells grown on the medium used for the selection of the suppressors – LB low agar plates – was sequenced. When comparing Δ relA to the WT, we found 304 (8.09%) differentially expressed genes (DEGs) (absolute log 2 Fold Change |log 2 FC| ≥ 2 and FDR–adjusted p -value ≤ 0.005), with 115 (3.06%) up-regulated genes and 189 (5.03%) down-regulated genes ( Table S1 ). As expected, the ABUW_3766-3773 operon known to be critical for surface motility ( 24 , 25 ) was found among the most up-regulated genes in the Δ relA strain ( Fig. 7A ). Next to this operon, the abaRMI ( ABUW_3774-3776 ) quorum-sensing locus was also highly up-regulated. Genes related to iron homeostasis (acinetobactin gene cluster and several TonB-dependent siderophore receptor) and fatty acid (FA) biosynthesis were also up-regulated in the Δ relA mutant ( Fig. 7A and Table S1 ). Several genes known to be regulated by (p)ppGpp in E. coli ( ostAB , raiA and fis ) were found to be down-regulated in Δ relA . In addition, the expression of the locus ABUW_2433-2443 , containing stress related genes such as cinA1 , katE, dtpA and dtpB was also found to be down-regulated in Δ relA . Apart from these, most of the other down-regulated genes were of unknown functions ( Fig. 7A and Table S1). Download figure Open in new tab Figure 7. The rpoB R557C mutation reverts back the transcriptomic profile of the Δ relA mutant close to a WT on low agar complex medium (A) Volcano plot representing the genes differentially expressed in the Δ relA mutant compared to the WT strain on low agar medium. Genes with a |log 2 FC| ≥ 2 and –Log 10 p -value ≥ 3 are represented in light pink or with a color code for group of genes associated with known functions (light green: surface motility; red: iron homeostasis; yellow: fatty acid biosynthesis; blue: quorum sensing (QS) system; black: stress related genes; brown: ubiquinol oxidation; purple: trehalose biosynthesis; dark green: catalases). (B) xy-plot representing the differential expression of genes between the Δ relA /WT vs Δ relA/ Δ relA rpoB R557C on motility complex medium. Each dot represents the log 2 FC value for the strains indicated under brackets (only genes with FDR-adjusted p ≤ 0.005 were kept). Interestingly, we only found 21 (0.56%) DEGs between the WT and the suppressor Δ relA rpoB R557C strain, with respectively 15 (0.40%) up- and 6 (0.16%) down-regulated genes. Figure 7B shows that the log 2 FC values of Δ relA vs WT and Δ relA vs Δ relA rpoB R557C have a linear correlation (R² = 0.9426). This indicates that the transcriptomic changes induced in the Δ relA mutant are almost entirely reverted to WT in the Δ relA rpoB R557C suppressor. We also determined the (p)ppGpp-dependent transcriptome for cells grown in liquid LB medium by comparing WT to Δ relA or Δ relA rpoB R557C strains. However, in these conditions only 153 genes (4.06%) were differentially expressed in Δ relA compared to WT (absolute log 2 Fold Change |log 2 FC| ≥ 2 and FDR–adjusted p -value ≤ 0.005), with 28 (0.74%) up-regulated genes and 125 (3.32%) down-regulated genes ( Fig. S7A and Table S2 ). In particular all the genes encoding the assembly machinery of Csu pili ( csu genes) were down-regulated in Δ relA ( Fig. S7A ). Importantly, there were still 165 DEGs when comparing the suppressor Δ relA rpoB R557C to the WT strain ( Table S2) , suggesting that the rpoB R557C restored the (p)ppGpp-dependent transcriptional profile more efficiently on low agar plate than in liquid medium. In agreement with this, the linear regression between the log 2 FC values of Δ relA and WT and Δ relA vs Δ relA rpoB R557C did not fit well for the liquid medium (R² = 0.4531; Fig. S7B ), showing a low correlation. These data suggest that the contact of A. baumannii onto a semi-solid surface could induce (p)ppGpp accumulation, which leads to a deep transcriptional reprogramming. The Δ relA mutant is highly sensitive to desiccation and have impaired H 2 O 2 detoxification Among the genes differentially expressed in the Δ relA mutant in both liquid and low agar media, we identified the TetR-type transcriptional regulator ABUW_1645 . It was the most down-regulated gene in liquid medium (log 2 FC = -7.91) and among the most down-regulated on low agar plates (log 2 FC = -5.27). This regulator has been previously described as being involved in the pleiotropic phenotypic switch in A. baumannii ( 26 ). Since its expression appears to be partially restored in the Δ relA rpoB R557C suppressor strain compared to the WT (log 2 FC of -0.61 in liquid and -2.32 in low agar medium), we wondered whether it could contribute to phenotypes observed in the Δ relA mutant. To test this hypothesis, a second copy of ABUW_1645 expressed from the IPTG-inducibe P tac promoter (P tac - ABUW_1645 ) was introduced in the Δ relA mutant as well as in the WT. However, this construct turned out to be lethal on IPTG containing plates, not only in the Δ relA mutant but also in the WT strain, while it did not impact the growth or the colony appearance without IPTG. Occasionally, WT and Δ relA clones with P tac - ABUW_1645 were able to grow in the presence of IPTG, but these clones harbored IS4 ISAba1 transposase inserted into ABUW_1645 , thereby disrupting its function. Although we cannot firmly exclude the possibility that ABUW_1645 participates to some extent to the phenotypes displayed by Δ relA , our data show that ABUW_1645 is toxic when highly expressed. On low agar medium, the most down-regulated gene in the Δ relA strain is dtpA (log 2 FC = -8.21), which encodes a hydrophilin involved in the tolerance of the Acinetobacter calcoaceticus–baumannii complex to high desiccation ( 15 ). Since the dtpA locus ( ABUW_2433–2443 ) is down-regulated in both liquid and low agar media, and includes several stress-related genes such as those encoding the second hydrophilin DtpB and the catalase KatE, we measured the tolerance to desiccation and the catalase activity in the WT, Δ relA and suppressors strains. The Δ relA strain showed a significant decrease ( p < 0.0001) in normalized catalase activity (37.4% ± 10.6%) compared to the WT strain (100% ± 8.0%). All the three Δ relA suppressors displayed a normalized catalase activity close to or above the WT, with 138.4% ± 11.5% ( rpoB R557C ), 97.0% ± 9.1% ( rpoB R460C ) and 122.2% ± 9.3% ( rpoC R436G ) ( Fig. 8A ). We then tested the survival of the same strains upon desiccation. After three days at 35% room humidity, 4.5% ± 2.5% of CFU were recovered for the WT strain while only 0.009% ± 0.005% CFU were recovered for the Δ relA mutant. Compared to the Δ relA mutant, all the three suppressors showed increased desiccation survival although with some differences between them in the CFU recovering (3.8% ± 3.7% for rpoB R557C , 2.4% ± 0.6% for rpoB R460C and 0.12% ± 0.08% for rpoC R436G ) ( Fig. 8B ). Download figure Open in new tab Figure 8. The Δ relA mutant has impaired catalase activity and desiccation resistance. (A) Catalase activity produced by overnight culture of the WT, Δ relA, Δ relA :: relA , Δ relA rpoB R557C , Δ relA rpoB R460C and Δ relA rpoB R436G strains. ANOVA and Dunnet’s multiple comparisons test (p < 0.01 and p < 0.0001). (B) Desiccation survival of the same strains after 3 days at 35% room humidity and 25 °C. Kruskal-Wallis and Dunn’s multiple comparisons test ( p < 0.05). Discussion In this study, we investigated and characterized the pleiotropic roles played by (p)ppGpp in A. baumannii . First, we identified potential new RSH enzymes including a putative orphan SAS ( ABUW_0769 ) that might synthesize (p)ppGpp. However, we did not find any correlation between its expression and (p)ppGpp production neither in E. coli nor in A. baumannii . Nevertheless, we cannot rule out the possibility that a specific condition is required to activate (p)ppGpp synthesis by ABUW_0769. We also identified a putative orphan gene ( ABUW_1957 ) coding for a SAH (SahA) that was able to hydrolyze (p)ppGpp both in E. coli and A. baumannii . However, under the tested conditions, its hydrolase activity alone was insufficient to counterbalance the absence of spoT and to counteract (p)ppGpp produced by RelA. During our phenotypic analysis of A. baumannii RSH enzymes, we did not identify a condition where the Δ sahA mutant behaved differently from the WT strain, including virulence in G. mellonella . Small alarmone hydrolases remain poorly characterized in bacteria but recently, two orphan SAHs have been described in Proteobacteria. In addition to (p)ppGpp, SAHs were shown to degrade another alarmone, (p)ppApp, and were suggested to act as a defensive mechanism during bacterial competition ( 27 – 29 ). SahA could potentially play a similar role during host colonization, but the involvement of small RSHs in bacterial competition is still an emerging field, requiring further investigation. Then, we extended our analysis to the long RSHs, RelA and SpoT. All attempts to generate a single spoT mutant failed, suggesting that SpoT is essential in A. baumannii , even in the presence of SahA. In all β- and γ-Proteobacteria studied so far, differences have been observed between a single Δ relA mutant and a double Δ relA Δ spoT mutant, essentially because SpoT is bifunctional. However, in A. baumannii , both RSHs are monofunctional, with RelA working as the sole (p)ppGpp synthetase and SpoT as a hydrolase ( 4 ). We found that both mutants exhibited the same phenotypes in all the tested conditions, both behaving as (p)ppGpp 0 strains as expected. Therefore, our results exclude any (p)ppGpp-independent role played by the SpoT protein in A. baumannii , at least in all our conditions. The absence of (p)ppGpp activated surface motility on low agar plates and altered colony morphology, likely reflecting a change of the cell surface properties. The lack of (p)ppGpp also had a detrimental effect on cellular morphology, leading to filamentation of a part of the population in stationary phase. Finally, as already observed with all pathogens studied so far, the virulence of A. baumannii in G. mellonella was also impacted in the (p)ppGpp 0 strains. To expand our analysis, we also generated a (p)ppGpp 0 strain in A. baumannii ATCC17978 and we obtained similar results in both AB5075 and ATCC17978 backgrounds, including the surface motility since both WT were not motile while (p)ppGpp 0 mutants were hyper-motile. This was surprising considering that A. baumannii ATCC17978 WT strain was often reported in the literature as motile and a Δ relA mutant has been shown in one report to be less motile than the WT strain ( 19 ). Aside from lab conditions and experimental setup, these differences could also be explained by the fact that there are at least two main laboratory variants of the A. baumannii ATCC17978 strain ( 21 ), the primary difference being the presence or absence of a 44-kb locus (AbaAL44) known to be responsible for several phenotypic changes including surface motility ( 21 ). In addition to the AbaAL44 locus, several SNPs have been also identified, including a mutation in obgE , a GTPase that may be involved in the SR and thus have an impact on surface motility ( 30 ). While challenging the (p)ppGpp 0 strains with stressful conditions known to be (p)ppGpp-dependent in other species, we found that in the absence of (p)ppGpp, A. baumannii remained prototrophic for amino acids when grown in synthetic minimal medium with xylose as sole carbon source. However, (p)ppGpp strongly accumulated upon SHX treatment, indicating that the hallmark of the SR is functional in A. baumannii . We also observed that FA and iron starvation both affected the growth of (p)ppGpp 0 cells whereas only iron limitation led to (p)ppGpp accumulation. In E. coli , FA starvation leads first to a SpoT-dependent (p)ppGpp synthesis ( 31 ). Given that the SpoT SD domain is degenerated and non-functional in A. baumannii , this could explain the lack of (p)ppGpp production upon triclosan exposure. In E. coli , a prolonged FA starvation ultimately leads to the depletion of pyruvate used as a precursor for lysine synthesis, which thereby triggers the (p)ppGpp synthesis by RelA ( 32 ). However, this regulation has only been described in E. coli and may differ in A. baumannii . Notwithstanding the absence of (p)ppGpp accumulation upon FA starvation, our transcriptomic analyses show that genes coding enzymes involved in FA biosynthesis are upregulated in Δ relA , suggesting that (p)ppGpp 0 cells behave like being starved for FA even in rich media. At least these data exclude the possibility that the growth defects of Δ relA cells in the presence of triclosan comes from low levels of FA biosynthetic enzymes. Iron deprivation also leads to (p)ppGpp accumulation in E. coli but in contrast to FA starvation, only SpoT is involved with its activity switching toward (p)ppGpp synthesis over hydrolysis ( 33 ). In A. baumannii however, (p)ppGpp production during iron starvation can only result from (p)ppGpp synthesis by RelA. Two not mutually exclusive scenarios are therefore possible. Either the SpoT-dependent regulation is conserved, which leads to a decrease of (p)ppGpp hydrolysis and a concomitant increase of (p)ppGpp levels due to basal synthetase activity of RelA, which cannot be hydrolyzed. Or iron starvation indirectly leads to amino acid(s) starvation, which in turn activates (p)ppGpp synthesis by RelA. During the characterization of surface motility, we observed an intriguing phenomenon since after prolonged incubation, white and shiny colonies appeared on the bacterial motility lawn of (p)ppGpp 0 cells, strikingly resembling to WT colonies. These extragenic suppressors are RNAP mutants that not only reverted surface motility but also suppressed most phenotypes associated with the loss of (p)ppGpp, including virulence in G. mellonella . Surprisingly, the rpoB R460C mutation was as efficient as rpoB R557C and rpoC R436G to suppress the lack of virulence of Δ relA despite the very strong filamentation observed in stationary phase. Of course, we cannot rule out the possibility that this filamentation was not induced inside G. mellonella . We applied our motility suppressor screen combined with high-throughput sequencing (Mut-Seq) to isolate as many mutations as possible in RNAP. With this approach, 48 putative missense mutations were identified in rpoB or rpoC , among which we found the three mutations we had previously isolated ( rpoB R460C , rpoB R557C and rpoC R436G ). In 2006, Trinh and colleagues undertook the enormous task of compiling most RNAP mutations reported so far across various organisms, including E. coli ( 23 ). Among these were also mutations mimicking (p)ppGpp effect, known as “RNAP stringent mutations”. We compared our list of mutations obtained with Mut-Seq to this reference list and found that more than 50% of the stringent mutations described in E. coli were also present under our conditions in A. baumannii , strongly suggesting that our screen was effective in selecting for stringent mutations, including putative new mutations that we believe have never been described before. With a total of 57 different substitutions identified within rpoB and rpoC in our screen, and in agreement with work done on E. coli , RNAP seems to be the primary target for mutations in the (p)ppGpp 0 background in A. baumannii . In support of that, by comparing the WT, Δ relA , and Δ relA rpoB R557C strains on surface motility medium, we found that (i) the expression of many genes (∼8%) is regulated by (p)ppGpp and (ii) their expression was mostly reverted close to the WT level with the rpoB R557C mutation. In contrast, the (p)ppGpp-dependent regulon was much smaller (∼4%) when strains were sampled from mid-exponential phase of growth in liquid complex medium and rpoB R557C poorly suppressed these transcriptional changes. Together, these data suggest that the low agar medium used for surface motility is “stressful” for A. baumannii , at least more than the liquid complex medium. In support of this, the ABUW_2433-2444 locus containing stress-related genes linked to oxidative stress and desiccation was highly up-regulated in the WT strain (mean log 2 FC locus = 5.34 ± 2.11) grown on semi-solid medium compared to liquid medium. On the contrary, the expression of the hydrophilins ( dtpA and dtpB ) and the catalase ( katE ) encoded in this locus was not induced in (p)ppGpp 0 cells, which led to a poor desiccation resistance and catalase activity. Strikingly, the suppressor mutations ( rpoB R460C , rpoB R557C and rpoC R436G ) were all able to suppress these phenotypes, which corroborates the high expression of the ABUW_2433-2444 locus observed in the Δ relA rpoB R557C strain. Altogether our data highlight a key role played by the second messenger molecule (p)ppGpp in the virulence and survival upon stress of the opportunistic pathogen A. baumannii . Materials and Methods Bacterial strains and growth conditions Strains, plasmids and oligonucleotides used in this study are listed in Table S3 . Details for plasmids construction are presented in supplementary methods. E. coli and A. baumannii strains were grown at 37 °C either aerobically with shaking at 175 rpm or on plate with 1.5% agar (Difco™). LB Broth Base (Invitrogen™) medium was used as complex medium for both E. coli and A. baumannii . M9 and MOPS were used as a synthetic minimal medium with xylose 0.3% (M9X and MOPSX, respectively) as carbon a source for A. baumannii . M9 was prepared using M9 salts (49 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 8.6 mM NH 4 Cl 18.6 mM NH 4 Cl) supplemented with 1 mM MgSO 4 and 0.1 mM CaCl 2 . When needed 0.5% glucose, 0.5% arabinose or 500mM/1mM IPTG were added. Growth was monitored by measuring absorbance at OD 600 nm in liquid cultures using an automated plate reader (Biotek, Epoch 2) with continuous linear shaking (700 rpm) for at least 20h at 37 °C. For E. coli , antibiotics were used at the following concentrations (μg/mL; in liquid/solid medium): chloramphenicol (30/30), apramycin (30/30) while for A. baumannii , media were supplemented with apramycin (30/30) or rifampicin (20/20) when appropriate. Plasmid delivery into A. baumannii was achieved by natural transformation as described in ( 34 ). In-frame deletions were created by using the pK18- mobsacB derivative plasmids as follows. Integration of the plasmids in the A. baumannii genome after single homologous recombination were selected on LB plates supplemented with apramycin. Independent recombinant clones were then inoculated in LB medium without antibiotics and incubated overnight (ON) at 37 °C. Then, dilutions were spread on LB agar plates without NaCl and supplemented with 10% sucrose and incubated at 30 °C. Single colonies were picked and transferred onto LB agar plates with and without apramycin. Finally, to discriminate between mutated and wild-type loci, apramycin-sensitive clones were tested by PCR on colony using locus-specific oligonucleotides. Microscopy experiment Bacteria were grown ON directly inoculated from -80 °C stocks. Five microliters of ON culture diluted to ½ were spotted on a LB agar pad. Phase contrast images were obtained using Axio Observer 7 microscope (ZEISS, Germany), Orca-Flash 4.0 camera (Hamamatsu, Japan) and Zen 3.9 software (ZEISS, Germany). Analysis was done with the MicrobeJ plugin ( 35 ) on Fiji software ( 36 ) in order to determine the cell length. Motility assay Motility medium containing LB supplemented with 0.5% agar (Difco™) was prepared the day prior of the experiment and plates were poured extemporaneously. A single isolated colony was used to inoculate a single plate with a sterile toothpick. Plates were incubated during 24 h at 37 °C. Measurement of (p)ppGpp Bacteria were grown ON directly inoculated from -80 °C stocks in MOPSX and diluted in MOPSX low phosphate to OD ∼ 0.1 and grown until OD = 0.4-0.6 and then diluted again to OD 0.1 in the same medium. Then radiolabeled phosphate [γ32P]-KH 2 PO 4 was added (final concentration: 40 µCi) and cells were incubated at 37 °C - 600 rpm. After 15 min, stressors (final concentrations: 3.4 mM serine hydroxamate, 400 µM 2,2’-dipyridyl, 250 µg/mL cerulenin, 250 µg/mL triclosan and 2 mg/mL polymyxin B), their solvent alone (final concentration: 2% ethanol) or MOPSX low phosphate was added and cells were incubated again during 10 to 30 min. To extract nucleotides, 200 µL of cells were added to 80 µL ice cold 50% formic acid and kept and ice for 20 min. Then, tubes were kept at -20 °C during at least 1 h. Nucleotides were migrated on polyethyleneimine (PEI) cellulose plate (Macherey-Nagel, Duren, Germany) in 1.5 M KH 2 PO 4 (pH 3.4) at room temperature. TLC plates were air dried and exposed against MS Storage Phosphor Screen (GE Healthcare) overnight. Phosphor screens were finally visualized using an Amersham Typhoon (Cytiva). Virulence Assay The animal model used in this study is based on the larvae of the insect Galleria mellonella. Bacterial strains of A. baumannii were cultured ON in LB broth at 37 °C, harvested 5 min at 4500 rpm and washed twice in saline solution (0.9% NaCl). Cultures were adjusted to reach a density of ∼10 6 CFU/larva and injected into the last proleg of G. mellonella larvae of ∼0.215 g each, using a 26 G needle (Terumo, Belgium) and a syringe pump (Thermofisher, MA, USA). The bacterial inoculum size was verified by plate counting on LB agar. Larvae were housed five per Petri dish and incubated at 37 °C for 6 days. Live and dead larvae were counted every 4 h for 48 h post-infection and then once a day for up to 6 days. Three independent experiments were performed to treat a total of 80 animals per condition. Data are presented on Kaplan-Meier curves, and log-rank statistical analysis was performed with GraphPad Prism 9.0 software. Suppressors and Mut-Seq Suppressors were selected on surface motility (LB low agar) plates by incubating at least 96 h at 37 °C. Then single colonies were isolated, incubated in LB, grown ON at 37 °C. ON cultures were used to extract genomic DNA (NucleoSpin Tissue, Macherey-Nagel, Duren, Germany). For the Mut-Seq, six isolated colonies were used to inoculate 30 surface motility plates. Ten putative suppressors were picked on each plate and inoculated in 96 well plates. Each well was tested for surface motility, growth on LB agar plate with or without rifampicin. Overnight cultures of ∼300 putative suppressors were done in 96 well plates, then 10 µL culture for each clone was pooled and used to extract genomic DNA. The rpoB and rpoC genes were amplified with pair of primers 4152/4153 and 4154/4155 respectively, on the genomic DNA mix, with Q5 ® High-Fidelity DNA polymerase (New England BioLab) according manufacturer recommendation. Both templates were mixed at a 1:1 ratio. Both Whole Genome Sequencing (suppressors) and DNA sequencing (Mut-Seq) were performed using an Illumina NextSeq 2000 (paired-end 2 x 100) instrument (Seqalis, Gosselies, Belgium). Data analysis was performed using HISAT2 and Rsamtools on Galaxy and R software, respectively. Transcriptomic analysis Total RNAs were extracted either from growing bacteria in liquid culture at comparable cell density (OD 600 nm ∼ 0.5) or from bacterial colonies on motility medium. At least two independent biological replicates were made for each strain. Extraction was performed using RNeasy mini kit (QIAGEN) according to the manufacturer’s instructions. RNASeq TTRNA libraries were prepared and sequenced with Illumina NextSeq 2000 (paired-end 2x100) instrument (Seqalis, Gosselies, Belgium). NGS data were analysed using Galaxy ( https://usegalaxy.org ) ( 37 ) as described in ( 38 ). Briefly, FastQC/Falco was used to evaluate the quality of the reads; HISAT2 was used to map the reads onto the AB5075 reference genome (NZ_CP008706) and generate bam files; featureCounts was used to generate counts tables using bam files and DESeq2 was used to determine differentially expressed genes. The Volcano plot was generated using GraphPad Prism 9 software. Catalase Assay Catalase assay was performed as described in ( 39 ). Briefly, bacteria were grown ON directly inoculated from -80 °C stocks. Cells were then washed and normalized to OD ∼ 4. In a glass tube (15 mm diameter x 130 mm height), 100 µL of cells, 100 µL of 1% Triton X100 and 100 µL of undiluted hydrogen peroxide (27%) were mixed. The heigh of the foam was measured after 5 min and subtracted by the height of the control without cells. Desiccation Assay Bacteria were grown ON directly inoculated from -80 °C stocks. On a 24-well plate, for each strain, 25 µL of cells was deposited in 3 different wells in a randomized manner and allowed to dry during 30 min in a microbiological safety cabinet and then incubated at 25 °C with 35% room humidity. Cells were harvested either directly after drying for the inoculum or after 3 days. To harvest cells, 300 µL of M9 salt was added in each well during 30 min to allow cell rehydration. Then cells were diluted and plated on LB medium to determine the number of colony forming units. Statistical analyses All the statistical analyses were performed using GraphPad Prism 9 software. Data availability RNA-Seq and Mut-Seq data have been deposited to the Gene Expression Omnibus (GEO) repository. Authors contribution A.P. and R.H. conceived and designed the experiments. A. B-V performed the virulence assays in Galleria mellonella ( Fig. 5 and Fig. S6 ). A.P. performed all the other experiments presented in this manuscript. A.P. and R.H. wrote the manuscript. Conflict of Interest The authors declare that they have no conflict of interest. Supplemented Figure legends Download figure Open in new tab Figure S1. SahA can hydrolyze (p)ppGpp in vivo in E. coli while putative SAS does not seem to produce (p)ppGpp in E. coli . ( A ) Growth on LB plates supplemented with Kanamycin and Arabinose after P1 mediated transduction of a spoT :: kan R lysate into E. coli MG1655 expressing spoT EC , spoT AB or sahA from a pBAD33 vector. ( B ) Viability of E. coli MG1655 WT (hydrolase +) or Δ relA Δ spoT (hydrolase -) strains expressing relA EC or ABUW_0769 (putative SAS) from a pBAD33 vector. Overnight cultures were serial diluted (1:10), spotted on LB plates with 0.5% of glucose or arabinose and incubated overnight at 37 °C Download figure Open in new tab Figure S2. RelA seems to be the sole (p)ppGpp synthetase in A. baumannii . Nucleotides extracted from cells treated with serine hydroxamate (SHX) for 15min were analyzed on thin layer chromatography. A. baumannii AB5075 WT, Δ relA , Δ relA Δ spoT are represented. Download figure Open in new tab Figure S3. The Δ sahA and Δ relA Δ spoT mutants show similar phenotypes compared to the WT and Δ relA strains, respectively. (A) Representative growth of WT, Δ relA , Δ relA Δ spoT and Δ sahA at 37 °C in complex (LB) or minimal synthetic (MOPSX) medium for 20 h in 96-well plates. (B) Microscopic analysis of WT, Δ relA , Δ relA Δ spoT and Δ sahA cells grown overnight at 37 °C in liquid LB complex medium. A representative picture (scale bar = 10 µm) is shown for each strain accompanied by a violin plot showing cell size distribution; the median size (µm) is indicated in red. (C) Surface motility of cells described in (B) on low agar (0.5%) LB complex medium. The appearance and the color of the colonies can also be observed on the picture in the box in bottom left corners. Pictures for both experiments were taken after 24h incubation at 37 °C. (D) Viability of WT, Δ relA , Δ relA Δ spoT, Δ sahA, rpoB R557C , Δ relA rpoB R460C and Δ relA rpoB R436G strains on minimal synthetic medium (M9X) with or without supplementation [triclosan (0.125 µM); DIP (150 µM); FeSO 4 (50 µM)]. Overnight cultures were serial diluted (1:10), spotted on plates and incubated overnight at 37 °C. Download figure Open in new tab Figure S4. Triclosan does not induce (p)ppGpp production A. baumannii AB5075. Nucleotides extracted from WT cells exposed to either serine hydroxamate (SHX) or ethanol (EtOH) for 10 and 30 min respectively or Triclosan (250 µg/mL) were analyzed on thin layer chromatography. Download figure Open in new tab Figure S5. Putative motility suppressors easily selected on low agar medium in (p)ppGpp 0 backgrounds. The WT, Δ relA , Δ relA Δ spoT and Δ relA Δ spoT Δ sahA strains were inoculated on low agar medium. After 6 days at 37 °C, white non-motile colonies growing on top of the motility lawn can be observed. Download figure Open in new tab Figure S6. The small alarmone hydrolase sahA is not required for the virulence in the Galleria mellonella model. Kaplan-Meier survival analysis of Galleria mellonella larvae infected with the WT and Δ sahA strains. Each curve represents the average of three independent experiment for a total of 80 larvae per condition. No significant differences between the survival curve of Δ sahA mutant and the WT one (Log-rank (Mantel-Cox) test; p = 0.4022). Download figure Open in new tab Figure S7. Lack of (p)ppGpp during exponential growth phase in complete medium has only on mild effect on the transcriptome profile. (A) Volcano plot representing the genes differentially expressed in the Δ relA mutant compared to the WT strain on liquid complete medium. Genes with a |log 2 FC| ≥ 2 and –Log 10 p -value ≥ 3 are represented in light pink or with a color code for group of genes associated with known functions (blue: csu pili; black: stress related genes; purple: trehalose biosynthesis; dark green: catalases). (B) xy-plot representing the differential expression of genes between the Δ relA /WT vs Δ relA/ Δ relA rpoB R557C in liquid complex medium. Each dot represents the log 2 FC value for the strains indicated under brackets (only genes with FDR-adjusted p ≤ 0.005 were kept). Acknowledgements We thank Charles Van der Henst for supplying A. baumannii WT strains and Paul Guiraud for help with the microscopy experiments. This work was supported by a Welbio Starting Grant (WELBIO-CR-2019S-05) to R.H. A.P. was a postdoctoral Research Fellow (CR) and R.H. is a senior Research Associate (MR) of the F.R.S. – FNRS. Funder Information Declared FNRS - Welbio Starting Grant , WELBIO-CR-2019S-05 Reference 1. ↵ Cashel M , Gallant J . Two compounds implicated in the function of the RC gene of Escherichia coli . Nature . 1969 Mar 1 ; 221 ( 5183 ): 838 – 41 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Potrykus K , Cashel M . (p)ppGpp: Still Magical? Annu Rev Microbiol . 2008 Oct ; 62 ( 1 ): 35 – 51 . OpenUrl CrossRef PubMed Web of Science 3. ↵ Atkinson GC , Tenson T , Hauryliuk V. The RelA/SpoT Homolog (RSH) Superfamily: Distribution and Functional Evolution of ppGpp Synthetases and Hydrolases across the Tree of Life . Stiller JW , editor. PLoS ONE . 2011 Aug 9 ; 6 ( 8 ): e23479 . OpenUrl CrossRef PubMed 4. ↵ Tamman H , Ernits K , Roghanian M , Ainelo A , Julius C , Perrier A , et al. Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint . Nat Chem Biol . 2023 Mar ; 19 ( 3 ): 334 – 45 . OpenUrl CrossRef PubMed 5. ↵ Ross W , Vrentas CE , Sanchez-Vazquez P , Gaal T , Gourse RL . The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation . Mol Cell . 2013 May 9 ; 50 ( 3 ): 420 – 9 . OpenUrl CrossRef PubMed Web of Science 6. Zuo Y , Wang Y , Steitz TA . The mechanism of E. coli RNA polymerase regulation by ppGpp is suggested by the structure of their complex . Mol Cell . 2013 May 9 ; 50 ( 3 ): 430 – 6 . OpenUrl CrossRef PubMed Web of Science 7. ↵ Mechold U , Potrykus K , Murphy H , Murakami KS , Cashel M . Differential regulation by ppGpp versus pppGpp in Escherichia coli . Nucleic Acids Res . 2013 Jul ; 41 ( 12 ): 6175 – 89 . OpenUrl CrossRef PubMed Web of Science 8. ↵ Ross W , Sanchez-Vazquez P , Chen AY , Lee JH , Burgos HL , Gourse RL . ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response . Mol Cell . 2016 Jun 16 ; 62 ( 6 ): 811 – 23 . OpenUrl CrossRef PubMed 9. ↵ Wang B , Dai P , Ding D , Del Rosario A , Grant RA , Pentelute BL , et al. Affinity-based capture and identification of protein effectors of the growth regulator ppGpp . Nat Chem Biol . 2019 Feb ; 15 ( 2 ): 141 – 50 . OpenUrl CrossRef PubMed 10. ↵ Hernandez VJ , Cashel M . Changes in conserved region 3 of Escherichia coli sigma 70 mediate ppGpp-dependent functions in vivo . J Mol Biol . 1995 Oct 6 ; 252 ( 5 ): 536 – 49 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Zhou YN , Jin DJ . The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like “stringent” RNA polymerases in Escherichia coli . Proc Natl Acad Sci U S A . 1998 Mar 17 ; 95 ( 6 ): 2908 – 13 . OpenUrl Abstract / FREE Full Text 12. ↵ WHO Bacterial Priority Pathogens List, 2024 : bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance . Geneva : World Health Organization ; 2024 . Licence: CC BY-NC-SA 3.0 IGO. 13. ↵ Monem S , Furmanek-Blaszk B , Łupkowska A , Kuczyńska-Wiśnik D , Stojowska-Swędrzyńska K , Laskowska E . Mechanisms Protecting Acinetobacter baumannii against Multiple Stresses Triggered by the Host Immune Response, Antibiotics and Outside-Host Environment . Int J Mol Sci . 2020 Jul 31 ; 21 ( 15 ): 5498 . OpenUrl CrossRef PubMed 14. ↵ Jawad A , Seifert H , Snelling AM , Heritage J , Hawkey PM . Survival of Acinetobacter baumannii on dry surfaces: comparison of outbreak and sporadic isolates . J Clin Microbiol . 1998 Jul ; 36 ( 7 ): 1938 – 41 . OpenUrl Abstract / FREE Full Text 15. ↵ Green ER , Fakhoury JN , Monteith AJ , Pi H , Giedroc DP , Skaar EP . Bacterial hydrophilins promote pathogen desiccation tolerance . Cell Host Microbe . 2022 Jul 13 ; 30 ( 7 ): 975 – 987 .e7. OpenUrl CrossRef PubMed 16. ↵ Maharjan RP , Sullivan GJ , Adams FG , Shah BS , Hawkey J , Delgado N , et al. DksA is a conserved master regulator of stress response in Acinetobacter baumannii . Nucleic Acids Res . 2023 Jul 7 ; 51 ( 12 ): 6101 – 19 . OpenUrl CrossRef PubMed 17. ↵ Pérez-Varela M , Tierney ARP , Kim JS , Vázquez-Torres A , Rather P . Characterization of RelA in Acinetobacter baumannii . J Bacteriol . 2020 May 27 ; 202 ( 12 ): e00045 – 20 . OpenUrl PubMed 18. ↵ Jung HW , Kim K , Islam MM , Lee JC , Shin M . Role of ppGpp-regulated efflux genes in Acinetobacter baumannii . J Antimicrob Chemother . 2020 May 1 ; 75 ( 5 ): 1130 – 4 . OpenUrl CrossRef PubMed 19. ↵ Kim K , Islam M , Jung HW , Lim D , Kim K , Lee SG , et al. ppGpp signaling plays a critical role in virulence of Acinetobacter baumannii . Virulence . 2021 Dec ; 12 ( 1 ): 2122 – 32 . OpenUrl CrossRef PubMed 20. ↵ Robins WP , Faruque SM , Mekalanos JJ . Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene . Proc Natl Acad Sci U S A . 2013 Feb 26 ; 110 ( 9 ): E848 – 857 . OpenUrl Abstract / FREE Full Text 21. ↵ Wijers CDM , Pham L , Menon S , Boyd KL , Noel HR , Skaar EP , et al. Identification of Two Variants of Acinetobacter baumannii Strain ATCC 17978 with Distinct Genotypes and Phenotypes . Infect Immun . 2021 Nov 16 ; 89 ( 12 ): e0045421 . OpenUrl CrossRef PubMed 22. ↵ Cortay JC , Cozzone AJ . Accumulation of guanosine tetraphosphate induced by polymixin and gramicidin in Escherichia coli . Biochim Biophys Acta . 1983 Feb 22 ; 755 ( 3 ): 467 – 73 . OpenUrl PubMed 23. ↵ Trinh V , Langelier MF , Archambault J , Coulombe B . Structural perspective on mutations affecting the function of multisubunit RNA polymerases . Microbiol Mol Biol Rev MMBR . 2006 Mar ; 70 ( 1 ): 12 – 36 . OpenUrl CrossRef PubMed 24. ↵ Rumbo-Feal S , Pérez A , Ramelot TA , Álvarez-Fraga L , Vallejo JA , Beceiro A , et al. Contribution of the A. baumannii A1S_0114 Gene to the Interaction with Eukaryotic Cells and Virulence . Front Cell Infect Microbiol . 2017 ; 7 : 108 . 25. ↵ Clemmer KM , Bonomo RA , Rather PN . Genetic analysis of surface motility in Acinetobacter baumannii . Microbiology . 2011 Sep ; 157 (Pt 9 ): 2534 – 44 . OpenUrl CrossRef PubMed Web of Science 26. ↵ Chin CY , Tipton KA , Farokhyfar M , Burd EM , Weiss DS , Rather PN . A high-frequency phenotypic switch links bacterial virulence and environmental survival in Acinetobacter baumannii . Nat Microbiol . 2018 May ; 3 ( 5 ): 563 – 9 . OpenUrl CrossRef PubMed 27. ↵ Ahmad S , Wang B , Walker MD , Tran HKR , Stogios PJ , Savchenko A , et al. An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp . Nature . 2019 Nov ; 575 ( 7784 ): 674 – 8 . OpenUrl CrossRef PubMed 28. Steinchen W , Ahmad S , Valentini M , Eilers K , Majkini M , Altegoer F , et al. Dual role of a (p)ppGpp- and (p)ppApp-degrading enzyme in biofilm formation and interbacterial antagonism . Mol Microbiol . 2021 Jun ; 115 ( 6 ): 1339 – 56 . OpenUrl CrossRef PubMed 29. ↵ Fung DK , Bai K , Yang J , Xu X , Stevenson DM , Amador-Noguez D , et al. Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation . mBio . 2022 Dec 20 ; 13 ( 6 ): e0242222 . OpenUrl CrossRef PubMed 30. ↵ Powers MJ , Simpson BW , Trent MS . The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport . eLife . 9 : e56571 . 31. ↵ Battesti A , Bouveret E . Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism . Mol Microbiol . 2006 Nov ; 62 ( 4 ): 1048 – 63 . OpenUrl CrossRef PubMed Web of Science 32. ↵ Sinha AK , Winther KS , Roghanian M , Gerdes K . Fatty acid starvation activates RelA by depleting lysine precursor pyruvate . Mol Microbiol . 2019 Oct ; 112 ( 4 ): 1339 – 49 . OpenUrl CrossRef PubMed 33. ↵ Vinella D , Albrecht C , Cashel M , D’Ari R . Iron limitation induces SpoT-dependent accumulation of ppGpp in Escherichia coli . Mol Microbiol . 2005 May ; 56 ( 4 ): 958 – 70 . OpenUrl CrossRef PubMed Web of Science 34. ↵ Godeux AS , Lupo A , Haenni M , Guette-Marquet S , Wilharm G , Laaberki MH , et al. Fluorescence-Based Detection of Natural Transformation in Drug-Resistant Acinetobacter baumannii . J Bacteriol . 2018 Oct 1 ; 200 ( 19 ): e00181 – 18 . OpenUrl PubMed 35. ↵ Ducret A , Quardokus EM , Brun YV . MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis . Nat Microbiol . 2016 Jun 20 ; 1 ( 7 ): 16077 . OpenUrl CrossRef PubMed 36. ↵ Schindelin J , Arganda-Carreras I , Frise E , Kaynig V , Longair M , Pietzsch T , et al. Fiji: an open-source platform for biological-image analysis . Nat Methods . 2012 Jul ; 9 ( 7 ): 676 – 82 . OpenUrl CrossRef PubMed Web of Science 37. ↵ Afgan E , Baker D , Batut B , van den Beek M , Bouvier D , Cech M , et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update . Nucleic Acids Res . 2018 Jul 2 ; 46 ( W1 ): W537 – 44 . OpenUrl CrossRef PubMed 38. ↵ Quintero-Yanes A , Mayard A , Hallez R . The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus . PLoS Genet . 2022 Dec ; 18 ( 12 ): e1010465 . OpenUrl CrossRef PubMed 39. ↵ Iwase T , Tajima A , Sugimoto S , Okuda K ichi , Hironaka I , Kamata Y , et al. A simple assay for measuring catalase activity: a visual approach . Sci Rep . 2013 Oct 30 ; 3 : 3081 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted July 06, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Functional interplay between (p)ppGpp and RNAP in Acinetobacter baumannii Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Functional interplay between (p)ppGpp and RNAP in Acinetobacter baumannii Anthony Perrier , Aurélie Budin-Verneuil , Régis Hallez bioRxiv 2025.07.04.663200; doi: https://doi.org/10.1101/2025.07.04.663200 Share This Article: Copy Citation Tools Functional interplay between (p)ppGpp and RNAP in Acinetobacter baumannii Anthony Perrier , Aurélie Budin-Verneuil , Régis Hallez bioRxiv 2025.07.04.663200; doi: https://doi.org/10.1101/2025.07.04.663200 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41910) Biophysics (21436) Cancer Biology (18576) Cell Biology (25480) Clinical Trials (138) Developmental Biology (13368) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15598) Genomics (22482) Immunology (17726) Microbiology (40360) Molecular Biology (17163) Neuroscience (88534) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)