A starvation-triggered AAA+ ATPase halts chromosome replication progression by disassembling the bacterial DNA sliding clamp

A starvation-triggered AAA+ ATPase halts chromosome replication progression by disassembling the bacterial DNA sliding clamp | 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 A starvation-triggered AAA+ ATPase halts chromosome replication progression by disassembling the bacterial DNA sliding clamp View ORCID Profile Surbhi , Arnab Kumar Shau , View ORCID Profile Feby Mariam Chacko , View ORCID Profile Sunish Kumar Radhakrishnan doi: https://doi.org/10.1101/2025.01.13.632716 Surbhi 1 Department of Biology, Indian Institute of Science Education and Research Pune , Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Surbhi Arnab Kumar Shau 1 Department of Biology, Indian Institute of Science Education and Research Pune , Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Feby Mariam Chacko 1 Department of Biology, Indian Institute of Science Education and Research Pune , Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Feby Mariam Chacko Sunish Kumar Radhakrishnan 1 Department of Biology, Indian Institute of Science Education and Research Pune , Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sunish Kumar Radhakrishnan For correspondence: sunish{at}iiserpune.ac.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Living cells should coordinate vital events such as DNA replication with the availability of nutrients. For example, when cells encounter starvation, to maintain genomic integrity they should harbour robust mechanisms to stop DNA replication. Mechanisms regulating the progression of DNA replication when bacterial cells encounter starvation remain largely unclear. Herein, we identify the role of IncA –a AAA+ ATPase homologous to the prokaryotic RarA and eukaryotic WRNIP1/Mgs1– in inhibiting the progression of chromosome replication in nutrient-starved stationary phase cells of Caulobacter crescentus . We show that the starvation-induced alarmone (p)ppGpp ensures the confinement of IncA production to the stationary phase cells. At the mechanistic level, IncA directly interacts with the β-sliding clamp protein DnaN and disassembles DnaN from the replisome, thereby stalling the progression of DNA replication. Furthermore, we reveal the requirement of IncA’s ATPase activity for disassembling DnaN. Remarkably, we demonstrate that the IncA homolog from E. coli is capable of inhibiting DNA replication in Caulobacter . We propose that IncA homologs serve a stress-dependent role in inhibiting DNA replication across diverse domains of life. Introduction Cells of prokaryotic and eukaryotic origin alike use various mechanisms to regulate vital cellular processes such as chromosome replication in response to nutrient availability. For example, bacterial cells encounter vast fluctuations in nutrient availability warranting the requirement for a robust mechanism to maintain genomic integrity and proliferation under nutrient-limiting conditions. In bacteria, depletion of the nutrient pool leads to the stringent response which regulates several cell cycle events and various developmental processes including DNA replication. The stringent response activates the RelA/SpoT homolog (Rsh) proteins that initiate the production of guanosine tetra- or penta-phosphate molecule, (p)ppGpp ( Bange et al , 2021 ). The production of (p)ppGpp influences transcription and translation causing an upregulation of stress-responsive factors and a downregulation of factors involved in growth and development during nutrient-replete conditions ( Hauryliuk et al , 2015 ; Irving et al , 2021 ; Travis & Schumacher, 2022 ; Voedts et al , 2024 ). One of the prominent processes that is affected by the increase in (p)ppGpp levels is chromosome replication. The bidirectional replication of the circular chromosome in bacteria is divided into initiation, elongation, and segregation events. The initiation event is triggered by the activity of the AAA+ ATPase DnaA ( Gorbatyuk & Marczynski, 2001 ; Sekimizu et al , 1987 ). Upon initiation, the replisome consisting of the helicase (DnaB), the single-stranded DNA-binding protein (SSB), and the DNA polymerase III holoenzyme is loaded onto the replication fork. The DNA polymerase III holoenzyme comprises the core polymerase enzyme and the β-sliding clamp ( Aakre et al , 2013 ; Lewis et al , 2016 ). The bacterial β-sliding clamp protein DnaN is important for tethering DNA polymerase III to the replicating DNA and modulating the activity of the polymerase ( Altieri & Kelman, 2018 ). Removal of DnaN leads to the collapse of the replication machinery, which instantaneously arrests DNA replication ( Maffeo et al , 2019 ). In the dimorphic model Caulobacter crescentus (henceforth Caulobacter ), the chromosome replication, and the segregation of the completely replicated chromosome happens only once per cell division cycle. The cell cycle in Caulobacter is tightly regulated and consists of a DNA replication-incompetent swarmer cell (G1), which is licensed to initiate chromosome replication only upon entry into the replicative stalked cell phase (S-phase). The primary purpose of the swarmer cell is to disperse off from a nutrient-deprived niche and colonize a new nutrient-rich environment. The swarmer to stalked cell transition (G1 to S) is proposed to be nutrient-dependent ( Barrows & Goley, 2023 ; Britos et al , 2011 ; Kirkpatrick & Viollier, 2012 ; Skerker & Laub, 2004 ). Interestingly, the abundance of (p)ppGpp varies during the cell cycle in Caulobacter . (p)ppGpp levels are higher in the swarmer cells and decrease upon entry into the replicative stalked cell phase ( Boutte et al , 2012 ; Gonzalez & Collier, 2014 ). In the swarmer cell, DNA replication is silenced by the overabundance of the master cell cycle regulator CtrA and low levels of ATP-bound DnaA - the DNA replication initiation-competent form of DnaA ( Bastedo & Marczynski, 2009 ; Gorbatyuk & Marczynski, 2005 ; Quon et al , 1998 ; Sanselicio et al , 2015 ). Upon transition of the swarmer cell into a stalked cell, CtrA is proteolyzed co-incident with the increase in abundance of ATP-bound DnaA which in turn leads to the initiation of chromosome replication ( Felletti et al , 2019 ; Quon et al ., 1998 ; Ryan et al , 2002 ; Taylor et al , 2011 ). Interestingly, the abundance of CtrA and DnaA in the swarmer cell is influenced by (p)ppGpp ( Lesley & Shapiro, 2008 ; Sanselicio et al ., 2015 ). A similar influence of (p)ppGpp on DnaA levels has been demonstrated in E. coli ( Chiaramello & Zyskind, 1990 ). However, an overproduction of DnaA is insufficient to trigger DNA replication at (p)ppGpp-abundant conditions ( Leslie et al , 2015 ; Riber & Lobner-Olesen, 2020 ; Sinha et al , 2020 ). Nevertheless, an expression of (p)ppGpp-blind RNA polymerase suffices to initiate replication in nutrient-starved (p)ppGpp-replete conditions ( Riber & Lobner-Olesen, 2020 ). Taken together, these observations allude to the presence of additional factors that are under the control of (p)ppGpp that influence DNA replication in nutrient-deprived conditions. Herein, we describe a (p)ppGpp-triggered mechanism of DNA replication elongation control in nutrient-starved stationary phase cells of Caulobacter . We identify a hitherto uncharacterized function for IncA, which shares homology with the functionally enigmatic AAA+ ATPases RarA (bacteria), Mgs1 (yeast), and WRNIP1 (human), all belonging to the clamp loader clade of proteins ( Carrasco et al , 2018 ; Hishida et al , 2002 ; Page et al , 2011 ; Yoshimura et al , 2017 ). We demonstrate that (p)ppGpp transcriptionally favors the production of incA specifically in the stationary phase cells. The protein IncA then directly binds to the β-sliding clamp DnaN. The binding of IncA to DnaN inhibits the assembly of DnaN. Furthermore, overexpression of IncA inhibits replication in exponentially growing wild-type cells of Caulobacter indicating that the high abundance of IncA is sufficient to inhibit replication. Finally, we show that the ATPase activity of IncA is required for the delocalization of DnaN but not for the binding of IncA to DnaN. Our results identify a conserved nutrient stress-dependent regulator of chromosome replication in bacteria. Results An overexpression screen identifies incA Several AAA+ ATPase domain harboring proteins have been shown to orchestrate essential cellular processes in all domains of life. Nevertheless, many remain functionally uncharacterized. Therefore, we performed an overexpression screen using AAA+ ATPase domain harboring proteins that are uncharacterized Caulobacter . Herein, we screened for those proteins that decreased viability upon overexpression in Caulobacter . Our overexpression screen led us to the identification of CCNA_01342, henceforth named incA ( in hibitor of c hromosome replication). Overexpression of incA from a vanillate inducible promoter on the high copy vector pBVMCS-4 (pP van - incA ) decreased the viability of wild-type ( WT ) Caulobacter cells ( Fig. 1C ). Moreover, a 3 h overproduction of incA from pP van - incA made the WT cells significantly filamentous ( Fig. 1A, B and Supplemental Fig. S1A-C). Together, these results suggested that IncA may be involved in regulating a crucial process during the growth of Caulobacter cells, and overproduction of IncA could interfere with this process leading to problems in cell cycle and proliferation. From the genome-wide transcriptome data, during the Caulobacter cell cycle, it was evident that incA transcripts are specifically abundant in the swarmer cells ( Schrader et al , 2016 ). This prompted us to investigate the regulatory mechanism that controlled the abundance of IncA in Caulobacter . Download figure Open in new tab Figure 1. IncA expression is (p)ppGpp-dependent and overexpression reduces cell viability. (A) Phase contrast micrographs of wild-type ( WT ) Caulobacter cells harbouring the high copy vector (pBVMCS-4) or ectopically expressing incA from the vanillate-inducible promoter (P van ) on pBVMCS-4. Cells were induced with 0.5mM vanillate for 5 hours. (B) Cell size distribution of cells from (A). Mean cell size of at least 150 cells per biological replicate were used for statistical analyses. (C) Growth of cells shown in (A). Ten-fold dilutions of the indicated strains were spotted on to the growth medium with or without the inducer vanillate (0.5 mM). Immunoblots showing the protein levels of IncA and MreB (loading control) in (D) WT and spoT null mutant (Δ spoT ) during exponential (exp) and stationary (sta) phase and (E) exponential phase cells expressing RelA’ or RelA*. Both RelA’ and RelA* were expressed from the xylose-inducible promoter (P xyl ) at the chromosomal xylX locus ( xylX ::P xyl ). β-galactosidase (lacZ) activity of the incA promoter (P incA ) fused to the lacZ reporter (P incA -lacZ ) in (F) WT and ΔspoT cells at exponential and stationary phase and (G) WT cells expressing relA ’ and relA *. The error bars in B, F and G represent the mean ± SD from at least three independent biological replicates. Statistical analyses were done using unpaired two-tailed t test; ****p < 0.0001, ***p < 0.001; ns, not significant. Scale bar: 2μm. The stationary phase-specific production of IncA is (p)ppGpp dependent Interestingly, a study to understand the transcriptional regulatory pattern during stringent response in Caulobacter had shown that incA transcript levels were up to fourfold less abundant in a spoT null (Δ spoT ) mutant ( Boutte & Crosson, 2011 ). The RelA/SpoT homologs (Rsh) are involved in the production of (p)ppGpp during nutrient stress or at the stationary phase ( Bange et al ., 2021 ). Caulobacter harbors only a single Rsh, spoT ( Boutte & Crosson, 2011 ). The Δ spoT cells of Caulobacter are incapable of making (p)ppGpp at the stationary phase ( Ronneau & Hallez, 2019 ). Therefore, we wondered if the production of incA happens at the stationary phase when nutrients are scarce and (p)ppGpp levels are high. To test this, we used polyclonal antibodies generated against IncA to compare the abundance of IncA in exponential and stationary phase cells. Immunoblot analyses indicated that IncA protein levels are indeed more abundant in the stationary phase cells ( Fig. 1D ). There was almost a two-fold higher amount of IncA at the stationary phase than in the exponential phase cells (Supplemental Fig. S1D). During the cell cycle in Caulobacter , (p)ppGpp levels are also found to be abundant in swarmer cells ( Boutte et al ., 2012 ; Lesley & Shapiro, 2008 ; Stott et al , 2015 ). This, together with the observation that incA transcript levels were abundant in the swarmer cells ( Schrader et al ., 2016 ), prompted us to wonder if the (p)ppGpp-dependent regulation of incA production happens at the level of transcription. To test this, we used a β-galactosidase ( lacZ) -based reporter fusion wherein the promoter of incA was fused to the promoter-less lacZ reporter gene (P incA - lacZ ). We tested the activity of P incA - lacZ in WT cells at exponential and stationary phase conditions. The P incA - lacZ activity was found to be significantly upregulated in the stationary phase cells ( Fig. 1F ). Moreover, the stationary phase-specific upregulation of the P incA - lacZ activity and the IncA protein abundance were absent in the Δ spoT cells ( Fig. 1D, F and Supplemental Fig. S1D). Next, we wondered if an artificial increase in (p)ppGpp levels could increase the production of incA in WT cells even during the nutrient-replete exponential phase conditions. To test this, we used a mutant form of E. coli RelA (RelA’) that lacks the C-terminal regulatory domain of RelA ( Gropp et al , 2001 ). The absence of the regulatory domain makes RelA’ constitutively active, inducing the production of (p)ppGpp even at normal conditions ( Bange et al ., 2021 ; Gonzalez & Collier, 2014 ; Gropp et al ., 2001 ). Furthermore, expression of RelA’ in WT Caulobacter from the xylose inducible promoter on the chromosome ( xylX:: P xyl - relA’ ) has been shown to increase the abundance of (p)ppGpp ( Gonzalez & Collier, 2014 ). Immunoblot and P incA - lacZ analyses indicated that the IncA protein levels and the P incA - lacZ activity were indeed increased in cells producing relA’ from xylX ::P xyl - relA’ ( Fig.1E, G and Supplemental Fig. S1E). It has been demonstrated that RelA’ becomes catalytically inactive if the E335 residue at its enzymatically active center is mutated (E335Q) ( Gonzalez & Collier, 2014 ; Harinarayanan et al , 2008 ). There was no difference in the IncA levels or P incA - lacZ activity when relA’ harbouring the E335Q mutation ( relA* ) was expressed from the xylX locus ( xylX:: P xyl - relA* ) ( Fig.1E, G and Supplemental Fig. S1E) suggesting that it is the (p)ppGpp production by RelA’ that influenced the synthesis of incA . Taken together, our results indicated that IncA production is significantly increased in the stationary phase cells, and that the stationary phase-specific increase of incA production was through the SpoT-dependent production of (p)ppGpp. IncA interacts with the DNA sliding clamp To understand the mechanism by which IncA overproduction induces cell filamentation and decreases the viability of WT Caulobacter cells, we decided to identify the interacting partners of IncA. Towards this, we carried out co-purification experiments using a C-terminal tandem affinity purification (TAP)-tagged IncA (IncA-TAP) expressed from the vanillate-inducible promoter on the pBVMCS-4 (pP van - incA - TAP ) in Δ incA cells. The co-purified samples were then analyzed using mass spectrometry. The relative abundance of co-purified proteins was quantified against affinity-purified samples from the Δ incA cells harboring the empty TAP vector (see methods). From our analyses we found that the replisome component proteins such as the β-sliding clamp DnaN, the nucleoid-associated protein HU, and the single-stranded DNA binding protein SSB co-purified with notable abundance in the IncA-TAP samples ( Fig. 2A , Supplemental Dataset 1). Among them, DnaN co-purified with at least two-fold abundance ( Fig. 2A , Supplemental Dataset 1). Download figure Open in new tab Figure 2. IncA interacts with and delocalizes the DNA sliding clamp DnaN. (A) Quantitative proteomics of tandem affinity purified (TAP) samples from incA -null mutants expressing the TAP-tag alone or IncA-TAP shown as volcano plots denoting the abundant proteins in the IncA-TAP samples. The threshold was set for log 2 (fold change) and -log10 (p-value) (see Supplemental Dataset 1). (B) Bacterial two-hybrid (BACTH) analysis between IncA fused to T25 fragment of adenylate cyclase at N-terminus (T25-IncA) and replisome components DnaN or HolB or SSB fused to T18 fragment of adenylate cyclase at C-terminus: DnaN-T18; HolB-T18; SSB-T18, respectively. The E. coli BTH101 cells containing the pair-wise combination of the plasmid constructs were used for the BACTH assay. Plasmids containing leucine zipper motifs of GCN4(pKT24-zip and pUT18C-zip) were used as a positive control. The interaction is denoted by the appearance of blue colour on plates containing X-gal. (C) Quantification of interactions in (B) represented as β-galactosidase (LacZ) activity. (D) Flow cytometry profiles showing DNA content in wild-type ( WT ) cells harbouring the empty vector pBVMCS-4 ( ev ) or overexpressing incA from P van on pBVMCS-4 (pP van -incA ). Cells were treated with 0.5mM vanillate for three hours to induce the expression of incA . (E) Micrographs denoting the co-localization of DnaN-mCherry and IncA-seGFP. Fluorescence profiles for DnaN-mCherry and IncA-seGFP are plotted for two representative cells. DnaN-mCherry was expressed from the native dnaN locus ( dnaN::dnaN-mCherry ) and IncA-seGFP was expressed from P van on PBVMCS-4 (pP van - incA - seGFP ). (F) Phase contrast and fluorescence micrographs denoting the localization of DnaN-YFP in WT cells harbouring the vector pBVMCS-4 ( ev ) or overexpressing incA from the vanillate-inducible promoter on pBVMCS-4 (pP van -incA ). DnaN-YFP was expressed from the native dnaN locus ( dnaN :: dnaN - yfp ). (G) Quantification of localized and delocalized DnaN-YFP in cells from (F) using at least one hundred cells from each biological replicate. The error bars in (C) and (G) represent mean ± SD from at least three independent biological replicates. Statistical analyses were done using a two-way ANOVA with Holm-Sidak’s multiple comparisons test in (C) and unpaired two-tailed t test in (G); ****p < 0.0001, ***p < 0.001; ns, not significant. Scale bar: 2μm. Furthermore, to confirm if IncA interacts directly with any of the replisome components, we resorted to bacterial-two-hybrid assays using the bacterial adenylate cyclase-based two-hybrid (BACTH) assay system in E. coli ( Karimova et al , 1998 ). The BACTH system relies on the assisted functional reconstitution of the two fragments, T18 and T25, of the adenylate cyclase from Bordetella pertussis to trigger cAMP-dependent LacZ expression ( Karimova et al ., 1998 ). The BACTH assay indicated that IncA directly interacts with DnaN ( Fig. 2B, C ) and not with SSB or the DNA polymerase III subunit HolB ( Fig. 2B, C ). Furthermore, localization experiments using cells producing DnaN-mCherry from the native dnaN locus ( dnaN :: dnaN-mCherry ) and IncA-seGFP from the vanillate-inducible promoter on pBVMCS-4 (pP van - incA-seGFP ) indicated that IncA-seGFP co-localized with DnaN-mCherry in 46% of cells with proper DnaN-mCherry foci ( Fig. 2E and Supplemental Fig. S2F). The above experiments established the interaction of IncA with DnaN and suggested that the IncA overexpression phenotype may be because of the effect of IncA on DnaN. IncA inhibits chromosome replication through DnaN The β-sliding clamp DnaN is an essential component of the replisome and is required for tethering and increasing the processivity of the DNA polymerase III complex while sliding along the DNA to ensure efficient DNA synthesis ( Johnson & O’Donnell, 2005 ; Yao & O’Donnell, 2021 ). Inhibition of DnaN leads to replication collapse in actively growing cells ( Maffeo et al ., 2019 ). From the BACTH experiments, it was evident that IncA directly binds to DnaN ( Fig. 2B ). Therefore, we wondered if the toxicity in the IncA overexpressing cells is due to the inhibition of DnaN activity by IncA leading to a DNA replication arrest. To test if DNA replication is inhibited in cells overproducing IncA, we resorted to flow cytometry analyses to monitor the replication status. Towards this, we used cells treated with rifampicin. The antibiotic rifampicin is known to specifically inhibit replication initiation while allowing the completion of replication elongation and segregation of replicated DNA. If DNA replication elongation is inhibited by IncA, then proper completion of replication and the segregation of the replicated DNA will be hindered in IncA overexpressing cells. From the flow cytometry analyses properly replicated 1N and 2N chromosome-harboring cells were observed in the rifampicin-treated WT harboring the empty vector ( Fig. 2D ). Strikingly, no such proper 1N and 2N chromosome-harboring cells were observed from the flow cytometry analyses of rifampicin-treated WT cells overexpressing incA from pP van - incA . This result suggested that DNA replication and segregation were not completed when IncA was overproduced ( Fig. 2D ). To further test if the chromosome replication has been initiated in the IncA overexpressing cells, we decided to monitor the localization of GFP-ParB. The GFP-tagged version of the chromosome partitioning protein ParB (GFP-ParB) is known to bind to the origin-proximal region and serves as a proxy to monitor replication initiation ( Narayanan et al , 2018 ). Due to the duplication of the origin-proximal region upon replication initiation, cells in which chromosome replication has been initiated will display two GFP-ParB foci. We overexpressed IncA in WT cells expressing GFP-ParB from the native parB locus ( parB::gfp-parB ). Two GFP-ParB foci were observed in 64% of cells overexpressing IncA (Supplemental Fig. S2A) indicating that chromosome replication has been initiated in the IncA overexpressing cells. Inhibition of chromosome replication progression induces SOS response ( Maslowska et al , 2019 ). Therefore, we wondered if SOS response is induced in IncA overexpressing cells. To test this, we decided to monitor the activation of the promoter of sidA , the SOS-specific inhibitor of cell division in Caulobacter , which is known to be activated only upon the induction of SOS response ( Modell et al , 2011 ). We overexpressed IncA in WT cells expressing YFP from the sidA promoter (P sidA ) at the chromosomal xylose ( xyl ) locus in Caulobacter ( xylX:: P sidA -yfp ). The production of YFP from P sidA was increased in cells overexpressing IncA indicating the induction of SOS response in these cells (Supplemental Fig. S2C). Together, these results suggested IncA directly interacts with DnaN and that the overabundance of IncA inhibited progression of DNA replication. Next, we wondered about influence of IncA on DnaN. Towards this, we overexpressed IncA in cells harboring a functional DnaN-YFP produced from the native dnaN locus ( dnaN::dnaN-yfp ). Fluorescence micrographs indicated the presence of intact DnaN-YFP foci in cells harboring the empty vector ( Fig. 2F, G ). Strikingly, upon overexpression of IncA, DnaN-YFP was delocalized and did not form prominent foci ( Fig. 2F, G , Supplemental Movie 1 and 2). Overexpression of IncA led to the delocalization of DnaN-YFP in about 60% of cells ( Fig. 2G ). Immunoblot analyses indicated that IncA overexpression did not affect the DnaN-YFP protein levels (Supplemental Fig. S2D). Furthermore, immunoblot analyses of co-purified samples from TAP-tagged IncA in DnaN-YFP expressing cells confirmed the in vivo interaction of IncA with DnaN-YFP (Supplemental Fig. S2E). Finally, overexpression of IncA also dislodged the delta prime subunit of the DNA polymerase III (HolB) further confirming that the delocalization of DnaN by IncA disrupted the replisome (Supplemental Fig. S2B). Expression analyses of incA indicated that IncA is profoundly produced in the stationary phase cells ( Fig. 1D, F ). This prompted us to wonder if the role of IncA is to inhibit replication in the stationary phase cells by inhibiting DnaN. To understand this, we localized and quantified DnaN-YFP foci in exponential and stationary phase cells of the WT and the incA null mutant (Δ incA ). Proper DnaN-YFP foci were comparable in WT and Δ incA cells at the exponential phase ( Fig. 3A, C ). However, at the stationary phase, DnaN-YFP was prominently delocalized in ∼60% of WT cells while a majority of Δ incA cells had intact DnaN-YFP foci ( Fig. 3A, D ). To test if this effect is specifically due to the absence of IncA, we ectopically expressed incA from its native promoter on a low copy vector (pP incA - incA ). Ectopic expression of incA in Δ incA cells destabilized DnaN-YFP at the stationary phase ( Fig. 3B, E ) confirming that it is the absence of IncA that allowed intact DnaN-YFP foci in stationary phase cells of Δ incA . Deletion of incA did not affect the DnaN-YFP protein levels (Supplemental Fig. S3B, C). Download figure Open in new tab Figure 3. IncA delocalizes DnaN in stationary phase cells. (A) Phase contrast and fluorescence micrographs of (A) stationary and exponential phase WT and ΔincA cells expressing DnaN-YFP from the native dnaN locus ( dnaN :: dnaN - yfp ) and (B) stationary phase ΔincA dnaN :: dnaN - yfp cells expressing incA from the incA promoter on the low copy vector plac290 . Exponential phase cells were collected at OD 600 of 0.6 and stationary phase cells were collected six hours after the exponential phase. (C-E) Quantification (%) of cells from (A) and (B) having localized or delocalized DnaN-YFP. The error bars represent mean ± SD of at least one hundred cells from each of three independent biological replicates. Statistical analyses were done using unpaired two-tailed t test; ***p < 0.001, **p < 0.01; ns, not significant. Scale bar: 2μm. The production of IncA is under the control of SpoT through (p)ppGpp ( Fig. 1D-G ). Therefore, we speculated that Δ spoT cells should have more intact DnaN-YFP foci due to their inability to produce IncA at the stationary phase. Our analyses indeed indicated the presence of intact DnaN-YFP signals at the stationary phase in Δ spoT cells (Supplemental Fig. S3A). Nevertheless, the levels of DnaN-YFP were found to be reduced in Δ spoT cells at the stationary phase (Supplemental Fig. S3B), which could be attributed to the pleiotropic effect that cells may encounter in the absence of (p)ppGpp ( Boutte & Crosson, 2011 ; Boutte et al ., 2012 ). Taken together, our analyses bolstered the idea that IncA directly interacts with DnaN. This interaction delocalizes DnaN specifically in the stationary phase cells that have an increased abundance of IncA. The ATPase activity of IncA is required to delocalize DnaN Moving forward, we set out to understand the role of the ATPase domain in IncA towards its activity on DnaN. The aspartate residue in the Walker B motif is a part of the active site in the ATPase domain. The conserved aspartate coordinates with the Mg 2+ to neutralize the negative charge of phosphate present in ATP. Mutations in the conserved aspartate residue eliminate the ATPase activity while maintaining ATP binding ( Puchades et al , 2020 ). Therefore, we mutated the corresponding aspartate residue at the 115 th (D115) position on IncA to alanine (IncA D115A ) (Supplemental Fig. S4A). Overexpression of incA D115A from the vanillate inducible promoter on pBVMCS-4 (pP van - incA D115A ) in Δ incA cells induced mild filamentation ( Fig. 4A, C , Supplemental Fig. S4B) but did not decrease the viability ( Fig. 4D ). Moreover, overexpression of IncA D115A did not delocalize DnaN-YFP ( Fig. 4A, B , Supplemental Fig. S4C) or inhibit the chromosome replication ( Fig. 4E ). It is possible that the ATPase activity is required to dislodge DnaN and does not have a role in determining its interaction with DnaN, which we tested using BACTH. Interestingly, our BACTH analyses indicated that IncA D115A could still interact with DnaN ( Fig. 4F, G ). Together, these experiments suggested that the ATPase mutant of IncA could interact with DnaN but cannot delocalize DnaN-YFP or inhibit replication, indicating that the ATPase activity is required for delocalizing DnaN from the replisome. Download figure Open in new tab Figure 4. The ATPase activity of IncA is required to inhibit DNA replication. (A) Phase contrast and fluorescence micrographs representing DnaN-YFP in ΔincA cells overexpressing wild-type IncA ( incA WT ) or Walker-B mutant of IncA ( incA D115A ) from a vanillate-induble promoter on pBVMCS-4 (pP van ). (B) Quantification of cells from (A) denoting the percentage of cells displaying localized and delocalized DnaN-YFP. At least one hundred cells from each of three independent biological replicates were used for quantification. (C) Cell size distribution, (D) growth and (E) Flow cytometry profiles of ΔincA cells harbouring the empty vector pBVMCS-4 ( ev ) or overexpressing incA WT or incA D115A from P van on pBVMCS-4 (pP van ). For growth assay, ten-fold dilutions of the indicated strains were spotted onto a growth medium with or without the inducer vanillate (0.5 mM). (F) Bacterial two-hybrid (BACTH) analysis between DnaN fused to the T18 fragment of adenylate cyclase at the C-terminus (DnaN-T18) and IncA WT or IncA D115A fused to T25 fragment of adenylate cyclase at the N-terminus: T25-IncA WT , T25-IncA D115A , respectively. The E. coli BTH101 cells containing the pair-wise combination of plasmid constructs were used for the BACTH assay. Plasmids containing leucine zipper motifs of GCN4(pKT24-zip and pUT18C-zip) were used as a positive control. The interaction is denoted by the appearance of blue colour on plates containing X-gal. (G) Quantification of interactions in (F) represented as β-galactosidase (LacZ) activity. The error bars represent mean ± SD from at least three independent biological replicates. Statistical analyses in (B), (C) and (G) were done using unpaired two-tailed t test; ****p < 0.0001, ***p < 0.001, **p < 0.01; ns, not significant. Scale bar: 2μm. The E. coli homolog of IncA inhibits DNA replication in Caulobacter IncA from Caulobacter shares 48% identity with RarA from E. coli . Furthermore, AlphaFold and ChimeraX-based structural alignment of monomeric IncA and RarA indicated that IncA shares a significant structural homology with RarA ( Fig. 5F ). Therefore, we wondered if RarA from E . coli has a similar function as IncA in Caulobacter . To understand this, we overexpressed E. coli RarA in Δ incA cells of Caulobacter from the vanillate inducible promoter on pBVMCS-4 (pP van - rarA ). Interestingly, overexpression of RarA induced severe filamentation ( Fig.5 A, C ) and viability defects ( Fig. 5D ) in Caulobacter . Furthermore, the overexpression of RarA partially delocalized DnaN-YFP ( Fig. 5A, B , Supplemental Fig. S5C). Flow cytometry analyses of rifampicin-treated RarA overexpressing Caulobacter cells indicated the inhibition of chromosome replication in these cells ( Fig. 5E ). The observation of DNA replication inhibition was further strengthened by the induction of the SOS response in RarA overexpressing Caulobacter cells (Supplemental Fig. S2C). Taken together, our results suggested that the IncA homolog from E. coli , RarA, harbors an IncA-like function in Caulobacter . Download figure Open in new tab Figure 5. RarA inhibits DNA replication in Caulobacter . (A) Phase contrast and fluorescence micrographs representing DnaN-YFP in ΔincA cells harbouring the vector pBVMCS-4 ( ev ) or overexpressing Caulobacter incA or E. coli rarA from the vanillate-inducible promoter on pBVMCS-4 (pP van ). (B) Quantification of cells from (A) denoting the percentage of cells displaying localized and delocalized DnaN-YFP. At least one hundred cells from each biological replicate was used for quantification. (C) Cell size distribution, (D) growth and (E) flow cytometry profiles of ΔincA cells harbouring the empty vector pBVMCS-4 ( ev ) or overexpressing incA or rarA. For growth assay, ten-fold dilutions of the indicated strains were spotted onto a growth medium with or without the inducer vanillate (0.5 mM). (F) Structural comparison of the IncA monomer (Purple) and RarA monomer (Pink). The monomeric structures were generated using Alphafold and docked using UCSF ChimeraX. The error bars in (B) and (C) represent mean ± S.D from at least three independent biological replicates. Statistical analyses were done using unpaired two-tailed t test; ****p < 0.0001, ***p < 0.001; ns, not significant. Scale bar: 2μm. Discussion Free-living cells often encounter nutrient-dependent stress that significantly impacts their survival and adaptability. When essential nutrients become scarce, cells modulate their metabolic processes, often entering a dormant state until nutrients become replete. In bacteria, the starvation response is escalated with the production of the alarmone (p)ppGpp, which affects several cell cycle and developmental processes including chromosome replication ( Bange et al ., 2021 ; Hauryliuk et al ., 2015 ; Ronneau & Hallez, 2019 ). Nutrient-poor conditions could cripple nucleotide availability which could in turn cause DNA lesions. Therefore, to prevent damage to the genetic material, it becomes imperative for cells to stop replication at starvation. The mechanisms that bacterial cells utilize to regulate DNA replication at nutrient-starved conditions are not well understood. Herein, we demonstrate the role of IncA, a highly conserved AAA+ ATPase homolog belonging to the clamp loader family, in inhibiting DNA replication at nutrient-starved stationary phase ( Fig. 6 ). Download figure Open in new tab Figure 6. Mechanism of IncA-dependent inhibition of DNA replication. At stationary phase, nutrient starvation enhances the activity of SpoT causing an increase in the abundance of (p)ppGpp. The increased (p)ppGpp levels license the activation of the incA promoter (P incA ) leading to an accumulation of the IncA protein, which then directly binds to, and delocalizes, the DNA sliding clamp causing replication arrest. At exponential phase, P incA remains less active due to the absence of (p)ppGpp. The stationary phase-specific transcriptional regulation of incA by (p)ppGpp The transcription of IncA is tightly linked to the levels of (p)ppGpp whose production is specifically enhanced during starvation. An increase in the (p)ppGpp level suffices to activate the promoter of incA leading to IncA production ( Fig. 1 ), suggesting the existence of a direct (p)ppGpp-dependent regulatory module controlling the production of incA . This module could work either by directly regulating the RNA polymerase activity or through the regulation of the activity of a transcriptional factor as has been shown in E. coli and Caulobacter ( Bange et al ., 2021 ; Boutte & Crosson, 2011 ; Irving et al ., 2021 ; Voedts et al ., 2024 ). By coupling incA production to (p)ppGpp, Caulobacter cells restrict the increased abundance of IncA to nutrient-poor conditions. Thereby, cells ensure that the inhibitory effects of IncA are confined only to starvation-induced stress. Such a type of control could check unwanted replication-induced damage in nutrient-poor conditions wherein carbon and nitrogen sources may become limited leading to a decrease in nucleotide availability ( Wang et al , 2020 ). The IncA-dependent effect on chromosome replication may also be prevalent in the swarmer cells. Swarmer cells of Caulobacter are replication-incompetent and stay in a G1-like state until they encounter nutrient-favorable conditions ( Barrows & Goley, 2023 ). The inhibition of replication initiation in the swarmer cells is primarily mediated through an over-abundance of CtrA, which binds to the chromosomal origin to inhibit replication initiation ( Quon et al ., 1998 ). Interestingly, (p)ppGpp levels are enhanced in the replication incompetent swarmer cells of Caulobacter ( Boutte et al ., 2012 ; Gonzalez & Collier, 2014 ). Furthermore, from the cell cycle RNA abundance data, it is evident that incA levels are enriched in the swarmer cells ( Schrader et al ., 2016 ). Therefore, it is likely that Caulobacter cells might utilize incA as an additional control to ensure inhibition of replication in swarmer cells until favorable nutrient conditions are encountered leading to the initiation of S-phase. A direct (p)ppGpp-dependent control of replication elongation has been shown in Bacillus subtilis . In Bacillus , under nutrient starvation, (p)ppGpp directly binds to the primase (DnaG) resulting in the inhibition of its primer extension activity that leads to stalling of replication elongation ( Giramma et al , 2021 ; Wang et al , 2007 ). Nevertheless, similar starvation-dependent replication elongation control has not been reported in Gram-negative bacteria. Replisome disassembly by a clamp loader clade protein The process of DNA replication is carried out by a multiprotein complex, the replisome. The homodimeric β-sliding clamp DnaN plays a crucial role in replication progression by tethering the DNA polymerase complex and sliding along the replicating DNA ( Simonsen et al , 2024 ). Removal of DnaN could lead to destabilization of the polymerase complex and collapse of replication ( Maffeo et al ., 2019 ; Simonsen et al ., 2024 ). IncA directly binds to, and delocalizes, DnaN thereby arresting DNA replication progression ( Fig. 2 ). The presence of cells harboring partially replicated chromosomes upon IncA overexpression ( Fig. 2E ), supports this possibility. A mutation in the aspartate residue of the Walker B motif, that could affect the ATPase activity of IncA (IncA D115A ), renders IncA incapable of inhibiting DNA replication ( Fig. 4A-D ). Nevertheless, the IncA D115A mutant could bind to DnaN ( Fig. 4E-F ). Therefore, it is conceivable that the ATPase activity in IncA is required to dislodge DnaN from the active replisome after the binding of IncA to DnaN. We speculate that the ATPase activity could be required for IncA to slide along with DnaN or for its disassembly. However, the molecular mechanism that IncA utilizes to delocalize DnaN remains to be resolved. IncA shares homology with AAA+ ATPases like RarA (Replication associated recombination A) from E. coli ( Page et al ., 2011 ) ( Fig. 5F ), Mgs1 (Maintenance of genomic stability 1) from S. cerevisiae ( Hishida et al ., 2002 ) (Supplemental Fig. S5A) and WRNIP1 (Werner helicase-interacting protein 1) from humans ( Yoshimura et al ., 2017 ) (Supplemental Fig. S5B). These AAA+ ATPases belong to the clamp-loader clade of proteins ( Ammelburg et al , 2006 ; Barre et al , 2001 ; Erzberger & Berger, 2006 ; Iyer et al , 2004 ). In E . coli RarA interacts with SSB and is speculated to be involved in resolving stalled replication forks ( Page et al ., 2011 ). Nevertheless, it has been suggested that RarA could have a loading or unloading function on ring proteins such as the sliding clamp ( Sherratt et al , 2004 ). Interestingly, overexpression of RarA from E. coli in Caulobacter induces replication arrest and decreased viability ( Fig. 5 ). Furthermore, IncA and RarA overexpression upregulates the SOS response in Caulobacter , which is a hallmark of replication collapse ( Janion, 2008 ; Maslowska et al ., 2019 ) (Supplemental Fig. S2C). These observations suggest that RarA possesses a DNA replication-inhibitory activity in Caulobacter . It has not escaped our attention that Mgs1 from budding yeast is structurally similar to the replication factor C (RFC), a eukaryotic clamp loader ( Hishida et al , 2006 ). Furthermore, during DNA damage stress, the ubiquitination of the eukaryotic sliding clamp PCNA facilitates the binding of Mgs1 to PCNA ( Saugar et al , 2012 ). The binding of Mgs1 to the ubiquitinated PCNA disrupts the interaction of PCNA with polymerase δ, leading to the disassembly of the replisome ( Branzei et al , 2002 ; Saugar et al ., 2012 ). In light of this, we speculate that Mgs1 might harbour an IncA-like effect on PCNA and may have a role in directly disassembling PCNA during stress, which warrants investigation. Author contributions Surbhi and SKR conceptualised and designed the study. Surbhi, AKS and FMC performed experiments and collected data. Surbhi, AKS and SKR analysed data. SKR procured funds. Surbhi and SKR wrote the manuscript. Materials and Methods Bacterial strains and growth media Caulobacter crescentus NA1000 ( Evinger & Agabian, 1977 ) and derivatives were grown on rich peptone yeast extract (PYE) media containing 0.2% peptone, 0.1% yeast extract, 1 mM MgSO 4 , 0.5 mM CaCl 2 and incubated at 29 °C, unless specifically mentioned. To induce expression from the P van promoter, media was supplemented with 0.5 mM vanillate for 3-5 h. The E. coli strains were grown in lysogeny broth (LB) media or minimal-A media containing 10.5 g l -1 K 2 HPO 4 , 4.5 g l -1 KH 2 PO 4 , 1 g l -1 (NH 4 ) 2 SO 4 , 0.5 g l -1 sodium citrate dihydrate, 0.2% Glucose, 1 mM MgSO 4 , 0.0001% thiamine ( Miller, 1992 ) at 37 °C unless specifically mentioned. Antibiotics were used at the following concentrations: kanamycin 20 μg ml -1 in solid medium and 5 μg ml -1 in liquid (50 μg ml -1 for E. coli ), gentamicin 2.5 μg ml -1 in solid medium and 1 μg ml -1 in liquid (25 μg ml -1 for E. coli ), tetracycline 1 μg ml -1 (10 μg ml -1 for E. coli ), spectinomycin 30 μg ml -1 in solid medium and 25 μg ml -1 in liquid, ampicillin 100 μg ml -1 (for E.coli ). Plasmids were introduced into C. crescentus by electroporation. Strains and plasmids used in this study are listed in Supplemental Tables S1-S3. Viability assay Overnight cultures of NA1000 containing vectors expressing IncA, IncA D115A or RarA from the vanillate inducible promoter (P van ) on the vector pBVMCS-4 ( Thanbichler et al , 2007 ) were normalized to OD 600 of 0.1 before being serially diluted. Aliquots (3 µl) of the dilutions were spotted onto PYE agar containing gentamycin (2.5 μg ml -1 ), with or without 0.5 mM vanillate. Plates were incubated at 30 °C for 2-3 days and imaged using Amersham ImageQuant 800 imager (Cytiva, USA).. IncA protein purification and antibody production To produce antibodies against IncA, the IncA protein with a N-terminal hexahistidine tag (His 6 -IncA) was expressed in E. coli BL21(DE3) cells. Cells were then harvested by centrifugation, and the recombinant protein was purified using Ni-NTA (nickel-nitrilotriacetic acid) resin (Qiagen). 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) was used for overexpression of His 6 -IncA.. The purified protein was then used to immunize New Zealand white rabbits (Bioklone Biotech, Chennai, India). Microscopy Phase contrast and fluorescence microscopy were performed using an Olympus IX83 inverted microscope (Evident Scientific, Japan) equipped with a U Plan X Extended Apo 100X (1.45 numerical aperture) objective and an ORCA-Flash4.0 V3 sCMOS camera (Hamamatsu, Japan). Cells were placed on a 1% agarose pad, or 1% agarose pad supplemented with PYE, for imaging. The images were processed and analysed using the Fiji image analysis platform ( Schindelin et al , 2012 ). Immunoblot analyses For immunoblot analyses Caulobacter cells were pelleted and resuspended in 1X SDS buffer (Tris-Cl (62.5 mM, pH 6.8), SDS (2%), Glycerol (10%), Bromophenol blue (0.004%), β-Mercaptoethanol (2.5%)). The proteins were resolved on Sodium Dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and blotted on to polyvinylidene difluoride (PVDF) Immobilon-P membranes (Merck Millipore). The membranes were then blocked in with TBST solution containing 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.2% Tween-20 and 5% non-fat dry milk followed by incubation with primary antibodies, anti-IncA (1:10,000), anti-MreB (1:30,000) ( Figge et al , 2004 ), anti-GFP (1:10,000) (Living Colors JL-8, Clontech Laboratories, CA, USA). The blots were then washed with TBS solution (20 mM Tris-HCl [pH 7.5], 150 mM NaCl), and detected with donkey anti-rabbit or donkey anti-mouse secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch, USA). The blots were visualized using Amersham ImageQuant 800 imager (Cytiva, USA) after treating with clarity western ECL substrate (Bio-Rad Laboratories, USA). Bacterial adenylate cyclase two-hybrid (BACTH) Assay BACTH system (Euromedex, France) was used to study the protein-protein interactions. The plasmids were constructed and co-transformed in E. coli BTH101 Δ cya competent cells as per manufacturer’s instructions. This system takes advantage of the catalytic domain of adenylate cyclase (CyaA) from Bordetella pertussis , which consists of two inactive fragments, T25 and T18, when separated. The heterodimerization of hybrid-proteins leads to the reassembly of the functional enzyme, enabling cAMP synthesis leading to lacZ gene expression. The proteins IncA, IncA D115A , DnaN, HolB, SSB, and RarA were cloned into pKT25 or pUT18 plasmids allowing fusion with T25 or T18 fragment of adenylate cyclase. Different combinations of T18 and T25 fusion plasmids were co-transformed in E. coli BTH101 Δ cya cells. Overnight cultures of BTH101 containing co-transformants were spotted (3 μl) onto LB kanamycin (50 μg ml -1 ), ampicillin (100 μg ml -1 ), IPTG (0.5 mM) and X-Gal (40 μg ml -1 ) plates. The plates were incubated at 30 °C for 24 h and then at 4 °C for few hours, at which point the plates were imaged. The LacZ activity was measured after the same overnight cultures were diluted in fresh LB media containing 0.5 mM IPTG, using the protocol mentioned below for promoter activity assay. Flow cytometry For flow cytometry analyses ( Siwach et al , 2021 ), Caulobacter cells were grown in the presence of 0.5 mM vanillate for 3 h. Cells were then exposed to 20 μg ml -1 of rifampicin for three h to stop initiation of DNA replication. 10 μl of this rifampicin treated cells were then fixed in 1.5 ml of 70% ice-cold ethanol. The samples were then vortexed and stored overnight at -20 °C. 500 μl of these samples were pelleted at 8000 rpm for 5 min. The pellets were washed thrice with 1 ml of FACS-staining buffer (10 mM Tris-HCl (pH-7.2), 1 mM EDTA, 50 mM sodium citrate, 0.01%Triton-X-100) by pelleting at 8000 rpm for 3 min. The washed pellets were resuspended in 1 ml of FACS-staining buffer containing 0.1 mg ml -1 RNase A (Roche, Switzerland), followed by incubation at 25 °C for 1 h. Cells were then pelleted at 8000 rpm for 3 min and resuspended in 1 ml FACS-staining buffer containing 0.5 μM SYTOX green nucleic acid stain (Invitrogen, USA) and incubated for 5 min in dark. The stained cells were then analyzed using BD Accuri C6 flow cytometer (BD Biosciences, CA, USA). The flow cytometry data was analysed using the FlowJo software (BD Biosciences). Tandem affinity purification The Tandem affinity purification experiments were carried out as described previously( Narayanan et al , 2015 ). Exponential phase cells (1 l) were harvested by centrifugation at 7000 rpm for 15 min and washed in buffer-1 (50 mM sodium phosphate [pH-7.4], 50 mM NaCl, 1 mM EDTA). Cells were then pelletised and resuspended in buffer-2 (buffer-1 containing 10 mM MgCl 2 , 0.5% n-dodecyl-β-D-maltoside (Sigma-Aldrich), 1x protease inhibitors (Complete EDTA-free, Roche, Switzerland) and lysed by addition of 5000 U of Ready-Lyse Lysozyme Solution (Biosearch Technologies, USA), followed by sonication at 60% amplitude, to reduce the culture viscosity. The sonicated lysate was then centrifuged at 10,500 rpm for 45 min at 4 °C to remove cell debris. The supernatant was then incubated with IgG Sepharose beads (Cytiva, USA) for 2 h at 4 °C with constant mixing. The beads were then washed thrice with IPP150 buffer (10 mM Tris-HCl [pH-8], 150 mM NaCl, 0.1% NP-40) and once with TEV-cleavage buffer (IPP150 buffer containing 0.5 mM EDTA and 1 mM DTT). The washed beads were then incubated overnight at 4 °C with TEV cleavage buffer containing 100 U ml -1 TEV protease (Promega, USA), with constant mixing, to release the tagged complex. 3 µM CaCl 2 was added to the eluate and incubated with Calmodulin-Sepharose beads (Cytiva, USA) for 1 h with constant mixing. The beads were then washed thrice with calmodulin binding buffer (10 mM β-mercaptoethanol, 10 mM Tris-HCl (pH-8), 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl 2 , 0.1% NP-40). The bound proteins from the beads were then eluted using calmodulin elution buffer (10 mM β-mercaptoethanol, 10 mM Tris-HCl (pH-8), 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM EGTA, 0.1% NP-40). The eluate was then concentrated using Amicon ultra centrifugal filters, 3 kDa MWCO (Merck-Millipore) and were analysed by immunoblots or mass spectrometry. Quantitative mass spectrometry For quantitative mass-spectrometry ( Kamat et al , 2015 ) 5 μg each of the TAP samples was processed for chloroform-methanol precipitation of proteins using 1:1:3 ratio of protein, chloroform, and methanol. The resultant white precipitate, was collected by centrifugation at 20,000 × g for 20 mins at 4 °C. The pellet was dried completely using a CentriVap vacuum concentrator (Labconco, USA). The dried pellet was resuspended in 50 μl of 8 M urea in 100 mM tetraethylammonium bicarbonate (TEAB) and sonicated using an ultrasonic cleaner (Spire, India) for 5 min. The degraded proteins were reduced using 5 mM dithiothreitol (DTT), followed by incubation at 60 °C for 30 min. The reaction was cooled down to room temperature before the addition of 15 mM iodoacetamide and incubated in dark for 15 min to alkylate the free sulfhydryl groups of cysteine residues. The mixture was supplemented with an additional 10 mM DTT and the urea was diluted by making up the volume of the reaction to 400 μl using 100 mM TEAB. The reaction was incubated at 37 °C for 16 h with constant shaking after addition of 4 μl of 0.5 mg ml -1 trypsin (Promega). After trypsin digestion, the samples were either labelled with 8 μl of 4% HCHO (light formaldehyde) or 8 μl of 4% DCDO (heavy formaldehyde). 8 μl of NaBH 3 CN was added to both reactions and were incubated at room temperature with constant shaking for 1 h, to promote labelling. The reactions were then quenched by the addition of 32 μl of 1% ammonia. Finally, the reactions were stopped by adding 5% formic acid and the supernatant was collected after centrifugation at 20,000 × g for 20 min. An equal volume of heavy-labelled and light-labelled peptide samples were mixed and processed for desalting via Empore C18 disks (Merck-Supelco). The samples were then subjected to LC-MS analysis using TripleTOF 6600 System (Sciex, USA). The data was then analysed using the ProteinPilot software (Sciex, USA) and plotted using VolcaNoseR ( Goedhart & Luijsterburg, 2020 ). Acknowledgments Anjana Badrinaraynan, Justine Collier, Vikas Jain, Sean Murray and Manjula Reddy for strains. Microscopy, Flow Cytometry and DST-FIST supported Proteomics Facility (SR/FST/LSII-043/2026) at IISER Pune. Research Fellowships from CSIR to Surbhi (09/0936(11651)/2021-EMR-1) and from IISER Pune to AKS. This work is supported by funds from the DBT-Wellcome Trust India Alliance through a Senior Fellowship to SKR (IA/S/20/2/505202). References ↵ Aakre CD , Phung TN , Huang D , Laub MT ( 2013 ) A bacterial toxin inhibits DNA replication elongation through a direct interaction with the beta sliding clamp . Mol Cell 52 : 617 – 628 OpenUrl CrossRef PubMed Web of Science ↵ Altieri AS , Kelman Z ( 2018 ) DNA Sliding Clamps as Therapeutic Targets . 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Share A starvation-triggered AAA+ ATPase halts chromosome replication progression by disassembling the bacterial DNA sliding clamp Surbhi , Arnab Kumar Shau , Feby Mariam Chacko , Sunish Kumar Radhakrishnan bioRxiv 2025.01.13.632716; doi: https://doi.org/10.1101/2025.01.13.632716 Share This Article: Copy Citation Tools A starvation-triggered AAA+ ATPase halts chromosome replication progression by disassembling the bacterial DNA sliding clamp Surbhi , Arnab Kumar Shau , Feby Mariam Chacko , Sunish Kumar Radhakrishnan bioRxiv 2025.01.13.632716; doi: https://doi.org/10.1101/2025.01.13.632716 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 (7622) Biochemistry (17648) Bioengineering (13871) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18561) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22475) Immunology (17713) Microbiology (40328) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)