Translation control by altered start codon usage as a means of modulating the general stress response and virulence in Listeria monocytogenes

Translation control by altered start codon usage as a means of modulating the general stress response and virulence in Listeria monocytogenes | 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 Translation control by altered start codon usage as a means of modulating the general stress response and virulence in Listeria monocytogenes View ORCID Profile Jialun Wu , View ORCID Profile Claire Kelly , Brenda Chanza , Ashley Reade , Catherine M. Burgess , View ORCID Profile Conor O’Byrne doi: https://doi.org/10.1101/2025.08.26.672274 Jialun Wu 1 Bacterial Stress Response Group, Microbiology, Ryan Institute, School of Biological and Chemical Sciences, University of Galway , H91 TK33, Galway, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jialun Wu For correspondence: conor.obyrne{at}universityofgalway.ie jialun.wu{at}universityofgalway.ie Claire Kelly 1 Bacterial Stress Response Group, Microbiology, Ryan Institute, School of Biological and Chemical Sciences, University of Galway , H91 TK33, Galway, Ireland 2 Teagasc Food Research Centre , Ashtown, D15 DY05 Dublin, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Claire Kelly Brenda Chanza 1 Bacterial Stress Response Group, Microbiology, Ryan Institute, School of Biological and Chemical Sciences, University of Galway , H91 TK33, Galway, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashley Reade 1 Bacterial Stress Response Group, Microbiology, Ryan Institute, School of Biological and Chemical Sciences, University of Galway , H91 TK33, Galway, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Catherine M. Burgess 2 Teagasc Food Research Centre , Ashtown, D15 DY05 Dublin, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Conor O’Byrne 1 Bacterial Stress Response Group, Microbiology, Ryan Institute, School of Biological and Chemical Sciences, University of Galway , H91 TK33, Galway, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Conor O’Byrne For correspondence: conor.obyrne{at}universityofgalway.ie jialun.wu{at}universityofgalway.ie Abstract Full Text Info/History Metrics Preview PDF Abstract In the food-borne pathogen Listeria monocytogenes , SigB is the central regulator of general stress response (GSR) and it mediates host entry by promoting acid resistance and epithelial cell attachment. The post-translational regulation of SigB activity is complex and is controlled by multiple genes. There is significant evidence that mutations can readily arise in these regulatory genes, or in sigB itself, leading to reduced SigB activity, which suggests that there is considerable genetic plasticity In the GSR. To further investigate this, we defined the complete genome sequence of a clinical isolate with attenuated SigB activity and investigated how it adapts to lethal acidic challenge (mimicking the selective pressure encountered during entry into the host). Acid resistance developed rapidly and 6 acid resistant derivatives (ARDs) were selected for further investigation. All 6 ARDs restored SigB activity due to mutations acquired in rsbW , which encodes an antagonist of SigB. These mutations resulted in non-canonical start codons ( rsbW ATG to rsbW ATA or rsbW ATT ) or premature translation termination ( rsbW - ). A translational reporter assay demonstrated distinct differences in translation efficiency between three start codons: ATG>ATA>ATT, suggesting that a perturbation of RsbW:SigB stoichiometry alters SigB activity. We then analysed start codon usage for all conserved genes in 60,692 L. monocytogenes genomes available in the NCBI database. This analysis revealed flexible usage of start codons associated with genetic clades in 39 conserved genes, 13 of which are involved in virulence and stress response. Further, we show that flexible use of canonical start codons (ATG and GTG) also mediates different levels of expression of virulence and stress response genes. Taken together, we show the genetic plasticity of GSR regulation in a model pathogen, and highlight the importance of translational control as a means of fine-tuning gene expression during short-term adaptation and long-term evolution for optimal fitness. Author Summary The general stress response (GSR) in foodborne pathogen Listeria monocytogenes is important for environmental stress response and for host entry, but GSR is also highly variable across wild isolates. In this study, we analysed the evolutionary trajectory of a clinical isolate with attenuated GSR and characterized the adaption by this strain to a host-mimicking stress (acidic condition). Under extreme selective pressure, mutations disabling a negative regulator of the GSR were enriched. Interestingly, several independently occurring mutations negatively affect the translation initiation by using non-canonical start codons. Prompted by this, we analysed the population-wide start codon usage by examining all available L. monocytogenes genomes available (n = 60,690). This analysis revealed differential start codon usage in 39 conserved genes that are associated with different genetic clades. Furthermore, we demonstrated differential translation efficiencies between the different canonical start codons. This work highlights the genetic plasticity of GSR in the important food-borne pathogen L. monocytogenes and shows that altered translational start codon can be used as means of regulatory control. Together the data suggest that genetic changes in the regulation of the GSR might confer niche-specific fitness advantages. Introduction The ethological agent of listeriosis, Listeria monocytogenes is a bacterial pathogen well-known to be capable of adapting to both saprophytic and pathogenic lifestyles. As a food and environmental microbe, L. monocytogenes is widely distributed and it is relatively resilient to low pH, low water activity, and low temperature [ 1 ]. Upon ingestion by humans, it can survive the stresses encountered in the gastro intestinal (GI) tract and cross the intestinal barrier, leading to systemic infection [ 2 ]. Symptomatic infections are typically associated with high mortality rates (estimated 20-30%) [ 3 ]. Despite the versatility of this bacterium, it has a relatively small (∼3 Mbp) and stable genome [ 4 , 5 ]. Most L. monocytogenes isolates are from one of two phylogenetic lineages, I and II. Lineage I strains are more often associated with infection while lineage II strains are more often isolated from food and food processing environments [ 5 ]. In particular, several clonal complexes (CC) from lineage I are classified as hypervirulent (e.g. CC1, CC2, CC4, CC6) clades because of their frequent association with clinical cases and more severe disease outcomes (e.g. meningitis) [ 6 ]. The alternative sigma factor sigma B (SigB) is a very well conserved stress response and virulence regulator in L. monocytogenes [ 7 ]. Its activity is tightly controlled by a group of proteins encoded in a same operon ( rsbRSTUVW - sigB - rsbX ). SigB is sequestered by the anti-sigma factor RsbW in unstressed conditions, which prevents SigB from associating with RNA polymerase and thus suppresses expression of the general stress response (GSR). Stress stimuli perturb the association of RsbT with a macromolecular complex known as the stressosome (which consists of RsbR and RsbS at a ratio of 2:1) [ 8 ]. The release of RsbT from the stressosome facilitates its interaction with the phosphatase RsbU [ 9 ]. RsbU then dephosphorylates the anti-anti sigma factor RsbV, which promotes its sequestration of RsbW, thereby releasing SigB [ 10 , 11 ]. Thus the partner switching that occurs between RsbW and RsbV and SigB in response to changes in environmental stress determines the activity of SigB and the expression of the GSR regulon. The last gene in the sigB operon, rsbX encodes a negatively acting regulator of SigB, which resets the sensing-ready state of the stressosome by dephosphorylation, allowing resequestration of RsbT [ 12 ]. Upon activation, SigB controls the expression of ∼300 genes, approximately 10% of the entire genome content [ 13 ]. This global gene expression profile results in increased resistance to an array of environmental stresses including acid, salt, light and bile [ 7 ]. SigB also plays an essential role in host-cell entry. During passage through the acidic gastric fluid, the SigB-dependent glutamate decarboxylase and arginine/agmatine deiminase are important for the survival of L. monocytogenes [ 14 , 15 ]. Additionally, in the intestine, SigB controls the expression of a surface adhesin called Internalin A (InlA), which mediates epithelial attachment through an interaction with E-cadherin [ 16 ]. Although SigB activity in a lineage II laboratory strain was shown to be dispensable for systemic infection in a guinea pig infection model [ 17 ], recent evidence demonstrates that hypervirulent CCs or lineages are generally associated with high SigB activity [ 18 ]. These data suggest that the importance of SigB in the pathogenic lifestyle of this species has previously been underestimated. In line with this are the high levels of conservation on the sigB operon [ 19 ]. Apparently in contradiction with these findings is the observation from several groups that mutations attenuating SigB activity arise readily both in vitro and in vivo in both laboratory strains and wild isolates [ 18 , 20 , 21 ]. Indeed, we have previously demonstrated that loss-of-function SigB mutations confer fitness advantages under mild-stress conditions, although they are detrimental for surviving lethal challenges [ 22 , 23 ]. Hence, we hypothesise that SigB functionality is evolutionarily conserved in the long-term, while SigB activity can be fine-tuned through genetic adaptation to confer short-term conditional fitness advantages in specific environmental conditions. To investigate this hypothesis the present study focused on a strain of L. monocytogenes CC1 (MQ140025) that was isolated from an ear swab in 2014 [ 24 ]. We previously reported that this strain displayed reduced acid survival and impaired SigB activity, likely due to lesions in the sigB operon ( rsbU Q317* and rsbX N77K) [ 21 ]. Given the high level of conservation of RsbX N77, it was proposed that N77K negatively affects RsbX function and results in elevated activation of SigB. While RsbU Q317* abolishes a C-terminal Mn 2+ binding site of RsbU and likely renders RsbU inactive [ 25 ]. Strain MQ140025 presents as a sigB - strain likely because RsbU functions downstream of RsbX in the SigB activation pathway. The fact that SigB is regulated by a multi-step signal transduction pathway, means that there are multiple genetic targets (at least 7) that can be mutated to produce changes in its activity. This study sought to investigate the plasticity of GSR, by adapting strain MQ140025 to an environment encountered within the gastric fluid of a host (acid stress challenge). We defined the complete genome of strain MQ140025 and subjected it to short-term adaption to a lethal acid challenge. The resistance mechanisms that conferred the stress adaptation were investigated. Arising from this analysis we exploited the NCBI database of L. monocytogenes genome sequences to investigate the evolution of start codon (SC) usage in conserved genes. The work presented reveals the genomic plasticity of the GSR in an intracellular pathogen and highlights altered translational initiation as a mechanism for fine-tuning gene expression during evolutionary adaption to environmental and host-related stresses. Results Clinical isolate shows rapid genetic adaption to lethal acidic stress To test whether clinical strain MQ140025 ( rsbU Q317*, rsbX N77K) could regain the SigB-mediated gastric acid survival, we designed an in vitro evolution experiment to select acid resistant mutants ( Figure 1A ). Cultures of strain MQ140025 were exposed to repeated cycles of acidic stress (pH 3.0 or pH 2.5) followed by growth over a period of four days ( Figure 1B ). A sample from each time point of the acid challenge was used as an inoculum to seed a tube of unacidified brain heart infusion (BHI) broth (1:2500). After recovery and incubation for 1 d at 37°C, the culture inoculated from the latest acid exposure time-point that successfully grew to stationary phase was re-exposed to acid challenge. An aliquot of each one of these cultures was also preserved as a −80°C permanent stock. After the first two cycles of acid challenge and growth, acid resistance rapidly developed ( Figure 1B ) and therefore the recovered cultures were exposed to both pH 2.5 and pH 3 during the third acid challenge to maintain a high selective pressure. The development of acid resistance occurred reproducibly across three independent experiments (parallel cultures, data not shown). These results show that acid resistance can be rapidly developed and selected in an acid sensitive strain of L. monocytogenes . Download figure Open in new tab Figure 1 Strain MQ140025 rapidly develop acid resistance during in vitro adaption. Schematic presentation of in vitro adaption experimental setup (A), three parallelly cultures of strain MQ140025 were repeated exposed to lethal acidic challenge and recovery cycles. The stress condition and sampling time points for in vitro adaption experiment are shown (B), sage and rose denotes recoverable and non-recoverable samples, respectively. The abilities of surviving under pH 3 (C) or pH 2.5 (D) are determined for 2-day (D2) and 4-day (D4) adapted cultures from three independent experiments (E1, E2, and E3). ARDs recovered from adapted cultures were examined for their ability to withstand 80 min pH 3 or pH 2.5 challenge (E). The six ARDs selected for further characterization were determined for survival under pH 3 (F) and pH 2.5 (G). Three independent experiments were performed for all survival assay, each with technical duplicates. To quantify the development of acid resistance in 2-day (D2) and 4-day (D4) adapted cultures from all three independently evolved cultures (designated E1, E2, and E3), they were challenged with either pH 3.0 ( Figure 1C ) or pH 2.5 ( Figure 1D ) in comparison with F2365, a reference strain that belongs to the same clonal complex (CC1) and sequence type (ST1), and the parental strain MQ140025 ( Table 1 ; Figure 1 C,D ). Results confirmed the acid sensitive phenotype of MQ140025 while all acid adapted cultures survived better than MQ140025, albeit not as well as F2365. All D4 cultures were more acid resistant than D2 cultures. To investigate these differences in acid resistance, for each evolved culture that was tested in pH 2.5 survival experiments ( Figure 1D ) one colony from the latest surviving time point was re-streaked to prepare permeant stocks (t = 90 min for D2 cultures; t = 120 min for D4 cultures). In total this yielded 18 acid resistant derivatives (ARDs) from three independent pH 2.5 survival experiments (1 st experiment: ARD1-6; 2 nd experiment: ARD7-12; 3 rd experiment: ARD13-18; Figure 1E ). All 18 ARDs displayed a comparable level of acid survival ( Figure 1E , even numbered ARDs were from day 2 and odd numbered ARDs were from day 4) that is higher than parental strain, confirming that these are inherited genetic effects. ARD1, 3, 5, 6, 10, and 12 were chosen for further characterization on acid resistance ( Table 1 ). They were fully resistant to pH 3 ( Figure 1F ) and all displayed equal, if not higher, level of acid resistance than the D4 adapted cultures ( Figure1 D,G ). These data show that the selected ARDs (1, 3, 5, 6, 10, and 12) represent the most acid resistant survivors from the adapted cultures. Taken together, MQ140025 variants with increased acid resistance naturally emerge and they are rapidly enriched during an in vitro evolution experiment at low pH. View this table: View inline View popup Download powerpoint Table 1 Strains used in this study ARD isolates display increased SigB activity During routine laboratory culturing on BHI agar, ARD3 and ARD5 formed smaller colonies ( Figure 2A ) that resembles those characterized as “small colony variants” in Staphylococcus aureus , which are often associated with increased SigB activity [ 26 , 27 ]. In L. monocytogenes , a ΔrsbX strain which has a hyperactive SigB phenotype forms smaller colonies and displays reduced motility [ 12 ]. Therefore, ARD1, 3, 5, 6, 10, and 12 were assayed for motility at 30°C ( Figure 2B ). While the parental strain MQ140025 displayed hyper-motility (HM); ARD1, ARD3, and ARD5 were characterized by smaller colony size and low motility (LM); ARD6, ARD10, and ARD12 were characterized by normal colony size and intermediate motility (IM) ( Figure 2A,B ). Based on these observations (decreased motility and increased acid resistance relative to MQ140025), we hypothesized that the ARDs had acquired mutations to restore SigB activity to different levels. To test this, the expression of the glutamate decarboxylase system and arginine deiminase system was examined by measuring the transcript levels of gadD3 , gadT2 , and aguA1 ( Figure 2C-E ). Among these three genes only the SigB-dependent gadD3 were upregulated in LM group. The changes of ArgR controlling aguA1 expression were not significant. The GadR-dependent gadT2 expression in MQ140025 and the ARDs was much lower than that of F2365, similar to the transcript level observed from ΔgadR strains [ 28 ], suggesting no involvement of GadR in the ARD phenotypes. To corroborate this finding, the transcript levels of SigB-dependent genes lmo2230 and inlA were examined. Significantly higher transcript levels of lmo2230 and inlA were found in most ARDs ( Figure 2F,G ). Interestingly, gadD3 , lmo2230 , and inlA were all induced to a higher level in LM group (ARD1, 3 and 5) than IM group (ARD6, 10 and 12; Figure 2 C,F,G ). This is in line with a slighter higher acid survival associated with LM group ( Figure 1G ). These observations indicate a positive correlation between SigB activity and acid survival and suggest that the ARDs have regained SigB activity. Download figure Open in new tab Figure 2 ARDs display altered colony morphology, motility, and increased SigB activity. Colony morphology at 37°C (A) and motility at 30°C (B) were assayed for strains F2365, MQ140025 and ARD. Transcripts levels of gadD3 (C), gadT2 (D), aguA1 (E), lmo2230 (F), and inlA (G) at stationary phase were measured for strains F2365, MQ140025 and ARDs and expressed relative to MQ140025 using 16S as reference gene. Three independent experiments were carried out and each with technical duplicates. Statistical significance was calculated between each strain to MQ140025 using paired two-tailed t -test (ns, not significant; *, P < 0.05; **, P < 0.01; and ***, P < 0.001). ARD isolates carry mutations in the anti-sigma factor gene rsbW Phylogenetic analysis placed strain MQ140025 in the most ancient genetic clade of ST1, within a branch that underwent significant population expansion ( Figure S1A ). To accurately determine the genetic changes in the ARDs, complete genome sequence of strain MQ140025 was determined by long-read Nanopore sequencing. Long read genome assembly yielded three circular sequences, representing the chromosome (2902539 bp) and two plasmids (50099 bp and 11340 bp). When the chromosomal sequence of strain MQ140025 was compared to CC1 reference strain F2365 ( Figure 3A ), no major genome rearrangement was detected except that strain MQ140025 exhibits a 3.5 kb deletion at a highly variable region encoding a type VII secretion system (T7SS) [ 29 , 30 ]. Close comparison between MQ140025 and F2365 revealed that: 1) MQ140025 along with other CC1 strains share premature stop codons (PMSC) with F2365 in lmo0140 , lmo0671 , and lmo2084 ; 2) MQ140025 doesn’t bear other PMSC identical to F2365 [ 31 ]; 3) MQ140025 acquired several additional PMSCs ( Figure 3A ). When comparing the complete chromosome sequence of MQ140025 with the earlier acquired Illumina genome sequence (NCBI accession: GCF002027925), we observed 2 polymorphisms with high confidence: rsbU *317Q and lmo1979 ΔE128. The reversion of rsbU lesion highlights the plasticity of loci involved in the GSR during routine laboratory passage. Of the two plasmids found in MQ140025, the larger one is virtually identical to a prevalent plasmid pLM7UG1 in L. monocytogenes ( Figure 3B ) [ 32 ]. The smaller plasmid does not resemble any plasmid previously reported from Listeria , although Blastn found regions that share similarity to a known Bacillus subtilis plasmid (pBS-02) and an unnamed Streptococcus xylosus plasmid (accession: CP066723 ). We assign the name pLMGC1 to this newly identified plasmid ( Figure 3C ). Download figure Open in new tab Figure S1 Phylogenetic inference of MQ140025. (A) Core-genome based phylogeny of ST1 5446 genomes. Genetic clade in which MQ140025 was placed is highlighted in colour. (B) Detailed illustration of MQ140025 and its close relatives within coloured branch in panel A. Download figure Open in new tab Figure 3 Genomic characterization of MQ140025 and ARD. Chromosomal sequence of MQ140025 was determined and mapped to reference genome sequence F2365 (A). Inner to outer rings represents: F2365 genome, genome GC skew, clockwise transcribing genes, MQ140025 genome, counter-clockwise transcribing genes. Outstanding gene disruption, substitution, insertion, and deletions (>3 bp) compared to F2365 are labelled in black. The rsbU mutation that occurred during long read sequencing comparing to original short read sequencing is labelled in grey. Purple labels denote mutations detected in ARD1 ( rsbV G111C rsbW M1M; lmo2586 H492Y), ARD3 ( rsbW R23*; lmo0640 **), and ARD5 ( rsbW Y37**). Cyan labels denote mutation shared by ARD6, ARD10, and ARD12 ( rsbV G111S rsbW M1M) and one additional mutation only in strain ARD12 at intergenic region ( lmo1611 :: lmo1612 ). A pLM7UG1-like plasmid in strain MQ140025 is mapped to pLM7G1 from L. monocytogenes serotype 7 strain SLCC2482 (B). Genomic architecture of a novel plasmid pLMGC1 is shown along with Bacillus subtilis plasmid pBS-02, and an unnamed Streptococcus xylosus plasmid (C). Sequence homologies within open reading frames are indicated in grey, and sequence homology detected in intergenic region is indicated in blue. To elucidate the genetic changes in the ARDs, Illumina sequencing reads from ARD1, 3, 5, 6, 10, and 12 were mapped to the MQ140025 genome. Surprisingly, mutations were found in rsbW from all ARDs. ARD3 and ARD5 each carries a distinct non-sense mutation in the rsbW coding sequence ( Figure 3A ), resulting in truncated variants of RsbW. Interestingly, all other ARDs carried SNPs affecting the rsbW start codon (SC). These mutations produce dual effects: they substitute RsbV glycine at position 111 with cysteine (ARD1) or serine (ARD 6, 10, 12) and they also change the rsbW start codon from a canonical ATG start codon (SC) to a non-canonical ATT (ARD1) or ATA (ARD 6, 10, 12) SC ( Figure 3A ). We reasoned that this could perturbate either the function of RsbV or, more likely the translation rate (and thus level) of RsbW. Although additional mutations were detected in ARD1, ARD3, and ARD12, they do not phenotypically distinguish these strains from other ARDs baring similar rsbW mutations. Therefore, we conclude it was the mutation within rsbW that led to the increased SigB activity and elevated acid resistance. Non-canonical SCs reduce rsbW translation efficiency Next we sought to understand the mechanism of SigB activation in ARD1, ARD6, ARD10, and ARD12. We reasoned that any effect produced by these SNPs on rsbW is likely to be prominent since RsbW acts downstream of RsbV by directly interacting with SigB. The presence of non-canonical SCs, albeit decoding as methionine, had the potential to reduce the RsbW translation efficiency. In strain EGD-e a canonical ATG was confirmed as the primary SC of RsbW [ 33 ], of note is the existence of a secondary SC of rsbW 6 bp downstream ( Figure 4A ). To test the influence of the non-canonical SCs on the RsbW translation efficiency, we designed three translational reporter constructs using the IPTG-inducible integrative expression vector pIMK3 [ 34 ]. In order to preserve the native RBS of rsbW , the 3’-end of rsbV sequence was fused to codon-optimized eGFP [ 35 ] to produce a 16 aa peptide tail of RsbV and a RsbW-eGFP fusion protein ( Figure 4A ). Three reporters were constructed using ATG, ATA, or ATT as the primary SC for rsbW - eGFP fusion proteins, and they were introduced to strain MQ140025 with pIMK3 as control. The induction of a fluorescence signal by IPTG was clearly decreased when using ATA as the SC compared to ATG, while there no detectable signal when using ATT as SC ( Figure 4B ). These data suggested that the relative translation rates from these three start codons were ATG > ATA > ATT. We then performed Western-blots using anti-eGFP antibodies on crude protein extracts from the same culture conditions. While eGFP expression was detected from both the ATA and ATT constructs it was at a reduced level compared to the canonical ATG SC, with the ATT construct again showing the lowest translation rate ( Figure 4C ). These results suggest that the primary SC of rsbW accounts for the functional expression under laboratory conditions, and the usage of an efficient SC is critical for RsbW expression. This supports a model in which ARD1, ARD6, ARD10 and ARD12 achieve elevated SigB activity through an altered translation rate of RsbW, which in turn is predicted to perturb the RsbW:SigB stoichiometry ( Figure 4D ). Download figure Open in new tab Figure 4 ARDs rescue SigB activity by reducing rsbW translation or abolishing RsbW function. Schematic presentation of translational reporter design for pIMK3 rsbW -ATG is shown (A), with 5’UTR hly partially omitted. Primary and secondary SCs of rsbW are outlined by black and grey rectangular, respectively. Normalized fluorescent signals are shown for rsbW translational reporter strains from stationary growth phase (18 h) with or without 1 mM IPTG (B). Western-blot quantification of GFP expression in reporter strains in the absence or presence of 1 mM IPTG (C). Schematic presentation of SigB activation pathway in strain MQ140025, and proposed modes of actions by which SigB is (in)activated in MQ140025 and ARD are shown (D). Occurrences of SC alterations in sigB operon by analysing 60 K L. monocytogenes genomes are shown (E). Canonical SC are highly preferred for genes within sigB operon The strong influence of rsbW start codon selection on SigB activity prompted us to investigate whether SC selection could play a general role in fine tuning SigB activity across the species. To investigate this, we compared the SC usage in the 8 genes comprising the sigB operon from 60,690 L. monocytogenes whole genome sequences available from the NCBI database. All sigB operon genes use ATG as a SC except RsbS (GTG), and this is generally conserved at the species level ( Figure 4E ). Translation initiation from GTG SCs was recently shown to be weaker than ATG in bacteria [ 36 ]. It seems plausible that GTG SC usage for rsbS might be a means of achieving the correct stressosome subunit stoichiometry, which is reported to be 2:1 for RsbR:RsbS [ 8 , 37 , 38 ]. The alteration of SCs in the sigB operon occurred at a very low frequency (n = 31, ∼0.05 %) and where they occurred other lesions were often present in the sigB operon (n = 5, data not shown). It is noteworthy that 8 out of 9 GTG → TTG SC substitutions in rsbS were from a rare genetic clade, CC380 ( Figure 4E ). Taken together, canonical SC are generally highly preferred in the sigB operon suggesting that efficient translation initiation is important for the integrity of the GSR. SC are under selection for stress response and virulence genes To systematically analyse the role of SC usage in the evolution and genetic adaption of L. monocytogenes , we extended the analysis from the sigB operon to the entire core-genome. We identified 2180 conserved genes among 60,690 L. monocytogenes genome analysed. The majority (98.2%; n = 2139) of these genes use an unchanging canonical start codon (ATG: 81.2%; TTG: 9.9%, GTG: 7.9%, Table S1). Interestingly, two conserved genes with fixed start codons use non-canonical SCs, infC (ATT) and rnhC (ATC). Both have been experimentally confirmed as translation initiation sites (TIS) in the reference strain EGD-e [ 33 ]. SC usage was found to be flexible for 39 conserved genes (1.8%). Seventeen of these were experimentally confirmed previously, while the remainder have not yet been experimentally tested ( Figure 5A ). Interestingly, one third of these 39 genes (n = 13) are known to be involved in virulence and/or stress response ( Figure 5A ). esaB encodes a cytoplasmic protein of a T7SS, which negatively influences virulence [ 30 , 39 ]. plcB encodes phosphatidylcholine-specific phospholipase C that is required for cell-to-cell spread during infection [ 40 ]. lmo1402 is co-transcribed with lmo1400 and ymdB ( lmo1401 ), and disruption of lmo1401 by transposon results in reduced haemolytic activity [ 41 ]. comEC encodes a late competence protein that is involved in phagosome escape [ 42 ]. Lmo1638 is a putative carboxypeptidase and it was shown to play a role during later stage infection [ 43 ]. lmo2445 encodes an internalin-like protein within an α-glucan metabolism operon that is required for full virulence [ 44 ]. Lmo0669 encodes a stress inducible SigB-dependent oxidoreductase. lmo0501 ( mltR ) encodes transcriptional regulator for mannitol metabolism that promotes cold, osmotic, and acid stress response [ 45 ]. Lmo0093 (KtrD) is a subunit of low affinity potassium transporter KtrCD which plays a role in osmolyte homeostasis [ 46 ]. lftR encodes a transcriptional repressor that influences virulence and antibiotic resistance [ 47 , 48 ]. MntC is a subunit of ABC-type manganese transporter which contributes to pathogenicity and stress response [ 49 , 50 ]. Lmo0946 (Sif) was recently shown to contribute to β-lactam antibiotic susceptibility, GSR and virulence [ 51 ]. The majority of these genes use different SCs between lineage I and lineage II, and some of them use different SCs between globally prevalent clonal complexes ( Figure S2 ). These data suggest an evolutionary preference for the usage of specific SC for these genes possibly associated with niche-specific selective pressures. Download figure Open in new tab Figure S2 SC are selected during evolutionary adaption to stress and pathogenesis. The SC usage in several clinical- or food- prevalent CC of L. monocytogenes are presented for 39 genes showing evidence of alternative SC usage, with lineage I and lineage II CCs in pink and blue backgrounds, respectively. Download figure Open in new tab Figure 5 SC selection as a means of modulation for stress and virulence genes expression. The SC usage in 39 conserved genes with flexible SC is shown for lineage I (LI) and II (LII) L. monocytogenes strains (A). The names of genes that are involved in stress response, virulence, or both are presented in green, red, and purple, respectively. The genes with experimentally confirmed SC are in bold [ 33 ]. Schematic presentations of translational reporter design for SC activity in plcB , lftR , and mntC are shown (B). Normalized fluorescent signals are shown for each reporter strain from stationary growth phase (18 h) with or without 1 mM IPTG (C). While the majority of SC flexibility in these 39 conserved genes are within canonical SCs, the level to which altered canonical SCs influences gene expression is unknown in L. monocytogenes . To test this, we designed eGFP-based translational reporters to compare the translation efficiency when using ATG or GTG as SC within the genomic context of plcB and lftR ( Figure 5B ). In both cases, a strong fluorescence signal was detected when using ATG as start codon, while a small reduction in the translation rate was detected when GTG was present ( Figure 5C ). These results suggest that for some genes SC flexibility might provide a genetic means of adjusting gene expression, potentially as a means of enhancing niche specific fitness. Discussion In this study, we characterized the genome of clinical L. monocytogenes strain MQ140025 and report its rapid genetic adaptation to extreme acid stress. This adaption occurred through increasing SigB activity by genetically disabling its antagonist, RsbW. Interestingly, in several adapted mutants this was achieved by using non-canonical SCs for rsbW translation initiation. This prompted us to investigate the overall SC usage in L. monocytogenes and the potential effect of SC usage on gene expression. These findings highlight the genetic plasticity of L. monocytogenes when under extreme selective pressure from environmental stress and demonstrate that alternative SC usage can be an effective means of regulatory control during niche adaptation. Stress responses occur during bacterial adaptation to ever-shifting environments. L. monocytogenes as a food-borne pathogen colonizes a wide range of niches, and understanding its stress adaptation is important for its successful control. Here, we applied extreme selective pressure by repetitively recovering the most acid resistant populations from parallel bacterial cultures. This was designed to mimic the bottleneck events in gastric fluid passage during infection, where the acid resistant bacteria are more likely to survive and access the intestine. The most acid resistant cells from the adapted cultures were isolated during this acid survival experiment. These strong selective pressures explain the enrichment and occurrence of rare SigB hyper-active mutations [ 22 ], which come with fitness burdens during growth ( Figure2A ). It is likely that mutants with a range of SigB activities are present in each acid adapted culture, but only those with the highest resistance were selected for by the conditions. Interestingly, the parental strain MQ140025 and ARDs derived from it displayed little expression of glutamate decarboxylase system encoded by gadT2D2 ( Figure 2D ) [ 52 ], which is controlled by the recently characterised RofA-like transcriptional regulator GadR [ 28 ]. A careful examination of the GadR sequence in MQ140025 revealed a substitution (R154Q) in its DNA binding domain. GadR-mediated gadT2D2 expression is critical for survival at pH 2.4 and this defect in gadT2D2 expression likely explains the reduced acid resistance in the ARDs when compared to the closely related strain F2365 (which encodes an otherwise identical GadR). It is noteworthy that when we used strain EGD-e (which carries a premature stop codon disrupting the gadR gene [ 28 ] in a similar acid adaption experiment but at a lower pH, it restored GadR activity after only two inactivation-recovery cycles (data not shown). Thus, increased SigB activity provides strain MQ140025 significant selective advantage for surviving pH 3 and pH 2.5 but it is likely that more severe acid stress would be necessary to select for increased gadT2D2 expression. These data suggest that the severity of the stress applied substantially shifts the types of genetic adaptations selected during in vitro evolution experiment. Phylogenetic analysis of MQ140025 allowed examination of the evolutionary trajectory of MQ140025. We compared the core genome mutations associated with bottlenecking events and population expansion events historical to strain MQ140025. The SigB operon mutations ( rsbU Q317* or rsbX N77K) were found specific to strain MQ140025. This suggested that these are transient mutations that confer niche-specific advantage. Interestingly, GadR R154Q occurred much earlier on in the strains ancestorial to the population expansion events ( Figure S1B ). The subsequent population expansion event was associated with Lmo0884 E111D and CodY R69D. Given the conservative nature of former (E111D) substitution, we reasoned that the CodY substitution (R69D) most likely confers the predicted fitness alteration. CodY is an important global regulator that controls amino acid metabolism, stress response and virulence in response to GTP and branch chain amino acids [ 53 ]. R69 resides within the isoleucine binding box of CodY and conceivably its substitution would impact isoleucine responsiveness and therefore overall fitness [ 54 , 55 ]. Strain MQ140025 emerged within this clade immediately following a further population expansion events, associated with four core-genome mutations including LiaR T66A and P prfA −88 T→A. LiaR is response regulator that mediates cell envelope stress response and the translation of PrfA is subject to extensive post-transcriptional control in 5’-UTR [ 56 , 57 ]. These observations highlight the changes within the core-genome during evolutionary selection that likely influence the stress response and virulence of the pathogen. The emergence of acid resistant mutants in the MQ140025 background after only 2 days of selection highlights the capacity for rapid genetic adaptation to extreme environmental stress. The finding that 6 independent isolates from this short-term IVEE were confirmed by whole genome sequence analysis to carry newly acquired lesions in the sigB operon demonstrates the importance of GSR to acid resistance, and therefore host entry. Previous studies from our group and others have reported on the prevalence of loss-of-function mutations in the sigB operon in public databases, under mild-stress or during laboratory culture [ 20 – 22 , 58 ]. Interestingly, we have also previously observed a rsbS SC alteration from a sigB - mutant following in vitro evolution experiment [ 20 ]. Collectively these studies highlight the plasticity of the sigB operon and suggest that environmental stress can be a significant driver of evolution in this pathogen. Gain of function mutations (that increase SigB activity) are not commonly reported, likely because these mutations are expected to come with a fitness cost as they can negatively impact cell growth and reproduction [ 12 , 59 ]. The rsbW mutations reported here are predicted to increase SigB activity by affecting the stoichiometry of RsbW to SigB, the principle interaction that limits SigB availability for participation in transcription. Thus, when a severe environmental stress (lethal acid pH) is encountered the survival advantage gained through increasing SigB activity likely outweighs any fitness cost associated with this change, at least in the short term. The finding that rsbW non-canonical SCs are rare (2 found in 60,690 genomes, Figure 4E ) in the reported genome sequences of L. monocytogenes suggests that in the long-term the cost does indeed outweigh the short-term survival advantage. Interestingly, one study that investigated the genetic adaption of L. monocytogenes to repeated murine passage following oral inoculation found that PMSCs frequently arose in the rsbW gene [ 60 ], perhaps indicating that the conditions in the mouse GI tract can provide significant selective pressure for the loss of RsbW function. A recent study has confirmed that there is a strong positive correlation between hyper-virulence and SigB activity, suggesting the host environment provides significant selective pressure for altered SigB activity [ 18 ]. The appearance of non-canonical SCs in rsbW in the acid resistant isolates raises the possibility that a reduced translation rate is serving a regulatory function in these strains (i.e, to increase SigB activity). When the start codon usage was examined across the entire genome for all available >60K genome sequences (via NCBI) a number of interesting points emerged. The vast majority of ORFs in the core genome use either ATG (81.6%), TTG (10.0%) or GTG (8.1%) as start codons (Table S1), with less than 0.4% using rare non-canonical SCs (ATA, ATC, ATT, CTG). When altered SC usage occurs for a given ORF it tends to occur along phylogenetic lines, either within an entire lineage or clonal complex. For example, the argB gene ( lmo1589 ) is initiated by ATG in lineage I but by GTG in lineage II stains, whereas lmo0315 is initiated by GTG in most clonal complexes, except for CC8, CC204, and CC121 where ATG is preferred ( Figure S2 ). This fact suggests that changes in SC usage may have been selected at some point, potentially offering some niche-specific advantage. The most likely route for a selectable advantage to altered SC stems from the impact of the SC on translation initiation rate. A recent study has shown that non-canonical SCs can provide a selective advantage in E. coli in the murine gut environment. When the initiation codon of the lacI gene, which encodes a repressor for lactose utilisation, was altered to GTG the translation rate of lacI was reduced thereby enhancing lactose consumption [ 36 ]. A systematic study of SC efficiency in E. coli showed that in addition to ATG, GTG and TTG, which had the highest initiation rates (in that order), CTG, ATA, ATT and ATC were also functional as SCs, albeit with reduced rates [ 61 ]. While the mechanism of translation initiation control in L. monocytogenes is not well studied it appears to be distinct from other well studied bacteria in that the Shine-Dalgarno strength does not correlate closely with translational efficiency [ 62 ]. The SC data presented in this study suggest that non-canonical SC selection could be a means of regulation the expression of some genes, including those playing a role in virulence and stress resistance. The variable usage of SCs in 39 conserved genes from different genetic clades raises the question of what are the evolutionary pressures driving these selections? Although the core genome content of L. monocytogenes species is highly conserved (despite two major lineages), some genetic groups (e.g. CC1, CC2, CC4, CC6, CC87) are more virulent than others (CC121, CC9, CC18, CC37) [ 18 ]. While differences in the accessory genome don’t fully account for hypervirulent phenotypes [ 63 ], it is now evident that polymorphisms in the core-genome contribute to differential stress response and virulence between hypervirulent and hypovirulent strains/clades [ 18 , 64 ]. Interestingly, the disruption of the lmo1622 CDS was recently shown to increase SigB activity in a hypervirulent clade [ 18 ]. Here we report the divergent SC usage of lmo1622 plausibly selected concurrently with the branching event that produced lineages I and II ( Figure 5 ), suggesting fine-tuning of lmo1622 expression might be associated with evolutionary niche-specific adaption. More evidence suggesting a role for alternative SC usage in virulence was found here ( Figure 5 ) for phospholipase C (PlcB) [ 40 ], competence component (ComEC) critical for intracellular growth [ 42 ], as well as cell invasion and environmental persistence related regulator (LftR) [ 47 , 48 , 65 ]. While changing between canonical SCs might not alter expression drastically, the fact that they are maintained within genetic clades suggests that they likely contribute to an overall genomic shift that provides an incremental fitness advantage during novel niche colonization. Deciphering the driving forces of these adaptations will be a challenge for future research in this area but it should contribute to the overall understanding of the biology of this important human pathogen. Materials and Methods General culture conditions L. monocytogenes frozen stocks were prepared using overnight cultures in BHI growth medium with 7% DMSO and stored at −80C°. For routine overnight culture, L. monocytogenes colonies were inoculated into 5 mL BHI (LAB M LAB048) in 50 mL conical centrifugation tubes and incubated for 18 h at 37C° with agitation (160 rpm). To revive acid stress adapted L. monocytogenes cultures, −80C° stocks were revived in BHI at 37C° for 24 h, then 2 μL culture was used for inoculating overnight cultures (18 h at 37C°). Where specified, kanamycin (50 μg/mL) was supplemented to growth media. For the motility assay, BHI with 0.25% (w/v) agar was seeded with 2 μL of overnight culture and incubated for 24 h at 30°C before pictures were acquired. in vitro acid adaption and acid survival experiments Protocols for acid challenge experiments were adapted from a previous study [ 28 ]. Briefly, 100 μL overnight culture was mixed with 900 μL acidified BHI (pH 2.5 or pH 3) and incubated at 37°C statically. For in vitro adaption experiments, at each indicated time point 2 μL of sample was removed and diluted into 5 mL BHI unacidified and incubated at 37°C with agitation (160 rpm) to recover for 24 h. For acid survival experiments, at each indicated time point 20 μL sample was taken and serially diluted in phosphate saline buffer. 10 μL from each dilution was spotted on BHI agar and incubated for 48 h before colony counts were recorded. The percentage survival at each time point was calculated as the colony counts divided by initial colony counts. All experiments were carried out three independent times, each with technical triplicates. Whole genome sequence analysis To characterize the complete genome sequence, one colony of strain MQ140025 revived in tryptic soy agar was used to prepare overnight culture in tryptic soy broth (18 h at 37C°). Genomic DNA was extracted using Qiagen DNeasy UltraClean Microbial Kit according to manufacturer’s instruction. A library was prepared using rapid barcoding kit V14 (Oxford Nanopore) following instructions. The constructed DNA library was loaded onto a PromethION 2 Integrated (P2i) sequencer (Oxford Nanopore) for sequencing and real-time base calling with MinKNOW (high-accuracy option). Yielding read files were first combined and then binned for genome assembly using Autocycler [ 66 ], by compressing the result from individual assemblers (canu, flye, metamdbg, and unicycler). The resulting genome sequence was annotated using Prokka [ 67 ]. Chromosome and plasmid comparison and visualization were carried out in BRIG [ 68 ] and geneviewer (van der Velden N 2025). The complete genome sequence of strain MQ140025 was deposited at NCBI database (Project accession: PRJNA1305691). To characterize the gain or loss-of-function mutations in strain MQ140025, chromosomal sequences from MQ140025 and F2365 were carefully compared. Firstly, the amino acid sequences from all genes in F2365 were extracted and used as query sequences to blast (tblastn) in MQ140025 chromosome. These tblastn results (hits) were examined when query coverage is less than 100% to check for premature stop codons. Then the two chromosomes were compared using Mauve to check for gaps in the alignment. These gaps present in coding sequences are manually examined for reading frame disruption or for significant insertion or deletion. The gaps greater than 10 bp within intergenic regions are also marked for further notice. Lastly, the loci where authentic premature stop codons were previously reported in strain F2365 were examined in strain MQ140025, along with other CC1 strains and reference strain EGD-e. In addition, the long read-assembled MQ140025 genome sequence was also compared with previously published short reads-assembled genome to check for mutations that arose during laboratory passages. To analyse derivatives of strain MQ140025, genomic DNA was extracted as previously described [ 21 ]. Briefly, 5 mL of an overnight culture was resuspended in 180 μL enzymatic lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100, and lysozyme 20 mg·mL -1 ) and incubated at 37°C for 30 min. Protease K and Buffer AL (Qiagen DNeasy Blood and Tissue kit) were then added to the samples and heated up to 56°C for 30 min. The remaining procedures were carried out following the manufacturer’s instructions (Qiagen DNeasy Blood and Tissue kit). Purified DNA samples were sent to Novogene and the resulting pair-end sequencing reads were mapped to the MQ140025 genome (Breseq) to check for mutations [ 69 ]. Phylogenetic analysis To analyse the phylogeny of ST1 strains, the multi-locus sequence type was determined for the aforementioned 66,690 genomes [ 70 ]. In total 5546 genomes were classified as ST1. The phylogeny of these ST1 genomes were inferred using Parsnp2 based on the core-genome SNP profile [ 71 ]. The SNPs associated with branching points of interests were examined using Parsnp2 alignment output. Molecular methods The codon-optimized eGFP sequence [ 35 ] was amplified by PCR using primers that were designed to include the regulatory regions under investigation ( Table 2 ). These fragments along with pIMK3 plasmid were digested (NcoI and BamHI, FastDigest, Thermofisher) and ligated then transformed to E. coli TOP10 to propagate. Then purified recombinant plasmids were electroporated into electrocompetent cells of L. monocytogenes then plated on BHI kan agar plates and incubated at 30°C to select transformants, as previously described [ 34 ]. View this table: View inline View popup Download powerpoint Table 2 Primers used in this study. Transcriptional analysis Methods for transcriptional analysis were adapted from Wu et al., (2023). Briefly, 1 mL of stationary culture was mixed with 5 mL RNAlater (Sigma) and incubated for 5 min at room temperature. Then cells were resuspended (8,000 rpm × 5 min, 4°C) using 700 μL cold buffer RLT (Qiagen RNeasy kit) in lysis matrix B tubes, for mechanical lysis (FastPrep, 40 s × 6 m·s -1 , twice). The rest of the protocol was carried out following the manufacturer’s instructions (Qiagen RNeasy kit). DNA contamination was first depleted from the resulting RNA preparation using TURBO DNase (Invitrogen), then reverse transcription and qPCR analyses were carried out as previously described [ 28 ]. Relative gene expression was calculated using 16S as a reference gene [ 72 ]. Fluorescence quantification Fluorescence levels from reporter strains were used as a measure of eGFP synthesis. Reporter strains were grown in BHI kan at 37°C for 18 h with or without 1 mM IPTG, then 1 mL culture was washed twice using 700 μL PBS with chloramphenicol (5 μg/mL). For each sample, 100 μL cell suspension was loaded to a 96 wells plate in duplicates. The fluorescence (489nm/518nm) and OD600nm at 20°C for each sample was measured using a microplate reader (Synergy H1, Agilent) with PBS containing wells used as blanks. Results were calculated and presented as 500 x (RFU/OD). Three independent experiments were performed. Immunoblotting method For crude protein extraction, cells were collected from 5 mL of 18 h overnight culture by centrifugation (8,000 rpm × 5 min). Chloramphenicol (10 μg/mL) was added at the point of harvesting to disable protein synthesis during the protein extraction procedure. Cell pellets were resuspended in 2 mL sonication buffer (13 mM Tris-HCl, 0.123 mM EDTA, and 10.67 mM MgCl 2 , pH 8.0) with lysozyme (1 mg/mL) and incubated at 37°C for 30 min. Then cells were resuspended in 200 μL ice-cold sonication buffer with protease inhibitor (cOmplete, Roche) and transferred to 2 mL tubes with 0.25 mL of 0.5 mm zirconia beads and 0.5 mL of 0.25 mm zirconia beads (Thistle Scientific) for mechanical lysis (FastPrep 6 m/s × 40 s, three times). Cell lysate was centrifuged at 4°C at 13,000 rpm for 30 min, and supernatant was collected. Protein samples were collected from three independent experiments. For immunoblotting, protein concentration was determined using the BCA assay (Pierce™ BCA Protein Assay Kits, ThermoFisher) according to manufacturer’s instruction. For each sample, 10 ng protein was separated in SDS-PAGE (12% acrylamide-bisacrylamide) and transferred to polyvinylidene difluoride membrane (PVDF). PVDFG membranes were blocked in Tris-buffered saline with 0.1 % tween (TBST) with 5% (w/v) skim milk powder at room temperature for 30 min. Primary antibody (rabbit anti-GFP, polyclonal, Invitrogen) was diluted 4000 × in TBST with 5% milk and incubated with membranes at 4°C overnight. The membranes were washed three times (TBST, 5 min at room temperature) before 1 h of incubation with secondary antibody at room temperature (mouse anti-rabbit, Santa Cruz, 5000 × diluted in TBST). After three additional washes (TBST, 5 min at room temperature), the membranes were incubated with chemiluminescence detection reagents (Amersham) for 5 min in the dark and visualized in a LI-COR C-DiGit chemiluminescence channel with “high sensitivity”. Start codon usage analysis A local blast DNA database was created for each L. monocytogenes genome sequence that was deposited at NCBI database (n= 60,690; access date: 24 th May 2024). Fasta format protein sequences of L. monocytogenes strain EGD-e were extracted from Listiwiki [ 73 ] to use as a query (each protein sequence was used as query in tblastn to search for matching DNA sequence in each DNA database). The “-seg” option was enabled for tblastn to obtain full length genes that have N-terminal amino acid sequences of low complexity. The subject sequence name and coordinate of each top tblastn hits was recorded, and subsequently used for extracting DNA sequence from fasta format genome sequences. A gene is considered present in a genome if tblastn retrieves full length protein sequence from the DNA database of this genome. The first three nucleotides in each extracted DNA sequence were recorded as the start codon. For each genome, in silico multi-locus sequence typing was performed using mlst [ 70 ]. CC and lineage was assigned by cross checking sequence types to the MLST typing scheme: “listeria_2”. 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