T2SS-peptidase XcpA associated with LasR evolutional phenotypic variations provides a fitness advantage to Pseudomonas aeruginosa PAO1

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa possesses hierarchical quorum sensing (QS) systems. The intricate QS network of P. aeruginosa synchronizes a suite of virulence factors, contributing to the mortality and morbidity linked to the pathogenicity of this bacterium. Previous studies have revealed that variations in the lasR gene are frequently observed in chronic isolates of cystic fibrosis (CF). Specifically, LasRQ45stop was identified as a common variant among CF, lasR mutants during statistical analysis of the clinical lasR mutants in the database. In this study, we introduced LasRQ45stop into the chromosome of P. aeruginosa PAO1 through allelic replacement. The social traits of PAO1 LasRQ45stop were found to be equivalent to those of PAO1 LasR-null isolates. By co-evolving with the wild-type in caseinate broth, elastase-phenotypic-variability variants were derived from the LasRQ45stop subpopulation. Upon further examination of four LasRQ45stop sublines, we determined that the variation of T2SS-peptidase xcpA and mexT genes plays a pivotal role in the divergence of various phenotypes, including public goods elastase secretion and other pathogenicity traits. Furthermore, XcpA mutants demonstrated a fitness advantage compared to parent strains during co-evolution. Numerous phenotypic variations were associated with subline-specific genetic alterations. Collectively, these findings suggest that even within the same parental subline, there is ongoing microevolution of individual mutational trajectory diversity during adaptation.

Through genetic mutations and modifications in its quorum sensing regulatory genes, P. aeruginosa can induce phenotypic changes, preparing the bacterium for diverse environments, including the CF lung (Chugani et al., 2012;Folkesson et al., 2012).One such adaptation in chronic CF isolates is the mutational inactivation of the lasR gene.Often, LasR loss-of-function mutations trigger AHL and LasB-negative phenotypes in chronic P. aeruginosa CF isolates (Seed et al., 1995;Kiratisin et al., 2002;Wang et al., 2018).When QS is vital for growth in the P. aeruginosa PAO1 background, LasR mutants gain a fitness advantage due to reduced metabolic costs when co-cultured with wild-type P. aeruginosa PAO1.This wild-type provides shared public goods for the entire cell population (Schuster et al., 2003).Yet, if the LasR mutant population surpasses a certain threshold, it collapses (Wang et al., 2015).Introducing C4-HSL or co-evolving bacteria that produce C4-HSL with PAO1 lasR mutants swiftly leads to populations with an active RhlIR QS system (Kostylev et al., 2019).Many lasR mutant clinical isolates still respond to AHL via the RhlRI system (Chen et al., 2019;Cruz et al., 2020).Today, advancements in high-throughput DNA sequencing technologies allow millions of DNA sequences from a single microbial sample.This technology has evolved into an efficient tool for determining genetic adaptations in infected host populations (Kostylev et al., 2019).
Previous studies noted that LasR locus analysis of P. aeruginosa isolates from CF patients identified LasR Q45stop as a frequent mutation site (Cruz et al., 2020).This research aims to explore the evolutionary pathways in lab conditions focusing on the highfrequency mutation site of the LasR Q45stop variation, which has a single substitution at base 133 in lasR.Laboratory-induced mutations of LasR Q45stop in strain PAO1 indicate it as a nonfunctional LasR.When co-cultured in casein broth with cooperator PAO1, LasR Q45stop spurred the swift emergence of proteolysis-associated mutant variants.Multiple variants displayed characteristics similar to CF clinical isolates, including a functional RhlI-RhlR system due to a mexT mutation.Our study identified the xcpA mutant exhibiting numerous virulence-related attributes, including reduced extracellular protein production and twitching motility.Additionally, we present lab experiments elucidating why mexT and xcpA are prevalent in the context of LasR Q45stop evolution.

Bacterial strains, media, and growth conditions
Bacterial strains and plasmids used are described in the Supplementary Table S1.P. aeruginosa was cultivated in Luria-Bertani [0.5% (w/v) yeast extract, 1% (w/v) tryptone, and 1% (w/v) sodium chloride] broth with a pH of 6.8, using 50 mM 3-(N-morpholino) propanesulfonic acid as a buffer (LB-Mops broth), PM broth with an addition of 1% sodium caseinate (casein broth) (Kostylev et al., 2019), or Pseudomonas P broth [0.14% (w/v) magnesium chloride, 2% (w/v) pancreatic digest of gelatin, and 1% (w/v) potassium sulfate].Several types of agar were used for plating purposes, including LB agar, and skim milk agar (Sandoz et al., 2007).Typically, 100 μg/mL of gentamicin, 50 μg/mL of kanamycin, or 300 μg/mL of carboxybenzylmycin was added to the medium.Bacteria were subcultured in 14 mm Falcon tubes with fresh LB or LB-Mops broth and incubated at 37°C with shaking at 250 rpm.Escherichia coli used in the cloning process was grown in LB broth.

Strain construction
Our investigations utilized the P. aeruginosa PAO1 strain (Stover et al., 2000), which differs from the currently sequenced strain due to an 8 bp deletion at the mexT gene.Mutants with deletions of lasR, lasR Q45stop , psdR, mexT, xcpA, and lasB were derived by cloning PAO1 using the suicide vector pEXG2, as previously described (Hmelo et al., 2015).The Supplementary Table S1, lists the primers used to construct the pEXG2 knockout plasmids.Reverse-transcriptase PCR of genomic DNA confirmed all deletion mutations.To compensate for the mutations, we integrated wild-type copies of genes from pUC18-mini-Tn7-Gm, as previously reported (Choi et al., 2005).

Assays for LasR activity, Cyanide and pyocyanin production
We measured promoter activity in cells cultured in LB-MOPS broth, as previously reported (Feltner et al., 2016), using lasI-gfp reporter strains in a 96-well plate.These were incubated at 37°C with continuous shaking in a Synergy H1 microplate reader (Biotec, America).GFP fluorescence (excitation at 485 nm, emission at 535 nm) and cell density (OD 600 ) were recorded at 2 h intervals for 28 h (refer to Supplementary Table S1 in the Supplementary material).For the detection of cyanide, we employed a cyanogenic test paper approach detailed previously (Chen et al., 2019).Pyocyanin activity was estimated using the modified protocol of O' Loughlin et al. (2013).The P. aeruginosa PAO1 strain was cultured overnight and subsequently diluted to an OD 600 of 0.01, followed by incubation in 3 mL of P broth medium for 24 h.

Motility assays
This study assessed swarming motility using a medium composed of nutritional broth [0.8% (w/v)] supplemented with D-glucose [0.5% (w/v)] and solidified with 0.5% (w/v) Bacto Agar.Overnight liquid broth cultures were adjusted to an optical density of 1.0 before 10.3389/fmicb.2023.1256785Frontiers in Microbiology 03 frontiersin.orginoculation onto plates.Growth was evaluated after 18-24 h of incubation at 37°C.After 24 h of growth in LB medium containing 1% solidified Bacto Agar at 37°C, the agar was gently detached, revealing a twitching motility zone adhered to the petri dish.A 10 min stain with 1% (wt/vol) crystal violet made the twitching zones visible, and their diameters were subsequently measured (Liu et al., 2022).

QS signal measurements
Quorum sensing signal extraction was carried out as described (Dong et al., 2008).The bacterium was cultivated in LB-Mops broth for 18 h, using 3 mL.Subsequently, an equal volume of acidified ethyl acetate (0.1% acetic acid) was combined with 3 mL of culture supernatant for signal extraction.Dried samples were reconstituted in 200 μL methanol and immediately filtered before LC-MS/MS analysis.We isolated and selected the prevalent signaling molecules for quantitative LC-MS/MS investigation.Modifications to the LC-MS procedure were made (Liu et al., 2022).HPLC was conducted using a Waters X Select HSS T3 1.8 μm phase column (2.1 × 100 mm) on the Dionex Ultimate 3000 system (Thermo Fisher Scientific).Metabolites identified included 3-oxo-C12-HSL (m/z 298.20128) and C4-HSL (m/z 172.09682).Data evaluation was executed with TraceFinder and Thermo Xcalibur, both from Thermo Fisher Scientific.

Assessment of extracellular protease production
Bacteria grown on LB broth milk agar plates were incubated for 30 h at 37°C.The amount of extracellular protease produced was quantified by measuring the area of the proteolytic zone surrounding the bacterial colony.

Competition experiments
Under these conditions, the gentamicin-resistance marker is neutral.We carried out competitive studies using P. aeruginosa strains both with and without this marker.The proportion of the competitor bearing the gentamicin-resistance marker was set at 1%, 10%, 50%, or 90% in casein broth for 48 h.To ascertain cell yields, plate counting, both with and without 10 μg/mL gentamicin, was employed to gauge proportional abundances after 48 h of incubation.The relative fitness was calculated by dividing the ratio of mutant to wild type after 48 h by the ratio at the time of inoculation.

Social evolution experiments
For evolution experiments (Supplementary Figure S2C), log-phase LB-Mops cultures of both cooperator and competitor were prepared, adding the competitor to the casein broth at a ratio of 0.1 (total 100 μL).Two days post primary inoculation, once visible proliferation was observed, a 60 μL volume of the transfer inoculum was introduced daily into a 3 mL casein broth.Serial dilutions of isolates were grown on LB agar plates both with and without 10 μg/mL gentamicin to determine the percentage of protease-deficient cells.Cultures were sampled from LB agar plates containing 10 μg/mL gentamicin, and colonies were then streaked daily onto skim milk agar plates to detect protease-altering variations.

Genome variant analysis
Bacteria were isolated from individual colonies and cultured in LB broth with a buffer for a duration of 24 h.Cells were collected for analysis via centrifugation.Bacteria were cultivated in LB-Mops broth, and genomic DNA was extracted using the Easy Pure Bacteria Genomic DNA Kit (TRAN).The genomic DNA of the four sublines (LasR Q45stop -1, LasR Q45stop -2, LasR Q45stop -3, and LasR Q45stop -4) was sequenced with the Illumina NovaSeq S4 PE-150 (Novogene, China).Whole Genomics Solution Corporation conducted the wholegenome sequencing and assembly.PCR testing was employed to validate the genomic sequencing results.

Statistical analysis
Results are depicted as mean ± SD.Experimental groups were evaluated using an unpaired student's t-test (one-tailed, unequal variance).Differences with a p-value <0.05 were considered significant.Experiments were repeated to confirm their consistency.

Results
The high-frequency mutant LasR Q45stop presents fitness advantages over WT The lasR C133T substitution has been identified as a recurring mutation site in P. aeruginosa CF isolates (Feltner et al., 2016).This single-base substitution introduces a premature stop codon into the lasR sequence, terminating protein translation prematurely.This mutation was henceforth referred to as LasR Q45stop .To validate the functionality of LasR Q45stop , we generated a knock-out deletion mutant LasR, an allelic replacement mutant LasR Q45stop , and a LasR complementation strain C-LasR Q45stop in the laboratory model strain P. aeruginosa PAO1.Comparing the QS-associated traits of the strains mentioned, we found that the homologous LasR Q45stop mutation in P. aeruginosa acts as a nonfunctional LasR in QS-associated phenotypes, such as elastase secretion, pyocyanin production, and 3OC12-HSL and C4-HSL levels (Figures 1A-F).
To ascertain earlier studies indicated that LasR mutants possess fitness advantages over WT cells when co-cultured in minimal medium with casein (casein broth) as the sole carbon source.However, this advantage was negated with the addition of adenosine to the casein medium (Feng et al., 2019).To determine if LasR Q45stop exhibited superior fitness to WT, we co-cultured the gentamicin-resistant PAO1 LasR Q45stop mutant alongside WT in casein broth.LasR Q45stop mutants were then distinguished and counted from WT using plates with or without gentamicin resistance.The schematic diagram (Supplementary Figure S2C) illustrates that 1% competitors (LasR Q45stop or LasR) were individually combined with 99% of WT in the initial casein broth, then passaged daily, and the frequency of mutant strains was subsequently monitored.Pyocyanin production during the initial days of the evolution process No noticeable alteration in the casein medium post-day 8 signified a population collapse (Figure 1G).Consistent with expectations, the proportion of LasR Q45stop mutants surged, peaking at nearly 70% in cultures (Figure 1H).This shift in frequency suggests that the LasR Q45stop mutant can act as a social cheater when co-cultured with WT in a caseinbased medium.

Mutations in mexT and xcpA are associated with the post-lasR-deficient evolution phenotype
To uncover the genetic variations responsible for the observed differences in virulence-associated phenotypes, we conducted a genome-wide association study among the parent LasR Q45stop and four protease-altered mutants (LasR Q45stop -1, LasR Q45stop -2, LasR Q45stop -3, and LasR Q45stop -4).This analysis identified three genes: mexT (PA2492), xcpA (PA4528), and psdR (PA4499) (Table 1).The single base indel or in-frame deletion in the xcpA gene (Table 1) was found in LasR Q45stop -1, LasR Q45stop -2, and LasR Q45stop -4.Conversely, a missense variant in the mexT gene was identified in LasR Q45stop -3 (Table 1).The LasR Q45stop -4 subline displayed additional structural variations (SVs), culminating in the deletion of the entire mexT gene and adjacent genes spanning from PA2259 to PA2544.This range is part of the 38.3 kb deletion observed in LasR Q45stop -4.Furthermore, we noted that all four sequenced isolates carried a nonsynonymous mutation in psdR, aligning with prior research that identified frequent psdR mutations in casein evolution experiments (Asfahl et al., 2015;Kostylev et al., 2019).Nevertheless, psdR has been demonstrated to enhance growth on casein independently of QS (Asfahl et al., 2015).We thus infer that MexT and XcpA are instrumental in the observed virulence-associated phenotypic variations in the evolved isolates.
To substantiate this, we engineered MexT-and XcpA-related mutants using LasR Q45stop -PsdR as the progenitor strain.Our results show that LasR Q45stop -PsdR-MexT and LasR Q45stop -PsdR-MexT A1044G mutants exhibited markedly elevated pyocyanin and elastase production (Figure 3), as well as augmented swarming motility (Figure 3).Deletion of xcpA from both LasR Q45stop -PsdR and LasR Q45stop -PsdR-MexT mutants led to reduced QS-dependent elastase phenotypes and twitching motility (Figure 3).Alterations in mexT have been highlighted as pivotal in reshaping the QS circuits and the fitness of P. aeruginosa PAO1 (Kostylev et al., 2019).XcpA is a prepilin peptidase that is part of the Xcp type II secretion system (T2SS) in P. aeruginosa.Furthermore, the Xcp T2SS is governed by the QS pathway, which P. aeruginosa utilizes for secreting major virulence factors, including LasA and LasB elastases (Li and Lee, 2019).Therefore, we conclude that the xcpA and mexT mutations underpin the evolved phenotype, at least under laboratory conditions.

The xcpA mutant gains a fitness advantage in caseinate
Mutants devoid of the metabolic cost of QS could possess a negative frequency-dependent fitness advantage over the parental strain when co-cultured in an environment where QS function is essential for growth.In previous research, LasR-MexT mutants exhibited a competitive advantage over the parental LasR mutant (Kostylev et al., 2019).Similarly, we postulated that XcpA and LasB mutants might also harbor a competitive edge.We co-cultured each identified mutant with the parental strain, starting at a frequency of 10%, to evaluate their relative fitness in PM-caseinate.Distinguished mutants were marked as gentamicin-resistant (Gm R ).Relative fitness was ascertained by taking the ratio of mutant-to-parent bacteria at the endpoint and dividing it by the initial mutant-to-parent bacteria ratio (Chen et al., 2019).A control was set up with unlabeled and labeled parental strains, yielding a relative fitness of 1 (Figure 4A).Conversely, the relative fitness of the XcpA mutant, when pitted against LasR Q45stop -PsdR-MexT or WT, exceeded 1, indicating a survival advantage in the XcpA mutant.However, the LasB mutant, which only lacked elastase production, exhibited a fitness comparable to the LasR Q45stop -PsdR-MexT parent strain (Figure 4A).Additionally, varying proportions of XcpA mutants were co-cultured with their parent strains in PM-caseinate for 48 h to determine their frequency-dependent fitness.When XcpA mutants were more abundant relative to LasR Q45stop -PsdR-MexT in the culture, their relative fitness was less than 1.However, when LasR Q45stop -PsdR-MexT bacteria predominated over the mutant in the starting inoculum, the relative fitness for XcpA exceeded 1 (Figure 4B).The relative fitness of the mutants declines as the mutant subpopulation expands (Diggle et al., 2007).These observations underscore that XcpA mutants can effectively invade their ancestral populations under conditions that require QS.

Discussion
P. aeruginosa, a well-known human pathogen, is particularly adept at infecting immunocompromised individuals, leading to severe infections (Moradali et al., 2017).Early work highlighted the complexity of interconnected quorum sensing (QS) regulatory circuits in controlling virulence phenotypes and adapting to complex environments (Willcox et al., 2008).The QS transcription factor LasR sits atop this hierarchy, regulating the expression of the RhlIR and PQS pathways (Cruz et al., 2020).LasR mutations have been shown to be prevalent in P. aeruginosa during both in vitro and in vivo evolution.Analysis of the associated lasR sequences from CF isolates (Feltner et al., 2016) indicated that LasR Q45stop is a high-frequency mutant (Supplementary Figure S1 and Supplementary Table S2).QS-regulated biological phenotypic assessments were diminished in LasR Q45stop mutants.However, these assessments were restored to wildtype levels upon the addition of functional LasR (Figure 1).This suggests that LasR Q45stop might function as a QS antagonist, inhibiting P. aeruginosa infection.Paradoxically, inactivating LasR mutations are frequently detected in isolates from chronic CF infections, suggesting an evolutionary selection favoring LasR mutants (Cruz et al., 2020).
Recent research indicates that defects in the LasIR system can result in the evolution of MexT and PqsR mutations under laboratory conditions, leading to reactivation of P. aeruginosa QS regulatory circuits through rewiring (Chen et al., 2019;Kostylev et al., 2019).Phenotypic and genotypic microevolution result from niche adaptation to the CF lung environment and competition among different microbiomes.Micro-evolutionary diversity changes dynamically over time, complicating the accurate diagnosis and treatment of P. aeruginosa infections (Hwang et al., 2021).The inherent genetic diversity of P. aeruginosa has been elucidated due to its suitability for adaptive laboratory evolution experiments.We identified LasR Q45stop evolutionary trajectories caused by acquiring stable mutant phenotypes, leading to rapid adaptation in stressful and fluctuating environments.Our findings substantiate the implications of LasR Q45stop microevolution.We employed a combination of molecular biology and bioinformatics analyses to study the evolution of LasR Q45stop cessation.We demonstrated that LasR Q45stop promotes an increased frequency of psdR, mexT, and xcpA mutations when co-cultured in our caseinate broth (Table 1).The psdR mutation enhances individual fitness by alleviating repression of intracellular metabolism, limiting growth.This mutation has been frequently observed in evolution experiments involving casein (Asfahl et al., 2015;Kostylev et al., 2019).Even with psdR deletion in both cooperators and cheaters, the pattern of invasion by the LasR mutant remains largely unaffected (Feng et al., 2019).The mexT mutation isolates have been identified both in the lab and clinical settings (Liu et al., 2022), while xcpA mutations frequently appear in environmental isolates (Supplementary Table S3).Moreover, our data highlight the role of MexT and XcpA in the adaptation of P. aeruginosa.Microbial cells exhibit substantial adaptation to environmental stress (Hogardt and Heesemann, 2010).Certain pivotal mutations in the genome can significantly bolster bacterial populations in securing a competitive edge in hostile environments (Feng et al., 2019).
For survival in the environment and adaptation to hosts for nourishment and pathogenicity, P. aeruginosa deploys a diverse array of virulence factors (Vidaillac and Chotirmall, 2021).Among these, the T2SS-peptidase XcpA is involved in the secretion of guanylate cyclase ExoA, proteases LasA/B, and several other factors, many of which have been earmarked as potential therapeutic targets (Swietnicki et al., 2019).Importantly, the lasB gene encodes elastase, a pivotal virulence component of P. aeruginosa.Its synthesis is orchestrated by the QS system, while its secretion modus operandi is overseen by the T2SS, also referred to as the general secretory pathway (GSP) (Filloux, 2004).The 12 identified Gsp components of the P. aeruginosa machinery were initially labeled as XcpA and XcpP to-Z and later renamed as XcpA O and XcpP C to-Z M , aligning with other GSP system nomenclature (Bleves et al., 2010).XcpA, recognized as a prepilin peptidase, was initially discovered and described in P. aeruginosa.It is believed to excise leader peptides endopeptidically, methylate the N-terminal amino acid residue, and cleave Xcp pre-pseudopilin substrates (Filloux, 2011).Few studies have explored the essential biological activities of prepilin peptidase in bacteria (LaPointe and Taylor, 2000).Tn insertions in the xcpA genes of P. aeruginosa strain PA14 manifested positive selection during fitness for gastrointestinal (GI) colonization but predominantly displayed attenuated fitness for systemic dissemination amidst neutropenia.This observation stemmed from a reliable mouse model mirroring the trajectory of human infections in cancer and bone marrow transplant patients (Koh et al., 2005).In Methanococcus voltae, FlaK operates as a preflagellin peptidase; membranes of the flaK mutant exhibited inability to process preflagellin in vitro (Bardy and Jarrell, 2003).In Bacillus subtilis, ComC functions as a prepilin peptidase; its inactivation results in the accumulation of pre-ComGC, likely due to the unprocessed protein's inability to translocate outside the membrane (Chung and Dubnau, 1995).The significance of XcpA, a class of prepilin peptidase, in processing secretion systems has been documented.Pathogenic bacteria and environmental entities could capitalize on the extracellular dispatch of an array of toxins, lipases, and enzymes adept at breaking down complex materials, mediated through the secretory system (Korotkov and Sandkvist, 2019).
This study sheds light on the role of XcpA in P. aeruginosa evolutionary paths.Our findings indicate that the XcpA mutation confers a fitness advantage.When mutant XcpA is present in PsdR-LasR Q45stop -MexT, it reduces exoprotein secretion and enhances individual fitness.Although the physiological value of xcpA-associated microevolution is not fully detailed in this study, we delve into its role in fitness and intraspecific competition in casein broth.Our evaluation scope is limited to studying individual LasR variants and their evolution with casein proteins under laboratory conditions.Notably, pathogenic adaptation mutations are not consistent across studies, suggesting diverse evolutionary paths of pathogenic adaptation in P. aeruginosa.Still, our findings pave the way for understanding LasR's evolutionary paths under lab conditions and potential further studies on XcpA in population evolution.
10.3389/fmicb.2023.1256785Frontiers in Microbiology 04 frontiersin.orghinted at the robust growth of the PAO1 co-culture.Pyocyanin levels tapered off from day 4 to day 6, culminating in a milky culture by day 7.

FIGURE 1
FIGURE 1 Characteristics in QS regulon of LasR Q45stop in solitary presence and co-culture states.(A) Protease production on casein agar.The PAO1 LasR mutant and the LasR Q45stop mutant produce relatively little protease.All data shown are the average values of three independent experiments ± SD. (B) Cyanide production by P. aeruginosa strains and mutants.Cyanide was monitored as described in Materials and methods.The blue coloration of the filter paper reflects cyanide production.(C) Swarming motility phenotype of PAO1 wild-type and LasR mutants.All data shown are the average values of three independent experiments ± SD. (D) Color changes of P. aeruginosa cultures in the presence of LasR mutant after 24 h, a reflection of the effect of LasR mutant on phenazine production.(E) LasR activity in the LasR mutant and PAO1 measured as GFP fluorescence (RFU).All strains contained the PlasIgfp fusion plasmid pProbe-GT-PlasIgfp.(F) Relative AHL levels of the indicated strain.The AHL levels produced by strain PAO1 are defined as 100%.AHL was measured after 18 h of growth in LB-Mops broth.All data shown are the average values of three independent experiments ± SD. (G) An example of one serial transfer experiment.Each photographic image was captured after 1 day of growth.The relative abundance of LasR mutant (as a percentage of the total population) in culture samples just before transfer (60 μL) is distinguished by resistance markers, and the daily passage number is shown below the tubes.Cultures that only partially degrade casein have a milky white appearance due to protein precipitation.(H) Competition between WT PAO1 and LasR mutant (black), or LasR Q45stop mutant (red).The initial frequency of LasR mutant was 0.01.Three biological replicates were performed.All data shown are the average values of three independent experiments ± SD.Two-tailed p-values were determined using unpaired ttests.Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, no significant difference (p ≥ 0.05).

FIGURE 2
FIGURE 2 (A) Protease production of isolates from co-cultured casein broth was measured on plates containing 1% (m/v) skimmed milk for 24 h.(B,C) Motility phenotypes of P. aeruginosa PAO1 and its isogenic QS LasR Q45stop strains.Image showing the comparative zone of twitching (B) and swarming motility (C) of PAO1 and its isogenic QS LasR Q45stop strains.All data shown are the average values of three independent experiments ± SD. (D,E) Cultures of P. aeruginosa PAO1, a LasR Q45stop mutant, and the evolved LasR Q45stop variants, grown in LP medium for 24 h (D) and casein broth for 48 h (E).The culture is visibly blue as a result of pyocyanin overproduction.Three biological replicates were performed.All data shown are the average values of three independent experiments ± SD.Two-tailed p-values were determined using unpaired t-tests.Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, no significant difference (p ≥ 0.05).Scale bar = 10 mm.

FIGURE 3 mexT
FIGURE 3 mexT and xcpA was related to the phenotype changes.(A) The influence of PsdR, MexT, and XcpA mutations on protease production in the P. aeruginosa LasR Q45stop mutant.The images are of colonies after about 24 h of incubation in milk agar plate.The influence of PsdR, MexT, and XcpA mutations on twitching (B) and swarming (C) motility phenotypes in the P. aeruginosa LasR Q45stop mutant.Three biological replicates were performed.All data shown are the average values of three independent experiments ± SD.Two-tailed p-values were determined using unpaired t-tests.Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, no significant difference (p ≥ 0.05).Scale bar = 10 mm.

TABLE 1
Summary of xcpA and mexT variants found in in protease-modified variants of PAO1 LasR Q45stop isolates that emerged during growth in caseinate.Variants LasR Q45stop -1 to LasR Q45stop -4 were identified in LasR Q45stop protease-modified isolates that emerged during growth in caseinate and were initially inoculated with the WT only."D" denotes base deletion; numbers specify the nucleotide position of the mutation.